Roles of testosterone and amygdaloid LTP induction in determining sex differences in fear memory magnitude.

Women are thought to form fear memory more robust than men do and testosterone is suspected to play a role in determining such a sex difference. Mouse cued fear freezing was used to study the sex-related susceptibility and the role of testosterone in fear memory in humans. A 75-dB tone was found to provoke weak freezing, while 0.15-mA and 0.20-mA footshock caused strong freezing responses. No sex differences were noticed in the tone- or footshock-induced (naïve fear) freezing. Following the conditionings, female mice exhibited greater tone (cued fear)-induced freezing than did male mice. Nonetheless, female mice demonstrated indistinctive cued fear freezing across the estrous phases and ovariectomy did not affect such freezing in female mice. Orchidectomy enhanced the cued fear freezing in male mice. Systemic testosterone administrations and an intra-lateral nucleus of amygdala (LA) testosterone infusion diminished the cued fear freezing in orchidectomized male mice, while pretreatment with flutamide (Flu) eradicated these effects. Long-term potentiation (LTP) magnitude in LA has been known to correlate with the strength of the cued fear conditioning. We found that LA LTP magnitude was indeed greater in female than male mice. Orchidectomy enhanced LTP magnitude in males' LA, while ovariectomy decreased LTP magnitude in females' LA. Testosterone decreased LTP magnitude in orchidectomized males' LA and estradiol enhanced LTP magnitude in ovariectomized females' LA. Finally, male mice had lower LA GluR1 expression than female mice and orchidectomy enhanced the GluR1 expression in male mice. These findings, taken together, suggest that testosterone plays a critical role in rendering the sex differences in the cued fear freezing and LA LTP. Testosterone is negatively associated with LA LTP and the cued fear memory in male mice. However, ovarian hormones and LA LTP are loosely associated with the cued fear memory in female mice.

D-cycloserine, sarcosine and D-serine diminish the expression of cocaine-induced conditioned place preference.

Reactivation of cocaine-associated memories plays a critical role in reinstating the cocaine-seeking behavior and causing relapse. Cocaine-induced conditioned place preference (CPP) was used as a behavioral paradigm indicative of cocaine-associated memory and repeated cocaine-free preference tests served as a behavioral procedure to retrieve such a memory in this study. Since D-cycloserine was reported to eradicate drug-associated memories, two other N-methyl-D-aspartate (NMDA) receptor agonists were assessed for their efficacy on facilitating the extinction of cocaine-induced CPP. Although D-cycloserine (30 mg/kg) abolished cocaine (10 mg/kg)-induced CPP, sarcosine (300 and 600 mg/kg) and D-serine (600 mg/kg) diminished the expression of such a cocaine memory. Sarcosine (600 mg/kg) and D-serine (600 mg/kg) did not affect the storage of this cocaine memory. It was of interest to note that D-cycloserine facilitated the extinction of cocaine-induced CPP in a fast and early-onset manner, while sarcosine and D-serine decreased cocaine-induced CPP expression in a delay-onset manner. D-cycloserine (30 mg/kg), D-serine (600 mg/kg) and sarcosine (600 mg/kg) did not affect the consolidation of cocaine (5 mg/kg)-induced CPP. Finally, sarcosine (at 600 mg/kg/day for 3 consecutive days) and D-serine (at 600 mg/kg/day for 3 consecutive days) did not produce observable aversive effect associated with their administration in a conditioned place aversion paradigm. Likewise, a similar dosing regimen of sarcosine or D-serine did not cause evident activity-impairing effect. In addition to D-cycloserine treatment, our results indicate that long-term treatment with D-serine and sarcosine may afford a therapeutic advance in suppressing the expression of cocaine-associated memory.

Companions reverse stressor-induced decreases in neurogenesis and cocaine conditioning possibly by restoring BDNF and NGF levels in dentate gyrus.

The presence of companions can reverse the stressor-induced decrease in neurogenesis in mouse dentate gyrus (DG). In this study, we decided to study the underlying mechanisms of the companions' protective effect and to assess whether two DG neurogenesis-related memories, cocaine-induced conditioned place preference (CPP) and spatial memory, can be affected by our stressor and companions. Neurotrophin levels in DG were measured, in this regard, to reveal their roles in mediating the stressors' and companions' effect. We found that the stressor did not affect NT-3 but acutely decreased NGF and BDNF levels in DG. The presence of companions abolished these stressor-decreased NGF and BDNF levels. Neither the stressor nor the presence of companions affected TrkA, TrkB or TrkC expression in DG. Pre-exposure to the stressor rendered deficits in cocaine-induced CPP and spatial memory, while companions reversed the stressor-decreased cocaine-induced CPP. Intra-ventricular infusion with K252a, a mixed TrkA and TrkB antagonist, did not affect the protective effects of companions on local NGF, BDNF levels in DG, but abolished the companions' protective effects against the stressor-decreased DG neurogenesis and cocaine-induced CPP. Systemic pretreatment with 7,8-dihydroxyflavone (DHF), a selective TrkB agonist, did not affect baseline, the stressor-stimulated corticosterone (CORT) secretion or local NGF, BDNF levels in DG, but in part mimicked companions' protective effects. These results, taken together, indicate that stressor-decreased NGF and BDNF levels in DG could be involved in the stressor-decreased DG neurogenesis and cocaine conditioning. The presence of companions reverses the stressor-decreased DG neurogenesis and cocaine conditioning possibly by restoring BDNF and NGF levels in DG.

Interactions between amyloid-ß and hemoglobin: implications for amyloid plaque formation in Alzheimer's disease.

Accumulation of amyloid-ß (Aß) peptides in the brain is one of the central pathogenic events in Alzheimer's disease (AD). However, why and how Aß aggregates within the brain of AD patients remains elusive. Previously, we demonstrated hemoglobin (Hb) binds to Aß and co-localizes with the plaque and vascular amyloid deposits in post-mortem AD brains. In this study, we further characterize the interactions between Hb and Aß in vitro and in vivo and report the following observations: 1) the binding of Hb to Aß required iron-containing heme; 2) other heme-containing proteins, such as myoglobin and cytochrome C, also bound to Aß; 3) hemin-induced cytotoxicity was reduced in neuroblastoma cells by low levels of Aß; 4) Hb was detected in neurons and glial cells of post-mortem AD brains and was up-regulated in aging and APP/PS1 transgenic mice; 5) microinjection of human Hb into the dorsal hippocampi of the APP/PS1 transgenic mice induced the formation of an envelope-like structure composed of Aß surrounding the Hb droplets. Our results reveal an enhanced endogenous expression of Hb in aging brain cells, probably serving as a compensatory mechanism against hypoxia. In addition, Aß binds to Hb and other hemoproteins via the iron-containing heme moiety, thereby reducing Hb/heme/iron-induced cytotoxicity. As some of the brain Hb could be derived from the peripheral circulation due to a compromised blood-brain barrier frequently observed in aged and AD brains, our work also suggests the genesis of some plaques may be a consequence of sustained amyloid accretion at sites of vascular injury.

Disruption of memory reconsolidation impairs storage of other, non-reactivated memory.

Two hypotheses were tested in this study. First, blockade of neural activity by lidocaine immediately following the retrieval of a memory may impair the reconsolidation and subsequent expression of that memory. Second, a non-retrieved memory would not be affected by this lidocaine treatment. Since the basolateral nucleus of the amygdala (BLA) is involved in emotion-related memory, an intra-BLA lidocaine infusion was used immediately after the retrieval of two emotion-related memories, the step-through passive avoidance response (PA) and cocaine-induced conditioned place preference (CPP). Intra-BLA lidocaine infusion immediately after cocaine-induced CPP retrieval diminished CPP magnitude in retests. However, intra-BLA lidocaine infusion alone did not affect cocaine-induced CPP performance. Intra-BLA lidocaine infusion immediately after PA retrieval decreased PA performance in retests. Omission of PA retrieval procedure, intra-BLA lidocaine infusion did not affect subsequent PA performance. Surprisingly, intra-BLA lidocaine infusion immediately following the retrieval of PA or cocaine-induced CPP diminished both PA and cocaine-induced CPP performance in the retests. Finally, Fos-staining results revealed that a number of BLA neurons were activated by the retrieval of both cocaine-induced CPP and PA. We conclude that inactivation of neural activity in BLA immediately following retrieval of a fear or cocaine-conditioned memory can impair subsequent expression of both memories. More importantly, retrieval of a memory does not seem to be an absolute condition for rapidly changing the memory.

Involvement of amygdaloid protein synthesis in early- and late-phase reconsolidation of emotion-related memories.

This study was undertaken to test whether de novo protein synthesis in the basolateral (BLA) and central (CeA) nucleus of amygdala was required for reconsolidation of emotion-related memories. Mice were trained to sequentially acquire both cocaine-induced conditioned place preference (CPP) and step through passive avoidance (PA) memories. Immediately following PA retrieval, intra-BLA anisomycin infusion was found to decrease subsequent PA performance in retests. Immediately following PA retrieval, intra-CeA anisomycin infusion did not acutely affect PA performance but decreased such a PA memory 5 days later. Given PA retrieval procedure was omitted, intra-BLA and intra-CeA anisomycin infusion did not affect PA memory. Likewise, intra-BLA anisomycin infusion immediately following cocaine-induced CPP retrieval was found to decrease cocaine-induced CPP magnitude in early and late retests. Immediately after cocaine-induced CPP retrieval, intra-CeA anisomycin infusion did not acutely affect cocaine-induced CPP but decreased this memory 5 days later. Given the cocaine-induced CPP retrieval procedure was omitted, intra-BLA and intra-CeA anisomycin infusion did not affect cocaine-induced CPP in subsequent retests. These results, taken together, imply that de novo protein synthesis in amygdala plays an important role in modulating reconsolidation of emotion-related memory. More importantly, de novo protein synthesis in the BLA is essential for early phase reconsolidation of retrieved emotion-related memories. Protein synthesis in the CeA is required for late phase reconsolidation of retrieved emotion-related memories.

Odors from proximal species reverse the stress-decreased neurogenesis via main olfactory processing.

Unconditioned foot shock followed by restraint in water was used as a stress regimen to induce decreases in neurogenesis in mouse dentate gyrus (DG). Presence of conspecific odors has been known to reverse the stress-induced decrease in DG neurogenesis. In this study, we found that the conspecific odors did not produce these protective effects in mice whose MOE was impaired by nasal zinc sulfate lavage. Moreover, we observed that the presence of odors from rats, hamsters, and guinea pigs throughout the stress procedure reversed the stress-induced decrease in cell proliferation and neurogenesis in mouse dentate gyrus, while these odors alone did not affect mouse dentate cell proliferation or neurogenesis. In contrast, the presence of rabbit, sugar glider, hedgehog, beetle odors did not affect cell proliferation, neurogenesis, the stress-decreased cell proliferation or neurogenesis in DG. Finally, the presence of fox urine odors decreased mouse dentate cell proliferation and neurogenesis but did not affect the stress-induced decrease in cell proliferation or neurogenesis. Taken together, we conclude that olfactory processing via activation of sensory neurons in MOE is responsible for the conspecific odor-produced protective effect against the stress-decreased cell proliferation and neurogenesis. Phylogenetic distances of the odor-generating species and mice might contribute to the odors' protective effects against the stress-induced decreases in cell proliferation and neurogenesis.

Accumbal 14-3-3ζ protein downregulation is associated with cocaine-conditioned memory.

Cocaine-conditioned memory has been known to cause cocaine craving and relapse, while its underlying mechanisms remain unclear. We explored accumbal protein candidates responsible for a cocaine-conditioned memory, cocaine-induced conditioned place preference (CPP). Two-dimensional gel electrophoresis in conjunction with liquid chromatography mass spectrometry analysis was utilized to identify accumbal protein candidates involved in the retrieval of cocaine-induced CPP. Among the identified candidate proteins, a downregulated 14-3-3ζ protein was chosen and confirmed by Western immunoblotting. A polymer-mediated plasmid DNA delivery system was then used to overexpress 14-3-3 protein in mouse nucleus accumbens before the CPP retrieval tests. Overexpression of accumbal 14-3-3ζ protein was found to diminish conditioned cue/context-mediated cocaine-induced CPP. In contrast, another isoform of 14-3-3 protein, 14-3-3ε protein, did not affect conditioned cue/context-mediated cocaine-induced CPP. Overexpression of accumbal 14-3-3ζ protein did not produce motor activity-impairing effect or alter local dopamine metabolism. Moreover, overexpression of accumbal 14-3-3ζ protein did not affect food-induced CPP. These results, taken together, indicated that overexpressed accumbal 14-3-3ζ protein specifically decreased conditioned cue/context-mediated cocaine memory. Further understanding of the function of accumbal 14-3-3ζ protein may shed light on the treatment of cocaine craving and relapse.

Repeated co-administrations of alcohol- and methamphetamine-produced anxiogenic effect could be associated with the neurotoxicity in the dentate gyrus.

To date, joint use of alcohol (EtOH) and methamphetamine (MA) represents a specific combination of polydrug abuse. Repeated administrations of EtOH, MA, and combined EtOH and MA were assessed for their effects on brain cell toxicity, cell mitosis and anxiety/depression-like behavior. We found that repeated co-administrations of EtOH and MA produced profound anxiogenic effects. Specifically, combined EtOH and MA decreased open arm exploratory responses in the elevated plus maze test. Moreover, combined EtOH and MA significantly decreased immobile responses in the tail suspension test. MA, EtOH, and their combination all reduced the number of NeuN-positive cells in amygdala (Amg), while neither of them altered the number of NeuN-positive cells in striatum (St) or prefrontal cortex (PFC). Combined EtOH and MA decreased the number of NeuN-positive cells in dentate gyrus (DG). EtOH, MA, and combined EtOH and MA all diminished comparable number of GFAP-positive cells in Amg, DG, and St. Neither of these treatment affected the number of GFAP-positive cells in PFC. EtOH, MA, and combined EtOH and MA generated comparable inhibiting effects on cell proliferation in the subventricular zone (SVZ) and DG. These results, taken together, suggest that repeated co-administrations of MA and EtOH may produce an observable anxiogenic effect. This combination-produced anxiogenic effect could be associated with neuronal loss in the dentate gurus. In contrast, such an anxiogenic effect is less likely related to this combination-caused glial toxicity in limbic regions or cell proliferation-inhibiting effect in the SVZ or DG.

Presence of conspecifics and their odor-impregnated objects reverse stress-decreased neurogenesis in mouse dentate gyrus.

Stress and corticosterone level are thought to negatively associate with neurogenesis in mammalian brains. Social support can diminish many adverse effects of stress. The present study examined the modulating effect of social support on stress-decreased cell proliferation and neuronal differentiation in a mouse model. A randomly-scheduled foot shock followed by restraint in water was used as a profound stress-provoking regimen. Bromodeoxyuridine (BrdU) staining was used to indicate newly mitotic cells and doublecortin (DCx) staining was used to reveal immature neurons. This stress-provoking regimen rapidly decreased BrdU- and BrdU/DCx-labeled cells in the dentate gyrus. However, such a stress-provoking regimen did not affect the number of these labeled cells in the subventricular zone. Familiar and unfamiliar mice' company throughout the stress regimen completely reversed the stress-decreased cell proliferation and neuronal genesis in the dentate gyrus. Likewise, both odor-familiar (from their home cages) and -unfamiliar (from cages other than their home cages) wooden blocks completely reversed the stress-decreased BrdU/DCx-labeled cells in the dentate gyrus. In contrast, wooden blocks free of any odor and camphor odor alone failed to affect the stress-decreased BrdU- or BrdU/DCx-labeled cells. Finally, we showed that conspecifics or their odors during the stress regimen reversed the stress-decreased cell proliferation and neuronal differentiation in the dentate gyrus via a corticosterone-independent mechanism. We conclude that stress and familiarity distinctively affect neurogenesis in the dentate gyrus and subventricular zone. Conspecific companions or presence of their odors reverse stress-decreased neurogenesis in the dentate gyrus, suggesting that social support during stress exposure may improve neurogenesis-related psychological functions.

Chronic treatment with monoamine oxidase-B inhibitors decreases cocaine reward in mice.

RATIONALE AND OBJECTIVE: Whether monoamine oxidase inhibitors (MAOIs) can be used to suppress the reinforcing effect of cocaine remains unknown. This study was undertaken to examine effects of a long-term dosing regimen with selective MAOIs on cocaine and food reward. MATERIALS AND METHODS: Since single dose of clorgyline (2 mg/kg), deprenyl (1 mg/kg), and pargyline (10 mg/kg) did not acutely affect mouse locomotor activity, these doses were chosen to treat the male C57BL/6j mice on a daily basis. RESULTS: Fourteen consecutive days of pretreatments with clorgyline, deprenyl, or pargyline (one injection per day) did not affect natural reward-supported operant behavior, since acquisition of the lever pressing responses for food pellets under an FR-1 protocol did not differ among these drug- and saline-treated mice. Likewise, 24 consecutive days of pretreatments with clorgyline did not alter acquisition of the cocaine (0.3 mg/kg per infusion)-supported operant responses under an FR-1 protocol. In contrast, 24 days of pretreatments with deprenyl and pargyline abolished the cocaine-supported operant responses under a similar protocol. Twenty-four days of clorgyline treatment enhanced serotonin contents in striatum, nucleus accumbens, and frontal cortex. Frontal cortical 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacidic acid concentrations were decreased following 24 days of pretreatments with deprenyl and pargyline. These changes were not evident in mice pretreated with clorgyline. CONCLUSIONS: We suggest that long-term treatments with MAO-B inhibitors may decrease cocaine-supported operant responses in cocaine-naïve mice by selectively decreasing frontal cortical metabolism of dopamine and serotonin.

Medial prefrontal cortex and nucleus accumbens core are involved in retrieval of the methamphetamine-associated memory.

Immunohistochemical Fos staining has proven to be a method to identify the neurons that are activated by stimulation. Although methamphetamine (MA)-conditioned place preference (CPP) memory was long-lasting, how this memory was established and retrieved remained unknown. We used the vehicle- and MA-conditioned environment (including cues and context) to reactivate the MA-CPP memory in mice. In the limbic system, Fos-positive neurons were examined following retrieval of the MA-CPP memory. We demonstrated that the current conditioning procedure produced reliable MA-CPP performance. Moreover, enhanced Fos expressions were found in the medial prefrontal cortex and the core of the nucleus accumbens after reactivation of the MA-CPP memory. Furthermore, familiarity with the environmental cues/context was found to significantly enhance Fos expressions in dorsal striatum and dentate gyrus. Nucleus accumbens shell, basolateral or lateral amygdala, in this regard, did not seem to be involved in retrieval of the MA-CPP memory. These results, taken together, suggest that the medial prefrontal cortex and the core of the nucleus accumbens are anatomical substrates responsible for reactivation of the MA-CPP memory.