Mapping parahippocampal systems for recognition and recency memory in the absence of the rat hippocampus

The present study examined immediate‐early gene expression in the perirhinal cortex of rats with hippocampal lesions. The goal was to test those models of recognition memory which assume that the perirhinal cortex can function independently of the hippocampus. The c‐fos gene was targeted, as its expression in the perirhinal cortex is strongly associated with recognition memory. Four groups of rats were examined. Rats with hippocampal lesions and their surgical controls were given either a recognition memory task (novel vs. familiar objects) or a relative recency task (objects with differing degrees of familiarity). Perirhinal Fos expression in the hippocampal‐lesioned groups correlated with both recognition and recency performance. The hippocampal lesions, however, had no apparent effect on overall levels of perirhinal or entorhinal cortex c‐fos expression in response to novel objects, with only restricted effects being seen in the recency condition. Network analyses showed that whereas the patterns of parahippocampal interactions were differentially affected by novel or familiar objects, these correlated networks were not altered by hippocampal lesions. Additional analyses in control rats revealed two modes of correlated medial temporal activation. Novel stimuli recruited the pathway from the lateral entorhinal cortex (cortical layer II or III) to hippocampal field CA3, and thence to CA1. Familiar stimuli recruited the direct pathway from the lateral entorhinal cortex (principally layer III) to CA1. The present findings not only reveal the independence from the hippocampus of some perirhinal systems associated with recognition memory, but also show how novel stimuli engage hippocampal subfields in qualitatively different ways from familiar stimuli.     

Qualitatively different modes of perirhinal–hippocampal engagement when rats explore novel vs. familiar objects as revealed by c‐Fos imaging

Expression of the immediate‐early gene c‐fos was used to test for different patterns of temporal lobe interactions when rats explore either novel or familiar objects. A new behavioural test of recognition memory was first devised to generate robust levels of novelty discrimination and to provide a matched control condition using familiar objects. Increased c‐Fos activity was found in caudal but not rostral portions of the perirhinal cortex (areas 35/36) and in area Te2 in rats showing object recognition, i.e. preferential exploration of novel vs. familiar objects. The findings are presented at a higher anatomical resolution than previous studies of immediate‐early gene expression and object novelty and, crucially, provide the first analyses when animals are actively discriminating the novel objects. Novel vs. familiar object comparisons also revealed altered c‐Fos patterns in hippocampal subfields, with relative increases in CA3 and CA1 and decreases in the dentate gyrus. These hippocampal changes match those previously reported for the automatic coding of object–spatial associations. Additional analyses of the c‐Fos data using structural equation modelling indicated the presence of pathways starting in the caudal perirhinal cortex that display a direction of effects from the entorhinal cortex to the CA1 field (temporo‐ammonic) when presented with familiar objects, but switch to the engagement of the direct entorhinal cortex pathway to the dentate gyrus (perforant) with novel object discrimination. This entorhinal switch provides a potential route by which the rhinal cortex can moderate hippocampal processing, with a dynamic change from temporo‐ammonic (familiar stimuli) to perforant pathway (novel stimuli) influences.     

Brain-derived neurotrophic factor enhances the expression of the monocarboxylate transporter 2 through translational activation in mouse cultured cortical neurons

MCT2 is the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate. It is suggested that MCT2 is upregulated to meet enhanced energy demands after modifications in synaptic transmission. Brain-derived neurotrophic factor (BDNF), a promoter of synaptic plasticity, significantly increased MCT2 protein expression in cultured cortical neurons (as shown by immunocytochemistry and western blot) through a translational regulation at the synaptic level. Brain-derived neurotrophic factor can cause translational activation through different signaling pathways. Western blot analyses showed that p44/p42 mitogen-activated protein kinase (MAPK), Akt, and S6 were strongly phosphorylated on BDNF treatment. To determine by which signal transduction pathway(s) BDNF mediates its upregulation of MCT2 protein expression, the effect of specific inhibitors for p38 MAPK, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), p44/p42 MAPK (ERK), and Janus kinase 2 (JAK2) was evaluated. It could be observed that the BDNF-induced increase in MCT2 protein expression was almost completely blocked by all inhibitors, except for JAK2. These data indicate that BDNF induces an increase in neuronal MCT2 protein expression by a mechanism involving a concomitant stimulation of PI3K/Akt/mTOR/S6, p38 MAPK, and p44/p42 MAPK. Moreover, our observations suggest that changes in MCT2 expression could participate in the process of synaptic plasticity induced by BDNF.     

Intracellular calcium and calmodulin link brain‐derived neurotrophic factor to p70S6 kinase phosphorylation and dendritic protein synthesis

The mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway plays an important role in brain‐derived neurotrophic factor (BDNF)‐mediated protein synthesis and neuroplasticity. Although many aspects of neuronal function are regulated by intracellular calcium ([Ca²⁺]_i) and calmodulin (CaM), their functions in BDNF‐induced phosphorylation of p70S6K and protein synthesis are largely unknown. Here, we report that BDNF, via TrkB‐dependent activation of mTOR, induces sustained phosphorylation of p70S6K at Thr389 and Thr421/Ser424. BDNF‐induced phosphorylation at Thr389 was dependent on PI3 kinase but independent of ERK‐MAPK. The previously identified MAPK phosphorylation site at Thr421/Ser424 required both PI3K and MAPK in BDNF‐stimulated neurons. Furthermore, we found that the reduction in [Ca²⁺]_i, but not extracellular calcium, blocked the BDNF‐induced phosphorylation of p70S6K at both sites. Inhibition of CaM by W13 also blocked p70S6K phosphorylation. In correlation, W13 inhibited BDNF‐induced local dendritic protein synthesis. Interestingly, sustained elevation of [Ca²⁺]_i by membrane depolarization antagonized the BDNF‐induced p70S6K phosphorylation. Finally, the BDNF‐induced p70S6K phosphorylation did not require the increase of calcium level through either extracellular influx or PLC‐mediated intracellular calcium release. Collectively, these results indicate that the basal level of intracellular calcium gates BDNF‐induced activation of p70S6K and protein synthesis through CaM. © 2009 Wiley‐Liss, Inc.     

Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling.     

Luteolin induces hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome

Studies have shown that the natural flavonoid luteolin has neurotrophic activity. In this study, we investigated the effect of luteolin in a mouse model of Down syndrome. Ts65 Dn mice, which are frequently used as a model of Down syndrome, were intraperitoneally injected with 10 mg/kg luteolin for 4 consecutive weeks starting at 12 weeks of age. The Morris water maze test was used to evaluate learning and memory abilities, and the novel object recognition test was used to assess recognition memory. Immunohistochemistry was performed for the neural stem cell marker nestin, the astrocyte marker glial fibrillary acidic protein, the immature neuron marker DCX, the mature neuron marker NeuN, and the cell proliferation marker Ki67 in the hippocampal dentate gyrus. Nissl staining was used to observe changes in morphology and to quantify cells in the dentate gyrus. Western blot assay was used to analyze the protein levels of brain-derived neurotrophic factor(BDNF) and phospho-extracellular signal-regulated kinase 1/2(p-ERK1/2) in the hippocampus. Luteolin improved learning and memory abilities as well as novel object recognition ability, and enhanced the proliferation of neurons in the hippocampal dentate gyrus. Furthermore, luteolin increased expression of nestin and glial fibrillary acidic protein, increased the number of DCX~+ neurons in the granular layer and NeuN~+ neurons in the subgranular region of the dentate gyrus, and increased the protein levels of BDNF and p-ERK1/2 in the hippocampus. Our findings show that luteolin improves behavioral performance and promotes hippocampal neurogenesis in Ts65 Dn mice. Moreover, these effects might be associated with the activation of the BDNF/ERK1/2 pathway.     

Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats

Ghrelin, an orexigenic hormone, is mainly produced by the stomach and released into the circulation. Ghrelin receptors (growth hormone secretagogue receptors) are expressed throughout the brain, including the hippocampus. The activation of ghrelin receptors facilitates high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in vitro, and also improves learning and memory. Herein, we report that a single infusion of ghrelin into the hippocampus led to long-lasting potentiation of excitatory postsynaptic potentials (EPSPs) and population spikes (PSs) in the dentate gyrus of anesthetized rats. This potentiation was accompanied by a reduction in paired-pulse depression of the EPSP slope, an increase in paired-pulse facilitation of the PS amplitude, and an enhancement of EPSP-spike coupling, suggesting the involvement of both presynaptic and postsynaptic mechanisms. Meanwhile, ghrelin infusion time-dependently increased the phosphorylation of Akt-Ser473, a downstream molecule of phosphoinositide 3-kinase (PI3K). Interestingly, PI3K inhibitors, but not NMDA receptor antagonist, inhibited ghrelin-induced potentiation. Although ghrelin had no effect on the induction of HFS-induced LTP, it prolonged the expression of HFS-induced LTP through extracellular signal-regulated kinase (ERK)1/2. The Morris water maze test showed that ghrelin enhanced spatial memory, and that this was prevented by pretreatment with PI3K inhibitor. Taken together, the findings show that: (i) a single infusion of ghrelin induced a new form of synaptic plasticity by activating the PI3K signaling pathway, without HFS and NMDA receptor activation; (ii) a single infusion of ghrelin also enhanced the maintenance of HFS-induced LTP through ERK activation; and (iii) repetitive infusion of ghrelin enhanced spatial memory by activating the PI3K signaling pathway. Thus, we propose that the ghrelin signaling pathway could have therapeutic value in cognitive deficits.     

Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in the rat

The phosphoinositide 3-kinase (PI3K)/Akt signalling cascade has classically been implicated in promoting cell survival but more recently has been shown to regulate a number of other cellular functions. In particular, studies have suggested that PI3K contributes to mechanisms associated with synaptic plasticity and memory processes but the function of this cascade in forms of synaptic plasticity, such as long-term potentiation, remains controversial and the PI3K substrates which mediate these effects are poorly understood. Here we report that the PI3K inhibitor LY294002 infused i.c.v. in vivo blocked maintenance of long-term potentiation induced in the dentate gyrus with a single tetanus to the perforant path but not with repeated tetani. This pattern of stimulation led to rapid and transient phosphorylation of the PI3K substrate Akt at Ser473 but not at Thr308. Functional readout of partial activation of Akt was demonstrated by an increase in phosphorylation of two downstream substrates, Forkhead (FKHR) and mammalian target of rapamycin (mTOR), in a delayed and prolonged manner at Akt-specific phosphorylation sites. LY294002 blocked phosphorylation of Akt and the prolonged phosphorylation of FKHR and mTOR but did not impair long-term potentiation-induced phosphorylation of extracellular receptor kinase. In addition, the same i.c.v. concentration of LY294002 impaired long-term consolidation of recognition memory but not short-term recognition memory or spatial learning and repeated training in the recognition memory task overcame the deficit in consolidation. These results suggest that activation of the PI3K/Akt pathway may contribute to the mechanisms of synaptic plasticity and memory consolidation by promoting cell survival via FKHR and protein synthesis via mTOR. Importantly, only partial activation of Akt at its Ser473 residue was necessary to mediate these effects.     

Blockade of intracellular Zn2+ signaling in the dentate gyrus erases recognition memory via impairment of maintained LTP

There is no evidence on the precise role of synaptic Zn²⁺ signaling on the retention and recall of recognition memory. On the basis of the findings that intracellular Zn²⁺ signaling in the dentate gyrus is required for object recognition, short‐term memory, the present study deals with the effect of spatiotemporally blocking Zn²⁺ signaling in the dentate gyrus after LTP induction and learning. Three‐day‐maintained LTP was impaired 1 day after injection of clioquinol into the dentate gyrus, which transiently reduced intracellular Zn²⁺ signaling in the dentate gyrus. The irreversible impairment was rescued not only by co‐injection of ZnCl₂, which ameliorated the loss of Zn²⁺ signaling, but also by pre‐injection of Jasplakinolide, a stabilizer of F‐actin, prior to clioquinol injection. Simultaneously, 3‐day‐old space recognition memory was impaired 1 day after injection of clioquinol into the dentate gyrus, but not by pre‐injection of Jasplakinolide. Jasplakinolide also rescued both impairments of 3‐day‐maintained LTP and 3‐day‐old memory after injection of ZnAF‐2DA into the dentate gyrus, which blocked intracellular Zn²⁺ signaling in the dentate gyrus. The present paper indicates that the blockade and/or loss of intracellular Zn²⁺ signaling in the dentate gyrus coincidently impair maintained LTP and recognition memory. The mechanism maintaining LTP via intracellular Zn²⁺ signaling in dentate granule cells, which may be involved in the formation of F‐actin, may retain space recognition memory. © 2015 Wiley Periodicals, Inc.     

Amyloid‐β interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in brain‐derived neurotrophic factor‐induced Arc expression in rat cortical neurons

The deposition of amyloid‐β (Aβ) contributes to the pathogenesis of Alzheimer's disease. Even at low levels, Aβ may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory. Brain‐derived neurotrophic factor (BDNF) is well known to be capable of inducing the synthesis of activity‐regulated cytoskeleton‐associated protein (Arc), which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar Aβ at a nonlethal level was sufficient to impair BDNF‐induced Arc expression in cultured rat cortical neurons. In this study, BDNF treatment alone induced the activation of the phosphatidylinositol 3‐kinase‐Akt‐mammlian target of rapamycin (PI3K‐Akt‐mTOR) signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the expression of Arc. Interrupting the PI3K‐Akt‐mTOR signaling pathway by inhibitors prevented the effects of BDNF, indicating the involvement of this pathway in BDNF‐induced 4EBP1 phosphorylation, p70S6K phosphorylation, eEF2 dephosphorylation, and Arc expression. Nonlethal Aβ pretreatment partially blocked these effects of BDNF. Double‐ immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2 dephosphorylation and Arc expression following BDNF treatment regardless of the presence of Aβ. These results reveal that, in cultured rat cortical neurons, Aβ interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in BDNF‐induced Arc expression. Moreover, this study also provides the first evidence that there is a close correlation between BDNF‐induced eEF2 dephosphorylation and BDNF‐induced Arc expression. © 2009 Wiley‐Liss, Inc.     

Intracellular Zn2+ signaling in the dentate gyrus is required for object recognition memory

The role of perforant pathway‐dentate granule cell synapses in cognitive behavior was examined focusing on synaptic Zn²⁺ signaling in the dentate gyrus. Object recognition memory was transiently impaired when extracellular Zn²⁺ levels were decreased by injection of clioquinol and N,N,N′,N′ ‐t etrakis‐(2‐pyridylmethyl) ethylendediamine. To pursue the effect of the loss and/or blockade of Zn²⁺ signaling in dentate granule cells, ZnAF‐2DA (100 pmol, 0.1 mM/1 µl), an intracellular Zn²⁺ chelator, was locally injected into the dentate molecular layer of rats. ZnAF‐2DA injection, which was estimated to chelate intracellular Zn²⁺ signaling only in the dentate gyrus, affected object recognition memory 1 h after training without affecting intracellular Ca²⁺ signaling in the dentate molecular layer. In vivo dentate gyrus long‐term potentiation (LTP) was affected under the local perfusion of the recording region (the dentate granule cell layer) with 0.1 mM ZnAF‐2DA, but not with 1 – 10 mM CaEDTA, an extracellular Zn²⁺ chelator, suggesting that the blockade of intracellular Zn²⁺ signaling in dentate granule cells affects dentate gyrus LTP. The present study demonstrates that intracellular Zn²⁺ signaling in the dentate gyrus is required for object recognition memory, probably via dentate gyrus LTP expression. © 2014 Wiley Periodicals, Inc.     

Brain-derived neurotrophic factor and its intracellular signaling pathways in cocaine addiction

Cocaine addiction is one of the severest health problems faced by western countries, where there is an increasing prevalence of lifelong abuse. The most challenging aspects in the treatment of cocaine addiction are craving and relapse, especially in view of the fact that, at present, there is a lack of effective pharmacological treatment for the disorder. What is required are new pharmacological approaches based on our current understanding of the neurobiological bases of drug addiction. Within the context of the behavioral and neurochemical actions of cocaine, this paper considers the contribution of brain-derived neurotrophic factor (BDNF) and its main intracellular signaling mechanisms, including mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K), in psychostimulant addiction. Repeated cocaine administration leads to an increase in BDNF levels and enhanced activity in the intracellular pathways (PI3K and MAPK/ERK) in the reward-related brain areas, which applies especially several days following withdrawal. It has been hypothesized that these neurochemical changes contribute to the enduring synaptic plasticity that underlies sensitized responses to psychostimulants and drug-conditioned memories leading to compulsive drug use and frequent relapse after withdrawal. Nevertheless, increased BDNF levels could also have a role as a protection factor in addiction. The inhibition of the intracellular pathways, ERK and PI3K, leads to a disruption in sensitized responses and conditioned memories associated with cocaine addiction and suggests new, potential therapeutic strategies to explore in the dependence on psychostimulants.     

Interfering with perirhinal brain‐derived neurotrophic factor expression impairs recognition memory in rats

The role of brain‐derived neurotrophic factor (BDNF) in recognition memory was investigated by locally infusing oligodeoxynucleotides (ODNs) into perirhinal cortex, a region of the temporal lobe essential for familiarity discrimination. Antisense but not sense BDNF ODN impaired consolidation of long‐term (24h) but not shorter‐term (20min) recognition memory. © 2010 Wiley‐Liss, Inc.     

Detecting and discriminating novel objects: The impact of perirhinal cortex disconnection on hippocampal activity patterns

Perirhinal cortex provides object‐based information and novelty/familiarity information for the hippocampus. The necessity of these inputs was tested by comparing hippocampal c‐fos expression in rats with or without perirhinal lesions. These rats either discriminated novel from familiar objects (Novel‐Familiar) or explored pairs of novel objects (Novel‐Novel). Despite impairing Novel‐Familiar discriminations, the perirhinal lesions did not affect novelty detection, as measured by overall object exploration levels (Novel‐Novel condition). The perirhinal lesions also largely spared a characteristic network of linked c‐fos expression associated with novel stimuli (entorhinal cortex→CA3→distal CA1→proximal subiculum). The findings show: I) that perirhinal lesions preserve behavioral sensitivity to novelty, whilst still impairing the spontaneous ability to discriminate novel from familiar objects, II) that the distinctive patterns of hippocampal c‐fos activity promoted by novel stimuli do not require perirhinal inputs, III) that entorhinal Fos counts (layers II and III) increase for novelty discriminations, IV) that hippocampal c‐fos networks reflect proximal‐distal connectivity differences, and V) that discriminating novelty creates different pathway interactions from merely detecting novelty, pointing to top‐down effects that help guide object selection. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.     

Mitogen-activated protein kinase pathway mediates effects of brain-derived neurotrophic factor on differentiation of basal forebrain oligodendrocytes

Previous studies indicate that brain-derived neurotrophic factor (BDNF), through the mediation of the trkB receptor, modulates the expression of differentiated traits in basal forebrain (BF) oligodendrocytes (OLGs). Specifically, BDNF up-regulates the expression of myelin basic protein (MBP), proteolipid protein (PLP), and myelin associated glycoprotein (MAG; Du et al. [2006] Mol. Cell. Neurosci. 31:366-375). However, the signaling cascades mediating the effects of BDNF have not been defined. The current study employs biochemical and molecular biological approaches to examine the involvements of the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3 kinase (PI3K) pathway, and the phospholipase C-gamma (PLC-gamma) pathway. Our results indicate that, in BF OLGs, BDNF activates the MAPK pathway and the PLC-gamma pathway but not the PI3K-Akt signaling cascade. By using specific inhibitors and mutated dominant negative or constitutively active forms of MAPK kinase, we demonstrate that the MAPK pathway is mediating the effects of BDNF on expression of differentiated traits in BF OLGs.