Control of the establishment of aversive memory by calcineurin and Zif268

Emotional memory is a rapidly acquired and persistent form of memory, and its robustness is in part determined by the initial strength of the memory. Here, we provide new evidence that the protein phosphatase calcineurin (CaN), a potent negative regulator of neuronal signaling that is known to constrain learning and memory, critically regulates the establishment of emotional memory through mechanisms involving the immediate early gene Zif268 (also known as Egr1). We found that CaN is inhibited in the amygdala during the establishment of aversive memory, but Zif268 is activated. Using inducible transgenesis in mice, we further saw that CaN inhibition and Zif268 overexpression during memory establishment strengthen the memory trace and enhance its resistance to extinction. We found that CaN inhibition correlates with increased Zif268 expression and that a common pool of proteins is regulated in the amygdala after CaN inhibition and Zif268 overexpression. Together, these findings reveal a previously unknown mechanism for the control of emotional memory that depends on CaN and Zif268.     

Chronic psychosocial stress decreases calcineurin in the dentate gyrus: a possible mechanism for preservation of early ltp

Chronic psychosocial stress impairs early long-term potentiation (LTP) in the hippocampal CA1 region but not in the dentate gyrus of anesthetized rats. Analysis of putative signaling molecules involved in the expression of LTP was performed to determine the possible reason(s) for the apparent resistance of the LTP of the dentate gyrus to chronic psychosocial stress. Immunoblotting was used to determine possible changes in the basal levels of various fractions of calcium-dependent calmodulin kinase II (CaMKII), phosphorylated CaMKII (P-CaMKII), calmodulin, protein kinase C gamma (PKCgamma) and calcineurin in the dentate gyrus of chronically stressed rats. Western blot analysis revealed that chronic stress significantly decreased the levels of the total CaMKII without affecting P-CaMKII levels. No significant change was detected in the levels of the upstream effectors, calmodulin and PKCgamma. However, chronic stress produced a significant decrease in calcineurin levels. The data suggest that the dentate gyrus of chronically stressed rats may have developed a compensatory mechanism whereby calcineurin levels are reduced to maintain normal P-CaMKII levels, which may be responsible for the normal early LTP of the dentate gyrus of chronically stressed rats.The results of this work will increase understanding of why certain brain regions are more resistant to deleterious effects of conditions that deteriorate learning and memory.     

Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation

Transcranial direct current stimulation (tDCS) can produce a lasting polarity-specific modulation of cortical excitability in the brain, and it is increasingly used in experimental and clinical settings. Recent studies suggest that the after-effects of tDCS are related to molecular mechanisms of activity-dependent synaptic plasticity. Here we investigated the effect of DCS on the induction of one of the most studied N-methyl-d-aspartate receptor-dependent forms of long-term potentiation (LTP) of synaptic activity at CA3-CA1 synapses in the hippocampus. We show that DCS applied to rat brain slices determines a modulation of LTP that is increased by anodal and reduced by cathodal DCS. Immediate early genes, such as c-fos and zif268 (egr1/NGFI-A/krox24), are rapidly induced following neuronal activation, and a specific role of zif268 in the induction and maintenance of LTP has been demonstrated. We found that both anodal and cathodal DCS produce a marked subregion-specific increase in the expression of zif268 protein in the cornus ammonis (CA) region, whereas the same protocols of stimulation produce a less pronounced increase in c-fos protein expression in the CA and in dentate gyrus regions of the hippocampus. Brain-derived neurotrophic factor expression was also investigated, and it was found to be reduced in cathodal-stimulated slices. The present data demonstrate that it is possible to modulate LTP by using DCS and provide the rationale for the use of DCS in neurological diseases to promote the adaptive and suppress the maladaptive forms of brain plasticity.     

Dissection of tumor-necrosis factor-alpha inhibition of long-term potentiation (LTP) reveals a p38 mitogen-activated protein kinase-dependent mechanism which maps to early-but not late-phase LTP

The pro-inflammatory cytokine tumor-necrosis factor-alpha (TNF-alpha) is elevated in several neuropathological states that are associated with learning and memory deficits. Previous work has reported that TNF-alpha inhibits the induction of LTP in areas CA1 [Neurosci Lett 146 (1992) 176] and dentate gyrus [Neurosci Lett 203 (1996) 17]. The mechanism(s) underlying this process of inhibition have not to date been addressed. Here, we show that perfusion of TNF-alpha prior to long-term potentiation (LTP) inducing stimuli inhibited LTP, and that in late-LTP (3 h post-tetanus) a depression in synaptic field recordings was observed (68 +/- 5%, n = 6 versus control 175 +/- 7%, n = 6, P < 0.001). We investigated the involvement of the mitogen-activated protein kinase (MAPK) p38 in the inhibition of LTP by TNF-alpha as p38 MAPK has previously been shown to be involved in interleukin-1beta inhibition of LTP in the dentate gyrus [Neuroscience 93 (1999b) 57]. Perfusion of TNF-alpha led to an increase in the levels of phosphorylated p38 MAPK detectable in the granule cells of the dentate gyrus. The p38 MAPK inhibitor SB 203580 (1 microM) was found by itself to have no significant effect on either early or late phase LTP in the dentate gyrus. SB 203580 was found to significantly reverse the inhibition of early LTP by TNF-alpha (SB/TNF-alpha 174 +/- 5%, n = 6 versus TNF-alpha 120 +/- 7%, n = 6, P < 0.001, 1 h post-tetanus) to values comparable to control LTP (control 175 +/- 7%, n = 6). Interestingly however, the depressive effects of TNF-alpha on late LTP (2-3 h) were clearly not attenuated by p38 MAPK inhibition (SB/TNF-alpha 132 +/- 5%, n = 6 versus control LTP 175 +/- 7%, n = 6, P < 0.001, 3 h post-tetanus). This work suggests that TNF-alpha inhibition of LTP represents a biphasic response, a p38 MAPK-dependent phase that coincides with the early phase of LTP and a p38 MAPK independent phase that temporally maps to late LTP.     

Simultaneous induction of long-term potentiation in the hippocampus and the amygdala by entorhinal cortex activation: mechanistic and temporal profiles

The medial temporal lobe, including the entorhinal cortex, the amygdala and the hippocampus, has an important role in learning and memory, and its circuits exhibit synaptic plasticity (long-term potentiation [LTP]). The entorhinal cortex is positioned to exert a potent influence on the amygdala and the hippocampus given its extensive monosynaptic projections to both areas. We therefore studied the effects of activation of the entorhinal cortex with simultaneous recording of LTP in the hippocampus and amygdala in the anesthetized rat. theta Burst stimulation of the lateral entorhinal cortex induced LTP simultaneously in the basal amygdaloid nucleus and in the dentate gyrus. However, the mechanisms involved in the induction of LTP in the two areas differed. The N-methyl-D-aspartate receptor antagonist 3-[(+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid delivered 1 h before LTP induction (10 mg/kg, i.p.), blocked LTP in the dentate gyrus but not in the amygdala. In addition we found that the basal amygdala as well as the dentate gyrus sustained late-phase LTP (10 h) which may participate in memory encoding and/or modulation processes. Overall, the results suggest a coordinating role for the entorhinal cortex by simultaneously modulating activity and plasticity in these structures, albeit through different mechanisms. Interactive encoding of this sort is believed to endow memories with a different, more integrative, quality than when either pathway is activated alone.     

Prenatal morphine exposure attenuates the maintenance of late LTP in lateral perforant path projections to the dentate gyrus and the CA3 region in vivo

Previously we reported that prenatal exposure to morphine twice daily during gestation decreases proenkephalin levels in adult progeny within the brain, including the dentate gyrus, and alters µ and opioid receptors in the hippocampal CA3 region. The lateral aspect of the perforant path contains and releases enkephalin-derived opioid peptides, and induction of long-term potentiation (LTP) in lateral perforant path projections to both the dentate gyrus and the hippocampal CA3 region is blocked by antagonists of opioid receptors. Thus LTP induction at these synapses involves opioid receptor activation mediated by the release of proenkephalin-derived opioid peptides with lateral perforant path activation. Here we show in adult behaving animals, neither LTP induction nor the early phase of LTP (E-LTP) maintenance is altered by prenatal morphine exposure in the lateral perforant path projections to the dentate gyrus and the CA3 region. However, maintenance and longevity of late LTP (L-LTP), as reflected in the magnitude of LTP over days, was attenuated in animals prenatally exposed to morphine. In contrast, in medial perforant path projections to the dentate gyrus and CA3 region, both LTP induction and the maintenance of E- and L-LTP were unaffected by prenatal morphine treatment. Thus a brief prenatal exposure to the opiate morphine produces sustained, and possibly permanent, alterations in L-LTP in the opioidergic lateral perforant path projection. This suggests that prenatal morphine exposure disrupts LTP via disruption of opioid mechanisms involved in LTP maintenance or via disruption of opioid receptor activation during LTP induction, which can subsequently alter LTP maintenance.     

Impairment of recognition memory and hippocampal long-term potentiation after acute exposure to clioquinol

Clioquinol (CQ) was associated with cases of transient global amnesia and with the neurodegenerative syndrome subacute myelo-optico-neuropathy (SMON) in humans. However, CQ forms lipophilic chelates with cations and has the potential as a scientific and clinical tool used for selective modulation of histochemically reactive zinc pools. The relationship among transient lack of synaptic zinc release, hippocampal long-term potentiation (LTP) induction and cognitive memory is poorly understood. To evaluate the role of synaptic zinc release, in the present study, hippocampal LTP induction and cognitive behavior were examined in young rats after i.p. injection of CQ (30 mg/kg). Intracellular zinc detected by Timm's stain and extracellular (synaptic cleft) zinc detected by ZnAF-2 were significantly decreased in the hippocampus 6 h after CQ injection. The molecular layer of the dentate gyrus, in which perforant path-granule cell synapses exist, was most responsive to CQ injection. Dentate gyrus LTP was induced similarly to the control 2 h after CQ injection, while significantly attenuated 6–24 h after CQ injection. In the training trial of the object recognition memory 2 h after CQ injection, there was no significant difference in learning behavior between the control and CQ-treated rats. In the test trial, CQ-treated rats showed normal recognition memory 1 h after the training, whereas recognition memory deficit 24 h after the training unlike the control rats. These results indicate that acute exposure to CQ impairs long-term (24 h) memory in the hippocampus of young rats. The CQ-mediated attenuation of dentate gyrus LTP, which may be associated with the transient lack of zinc release from zincergic neurons, seems to be involved in the impairment of the long-term memory.     

A specific role for group II metabotropic glutamate receptors in hippocampal long-term depression and spatial memory

Activity-dependent and sustained alterations in synaptic efficacy are widely regarded as the cellular correlates underlying learning and memory. Metabotropic glutamate receptors (mGluRs) are intrinsically involved in both hippocampal synaptic plasticity and spatial learning. Group II mGluRs are required for persistent hippocampal long-term depression (LTD), but are not required for long-term potentiation (LTP) in the hippocampal CA1 region in vivo. The role of these receptors in spatial learning, and in synaptic plasticity in the dentate gyrus in vivo has not yet been the subject of close scrutiny. We investigated the effects of group II mGluR antagonism on LTP and LTD in the adult rat, at medial perforant path-dentate gyrus synapses, and on spatial learning in the eight-arm radial maze. Daily application of the group 2 mGluR antagonist (2S)-alpha-ethylglutamic acid (EGLU) resulted in impairment of long-term (reference) memory with effects becoming apparent 6 days after training and drug-treatment began. Short-term (working) memory was unaffected throughout the 10-day study. Acute injection of EGLU did not affect either LTD or LTP in the dentate gyrus in vivo. Following six daily applications of EGLU a clear impairment of LTD but not LTP was apparent however. These data support that prolonged antagonism of group II mGluRs results in an impairment of LTD that parallels the appearance of spatial memory deficits arising from group II mGluR antagonism. These findings support the importance of group II mGluRs for spatial memory formation and offer a further link between LTD and the encoding of spatial information in the hippocampus.     

Inhibition of protein synthesis in the dentate gyrus, but not the entorhinal cortex, blocks maintenance of long-term potentiation in rats

We examined whether the critical protein synthesis for maintenance of perforant path long-term potentiation (LTP) takes place in the dentate gyrus or the entorhinal cortex. Field potential recordings were made of responses in the dentate gyrus to stimulation of the perforant path in urethane-anaesthetized rats. Anisomycin (10 micrograms) injected into the dentate gyrus, but not the entorhinal cortex, 1 h prior to tetanization led to nearly complete decay of perforant path LTP of the excitatory postsynaptic potential (EPSP) within 3 h. Intra-dentate injection of neither actinomycin D (a mRNA synthesis inhibitor) nor boiled anisomycin affected LTP maintenance over 6 h. These results suggest that the proteins necessary for the maintenance of LTP over 6 h are synthesized in the dentate gyrus from already existing mRNA without involving protein synthesis in the cell bodies of the afferent fibres.     

Activity-regulated cytoskeletal-associated protein is localized to recently activated excitatory synapses

Activity-regulated, cytoskeletal-associated protein (Arc) is an immediate early gene induced in excitatory circuits following behavioral episodes. Arc mRNA is targeted to activated regions of the dendrite after long-term potentiation (LTP) of the dentate gyrus, a process dependent on NMDA receptor activation. We used post-embedding immunogold electron microscopy (EM) to test whether synaptic Arc expression patterns are selectively modified by plasticity. Consistent with previous light microscopic observations, Arc protein was rapidly induced in the dentate gyrus following LTP-producing stimulation of the perforant path and was detectable in granule cell nuclei, somata and dendrites after two hours of high frequency stimulation. Post-embedding EM revealed Arc immunogold labeling in three times as many spines in the middle molecular layer of the stimulated dentate gyrus than in either the ipsilateral outer molecular layer or the contralateral middle and outer molecular layers. This upregulation did not occur with low frequency stimulation of the perforant path. Therefore Arc protein localization may be a powerful tool to isolate recently activated dendritic spines.     

4-Hydroxynonenal modulates the long-term potentiation induced by L-type Ca2+ channel activation in the rat dentate gyrus in vitro

Increased oxyradical production and membrane lipid peroxidation (MLP) occur under physiological and degenerative conditions in neurons. We investigated whether 4-hydroxynonenal (4HN), one of the membrane lipid peroxidation products, affects long-term potentiation (LTP) in the rat dentate gyrus in vitro. Treatment of hippocampal slices with 4HN (10 μM) enhanced LTP without affecting basal evoked potentials. The enhancement was completely inhibited by 2 μM nifedipine, a blocker of L-type Ca²⁺ channels. In cultured dentate gyrus neurons, treatment of the cells with 4HN for 24 h resulted in a significant amount of cell death that was detoxified by glutathione, whereas short-term treatment with 4HN (6 h) had no effect. Nifedipine partially but significantly suppressed the 4HN-induced cell death. These results suggest that 4HN modulates LTP and induces delayed cell death through L-type Ca²⁺ channel activation in the dentate gyrus. 4HN thereby plays an important role in both physiological and pathophysiological events in the hippocampus.     

Opposite effects of shell or core stimulation of the nucleus accumbens on long-term potentiation in dentate gyrus of anesthetized rats

Hippocampal long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy which is considered a cellular correlate of learning and memory. It has been shown that both, stimuli with emotional/motivational content and the electrical stimulation the basolateral amygdala, can modulate hippocampal LTP. The nucleus accumbens is part of the ventral striatum and is composed of two main regions: core and shell. Core and shell share a similar cellular composition, but differ in their connectivity with other brain areas. Considering that the nucleus accumbens is related to motivation and that it receives a strong projection from the basolateral amygdala, we have studied the effect of stimulating accumbens shell or core on medial perforant path-granule cells' LTP in anesthetized male Wistar rats. We found that electrical stimulation of the shell enhances the magnitude of LTP while the stimulation of the core completely prevents LTP induction. The stimulation of the accumbens shell or core alone produced no apparent, direct field potential in dentate gyrus. Additionally, the co-stimulation of the shell or core with the medial perforant path does not modify the input-output curves obtained using stimulation of the perforant path only. These results demonstrate that electrical stimulation of the accumbens shell or core has a bidirectional effect on LTP induction at the dentate gyrus.     

Interleukin-18 mediated inhibition of LTP in the rat dentate gyrus is attenuated in the presence of mGluR antagonists

Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory impairments. We have previously demonstrated the inhibition of long-term potentiation (LTP), a recognised model for memory, in the dentate gyrus region of the rat hippocampus, by interleukin-18. We have also previously shown that the inhibitory effect of TNF-alpha on LTP can be attenuated by inhibitors of metabotropic glutamate receptors (mGluRs). We therefore went on to investigate the effects of the mGluR antagonists MPEP and MTPG on the effect of IL-18 on LTP in the rat dentate gyrus in vitro. Recordings of field excitatory post-synaptic potentials (EPSPs) were made from the medial perforant path of rat hippocampal slices. IL-18 (100 ng/ml) applied for 20 min before-HFS had no significant effect on baseline EPSPs but significantly impaired LTP (IL-18 LTP 116+/-9%, versus control LTP 163+/-6% 1h post-tetanus, P<0.001, n=5). Perfusion of the mGluR5 specific antagonist MPEP (5 microM) for 40 min prior to application of IL-18 had no significant effect on baseline EPSPs but significantly attenuated the inhibitory effect of IL-18 on LTP at 30 min but not 1h (177+/-2% and 138+/-8%, respectively, compared to controls; n=5). Perfusion of the group II mGluR antagonist MTPG (50 microM) for 40 min prior to application of IL-18 had no significant effect on baseline EPSPs but was found to significantly reverse the inhibitory effect of IL-18 on LTP at 1h (164+/-6% compared to IL-18 alone, n=5). This study provides novel evidence of the involvement of mGluRs in the IL-18 mediated inhibition of LTP.     

Basolateral amygdala stimulation does not recruit LTP at depotentiated synapses

Hippocampal long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy considered to be the cellular basis of memory. LTP consists of an early, protein synthesis-independent phase (E-LTP) and a late phase that depends on protein synthesis (L-LTP). Application of a weak tetanus can induce E-LTP in the dentate gyrus (DG) which can be reinforced into L-LTP by direct stimulation of the basolateral amygdala (BLA) within 30 min before or after LTP induction (structural LTP-reinforcement, [1]). LTP can be depotentiated by low-frequency stimulation (LFS) to the same synaptic input if applied shortly after tetanization (< 10 min). Here, we addressed the question of whether stimulation of the BLA is able to recover LTP at depotentiated synaptic inputs. We hypothesized that E-LTP can activate synaptic tags, which were then reset by depotentiation. Stimulation of the BLA thereafter could beneficially act on tag-reactivation as well as on the activation of the synthesis of plasticity-related proteins (PRPs), normally captured by the tags and thus transforming E-LTP into L-LTP. Our results show, that BLA-stimulation was not able to reactivate the resetting of tags by depotentiation in the DG of freely moving rats.     

Requirement of β-adrenergic receptor activation and protein synthesis for LTP-reinforcement by novelty in rat dentate gyrus

Long-term potentiation (LTP) is supposed to be a cellular mechanism involved in memory formation. Similar to distinct types of memory formation, LTP can be separated into a protein synthesis-independent early phase (early-LTP) and a protein synthesis-dependent late phase (late-LTP). An important question is whether the transformation from early- into late-LTP can be elicited by behavioural conditions such as the attention to novel events. Therefore, we investigated the effect of exploration of a novel environment (novelty-exploration) on subsequently induced early-LTP in the dentate gyrus of freely moving rats. While a delay of 60 min between exploration onset and LTP induction had no effect, intervals of 30 or 15 min led to a reinforcement of early- to late-LTP. Exploration of a familiar environment failed to prolong LTP maintenance. The novelty-induced LTP reinforcement was blocked when the translation inhibitor anisomycin or the β-adrenergic antagonist propranolol were applied intracerebroventricularly before exploration onset. These findings support the hypothesis that the synergistic interplay of novelty-triggered noradrenergic activity and weak tetanic stimulation promotes the synthesis of certain proteins that are required for late-LTP. Such a cellular mechanism may underlie novelty-dependent enhancement of memory formation.