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Simultaneous Two-photon <em>In Vivo </em>Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Nencki Institute of Experimental Biology

Rats were subjected to two aversive learning protocols: either classical (fear conditioning) or instrumental (two-way active avoidance training). Next, immunocytochemical mapping of phosphorylated extracellular signal-regulated kinase (P-ERK) and c-Fos transcription factor protein was performed, and the expression pattern of both markers within the dorsal nucleus of lateral amygdala (LaD) was analyzed. As immunocytochemical studies revealed, aversive training induced ERK phosphorylation and c-Fos expression specifically in ventral but not dorsal tip of LaD. These data show for the first time molecular distinction between subdivisions of LaD as well as they also strengthen the idea of Repa that the neurons in the ventral tip of LaD are involved in storage of long-lasting changes associated with formation of fear memories.

Differential response of two subdivisions of lateral amygdala to aversive conditioning as revealed by c-Fos and P-ERK mapping.

It is known that acute cocaine administration activates the extracellular signal-regulated kinase (ERK) pathway in the striatum, and results in transcription and translation of immediate early genes (IEGs). In the present study we investigated a possible involvement of ERK in the regulation of IEG expression in the amygdala, another brain structure known to be related to an addicted state. The patterns of cocaine-induced c-Fos, JunB and Zif268 protein expression were investigated, using an immunohistochemical approach, within distinct nuclei of the amygdala, either in the presence or absence of a selective inhibitor of the ERK pathway, SL327. Although these IEGs were similarly activated in the various nuclei of the amygdala after acute administration of cocaine, they showed different patterns after chronic injections. They also showed selective sensitivities to ERK inhibition. In particular, whereas c-Fos and JunB expressions were augmented following chronic cocaine treatment, as compared with acute treatment, Zif268 expression was decreased by this chronic treatment. Additionally, chronic blocking of ERK activation affected cocaine-induced c-Fos and JunB but not Zif268 expression. Thus, the differential involvement of ERK in chronic vs. acute regulation of IEGs may account for its specific role in addiction-related behavioral alterations, such as sensitization and tolerance.

Extracellular signal-regulated kinases (ERKs) modulate cocaine-induced gene expression in the mouse amygdala.

Alcohol-related cues may induce relapse to heavy alcohol drinking and promote molecular adaptations in discrete brain regions. An exact nature of these molecular alterations is still unknown. In the present study, rats trained to self-administer ethanol were tested for cue-induced reinstatement of ethanol seeking after 30 days of abstinence. Next, a detailed immunocytochemical analysis of c-Fos activation was performed within seven nuclei of the amygdala. In the second experiment, c-Fos activation after reinstatement of ethanol seeking induced by discrete cues was compared with the activation pattern of its putative partner (c-Jun) and regulators (extracellular signal-regulated kinases and c-Jun N-terminal kinases). Reexposure to ethanol-associated context cues (an extinction session) potentiated c-Fos expression within the basolateral and central amygdala. Repeated presentation of ethanol-associated discrete cues in an extinction/reinstatement session led to even stronger c-Fos activation in the latter nuclei. In the second experiment, reexposure to the ethanol-associated context and discrete cues activated both c-Jun and extracellular signal-regulated kinases (ERK1/2) in the basolateral amygdala. Our observations suggest that the basolateral and central amygdala may be specifically involved in alcohol-seeking behavior induced by discrete cues.

Alcohol relapse induced by discrete cues activates components of AP-1 transcription factor and ERK pathway in the rat basolateral and central amygdala.

The amygdala is a heterogeneous brain structure implicated in processing of emotions and storing the emotional aspects of memories. Gene activity markers such as c-Fos have been shown to reflect both neuronal activation and neuronal plasticity. Herein, we analyze the expression patterns of gene activity markers in the amygdala in response to either behavioral training or treatment with drugs of abuse and then we confront the results with data on other approaches to internal complexity of the amygdala. c-Fos has been the most often studied in the amygdala, showing specific expression patterns in response to various treatments, most probably reflecting functional specializations among amygdala subdivisions. In the basolateral amygdala, c-Fos expression appears to be consistent with the proposed role of this nucleus in a plasticity of the current stimulus-value associations. Within the medial part of the central amygdala, c-Fos correlates with acquisition of alimentary/gustatory behaviors. On the other hand, in the lateral subdivision of the central amygdala, c-Fos expression relates to attention and vigilance. In the medial amygdala, c-Fos appears to be evoked by emotional novelty of the experimental situation. The data on the other major subdivisions of the amygdala are scarce. In conclusion, the studies on the gene activity markers, confronted with other approaches involving neuroanatomy, physiology, and the lesion method, have revealed novel aspects of the amygdala, especially pointing to functional heterogeneity of this brain region that does not fit very well into contemporarily active debate on serial versus parallel information processing within the amygdala.

Functional internal complexity of amygdala: focus on gene activity mapping after behavioral training and drugs of abuse.

The noradrenergic system plays and an important modulatory role in memory consolidation of emotionally arousing tasks. However, the molecular cascades regulated in the brain by norepinephrine and involved in memory formation are still largely unknown. The purpose of the present study was to evaluate the role of the noradrenergic system on the acquisition of a highly emotionally arousing task-two-way active avoidance training-and its molecular and cellular substrates. The selective norepinephrine neurotoxin N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine (DSP-4, 50mg/kg) was used. DSP-4-treated rats were trained in a shuttle box to avoid a footshock signaled by an auditory stimulus. Immunohistochemical mapping of the neuronal plasticity-related molecules c-Fos protein and the activated form of extracellular signal-regulated kinase (phosphorylated ERK [pERK]) was then employed. We found that DSP-4 treatment depleted the expression of the norepinephrine marker dopamine -hydroxylase (DBH) in the locus coeruleus and its projection area, the basolateral nucleus of the amygdala, confirming locus coeruleus noradrenergic lesion in the experimental animals. Furthermore, DSP-4 treatment impaired the acquisition of the avoidance reaction. We also found that acquisition of the active avoidance reaction induced c-Fos expression and ERK activation in the amygdala and piriform cortex. This upregulation was prevented by DSP-4 treatment. Thus, our data suggest that the noradrenergic system is involved in the acquisition of the active avoidance reaction by regulating ERK pathway activity and c-Fos expression in the amygdala and piriform cortex.

Central noradrenergic lesion induced by DSP-4 impairs the acquisition of avoidance reactions and prevents molecular changes in the amygdala.

The alpha-isoform of calcium/calmodulin-dependent kinase II (Î±CaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This Î±CaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the Î±CaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of Î±CaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of Î±CaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of Î±CaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that Î±CaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that Î±CaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM.

Properties of contextual memory formed in the absence of Î±CaMKII autophosphorylation.

L&#39;isoforme alpha de calcium/calmoduline-d&eacute;pendante kinase II (&alpha;CaMKII) est une kinase synaptique majeure qui subit une autophosphorylation apr&egrave;s l&#39;activation des r&eacute;cepteurs NMDA, la kinase de commutation dans un &eacute;tat d&#39;activit&eacute; ind&eacute;pendante du calcium. Cela &alpha;CaMKII autophosphorylation est essentielle pour la potentialisation &agrave; long terme (LTP), de d&eacute;pendant du r&eacute;cepteur NMDA induit par un t&eacute;tanos seul, dans la zone de l&#39;hippocampe CA1 et au n&eacute;ocortex. En outre, l&#39;autophosphorylation de &alpha;CaMKII est essentielle pour contextual formation &agrave; long terme de la m&eacute;moire (LTM) apr&egrave;s un proc&egrave;s de formation unique, mais pas apr&egrave;s une s&eacute;ance d&#39;entra&icirc;nement mass&eacute;. Ici, nous montrons que, en l&#39;absence d&#39;autophosphorylation &alpha;CaMKII conditionnement de peur contextuelle est hippocampe d&eacute;pendant et que multi-tetanus-d&eacute;pendante-LTP tardive ne peut &ecirc;tre induite dans la zone de l&#39;hippocampe CA1. De plus, nous montrons que, en l&#39;absence d&#39;autophosphorylation &alpha;CaMKII que LTM contextuelle persiste pendant 30 jours, le dernier point de temps test&eacute;. En outre, il semble que les contextuelle, mais pas signalis&eacute;e, LTM formation en l&#39;absence d&#39;autophosphorylation &alpha;CaMKII porter atteinte dans 18 mois souris. Ensemble, nos r&eacute;sultats sugg&egrave;rent que &alpha;CaMKII autophosphorylation ind&eacute;pendant plasticit&eacute; dans l&#39;hippocampe est suffisante pour la formation de LTM contextuelle et l&#39;autophosphorylation de &alpha;CaMKII peut-&ecirc;tre &ecirc;tre importante pour retarder les d&eacute;ficiences li&eacute;es &agrave; l&#39;&acirc;ge dans la formation de la m&eacute;moire hippocampe. En outre, ils proposent que LTP d&eacute;pendant du r&eacute;cepteur NMDA dans la zone de l&#39;hippocampe CA1 est essentielle pour contexte formation LTM apr&egrave;s un seul essai mais non apr&egrave;s entra&icirc;nement mass&eacute;. Enfin, nos r&eacute;sultats remettent en question la proposition que LTP d&eacute;pendant du r&eacute;cepteur NMDA dans le n&eacute;ocortex est requis pour la distance LTM contextuelle.

Propri&eacute;t&eacute;s de la m&eacute;moire contextuelle forment en l&#39;absence d&#39;autophosphorylation &alpha;CaMKII.

L&#39;alfa-isoforma di calcio/calmodulina-dipendente della chinasi II (&alpha;CaMKII) &egrave; una chinasi di synaptic principale che subisce autophosphorylation dopo l&#39;attivazione del recettore NMDA, la chinasi di commutazione in uno stato di attivit&agrave; indipendente dal calcio. Questo &alpha;CaMKII autophosphorylation &egrave; essenziale per il potenziamento di lunga durata dipendente dal recettore NMDA (LTP), indotta da un singolo tetano, nell&#39;area dell&#39;ippocampo CA1 e nella neocorteccia. Inoltre, il autophosphorylation &alpha;CaMKII &egrave; essenziale per contestuale formazione di memoria (LTM) a lungo termine dopo un processo di formazione unico, ma non dopo una sessione di allenamento ammassato. Qui, ci mostrano che in assenza di &alpha;CaMKII autophosphorylation contestuale paura condizionata &egrave; ippocampo dipendente e quello multi-tetanus-dipendente tardo-LTP non pu&ograve; essere indotta nell&#39;area dell&#39;ippocampo CA1. Inoltre, ci mostrano che in assenza di &alpha;CaMKII autophosphorylation che contestuale LTM persiste per 30 giorni, l&#39;ultimo punto di tempo testato. Inoltre, contestuale, ma non attirare, formazione di LTM in assenza di autophosphorylation &alpha;CaMKII sembra essere ridotta a 18 mese-vecchi topi. Presi insieme, i nostri risultati suggeriscono che plasticit&agrave; autophosphorylation indipendente &alpha;CaMKII nell&#39;ippocampo &egrave; sufficiente per la formazione di LTM contestuali e quello autophosphorylation &alpha;CaMKII pu&ograve; essere importante per ritardare la disabilit&agrave; legata all&#39;et&agrave; nella formazione della memoria ippocampale. Inoltre, essi propongono che LTP dipendente dal recettore NMDA nella zona dell&#39;ippocampo CA1 &egrave; essenziale per la formazione di LTM contestuale dopo un singolo processo, ma non dopo aver ammassato formazione. Infine, i nostri risultati sfidano la proposta che LTP dipendente dal recettore NMDA nella neocorteccia &egrave; richiesto per remoto LTM contestuali.

Propriet&agrave; di memoria contestuale formata in assenza di &alpha;CaMKII autophosphorylation.

Insulin has been shown to impact on learning and memory in both humans and animals, but the downstream signaling mechanisms involved are poorly characterized. Insulin receptor substrate-2 (Irs2) is an adaptor protein that couples activation of insulin- and insulin-like growth factor-1 receptors to downstream signaling pathways. Here, we have deleted Irs2, either in the whole brain or selectively in the forebrain, using the nestin Cre- or D6 Cre-deleter mouse lines, respectively. We show that brain- and forebrain-specific Irs2 knockout mice have enhanced hippocampal spatial reference memory. Furthermore, NesCreIrs2KO mice have enhanced spatial working memory and contextual- and cued-fear memory. Deletion of Irs2 in the brain also increases PSD-95 expression and the density of dendritic spines in hippocampal area CA1, possibly reflecting an increase in the number of excitatory synapses per neuron in the hippocampus that can become activated during memory formation. This increase in activated excitatory synapses might underlie the improved hippocampal memory formation observed in NesCreIrs2KO mice. Overall, these results suggest that Irs2 acts as a negative regulator on memory formation by restricting dendritic spine generation.

Insulin receptor substrate 2 is a negative regulator of memory formation.

Human studies indicate that high impulsivity, novelty seeking and anxiety predispose individuals to alcohol abuse. Unclear, however, is whether the same phenotypes can be observed in laboratory animals prone to uncontrolled alcohol drinking. To characterize a novelty-seeking trait, anxiety, impulsivity, compulsivity and the motivation for natural rewards in mice, numerous tests were performed in the automated IntelliCage learning system. The same mice then had extended access to alcohol for 70 days, followed by the evaluation of addiction-like behaviors, including (1) the motivation for alcohol in a progressive-ratio schedule of reinforcement; (2) persistent and compulsive alcohol seeking and taking during signaled 'no alcohol' periods and (3) when subjected to punishment; and (4) the intensity of relapse after alcohol withdrawal. Our data suggest that high levels of anxiety-related traits (i.e. low novelty seeking, low resistance to punishment and a high level of compulsive behaviors) and high impulsivity predict addiction-like alcohol drinking in mice. Future studies are, however, warranted to create a valid model of alcohol addiction in mice in the IntelliCage system.

Characterization of an alcohol addiction-prone phenotype in mice.

Long-term memory (LTM) formation has been linked with functional strengthening of existing synapses and other processes including de novo synaptogenesis. However, it is unclear whether synaptogenesis can contribute to LTM formation. Here, using Î±-calcium/calmodulin kinase II autophosphorylation-deficient (T286A) mutants, we demonstrate that when functional strengthening is severely impaired, contextual LTM formation is linked with training-induced PSD95 up-regulation followed by persistent generation of multiinnervated spines, a type of synapse that is characterized by several presynaptic terminals contacting the same postsynaptic spine. Both PSD95 up-regulation and contextual LTM formation in T286A mutants required signaling by the mammalian target of rapamycin (mTOR). Furthermore, we show that contextual LTM resists destabilization in T286A mutants, indicating that LTM is less flexible when synaptic strengthening is impaired. Taken together, we suggest that activation of mTOR signaling, followed by overexpression of PSD95 protein and synaptogenesis, contributes to formation of invariant LTM when functional strengthening is impaired.

Mechanism for long-term memory formation when synaptic strengthening is impaired.

Formation de la m&eacute;moire (LTM) &agrave; long terme a &eacute;t&eacute; li&eacute;e avec renforcement fonctionnel des synapses existantes et d&#39;autres processus, y compris de novo synaptogen&egrave;se. Toutefois, on ignore si synaptogen&egrave;se peut contribuer &agrave; la formation de LTM. Ici, &agrave; l&#39;aide de mutants de &alpha;-calcium/calmoduline kinase II autophosphorylation d&eacute;ficient (T286A), nous d&eacute;montrons que lorsque le renforcement fonctionnel est gravement alt&eacute;r&eacute;e, contexte formation LTM est li&eacute;e &agrave; PSD95 d&#39;induites par l&#39;entra&icirc;nement vers le haut-r&egrave;glement suivie de g&eacute;n&eacute;ration persistante d&#39;&eacute;pines multiinnervated, un type de synapse qui se caract&eacute;rise par plusieurs terminaisons pr&eacute;synaptiques, communiquant avec l&#39;&eacute;pine postsynaptique m&ecirc;me. Les PSD95 vers le haut-r&egrave;glement et contexte formation LTM T286A mutants n&eacute;cessaires signalisation par le mammalian target of rapamycin (mTOR). De plus, nous montrons que LTM contextuelle r&eacute;siste &agrave; la d&eacute;stabilisation en mutants T286A, indiquant que LTM est moins flexible lorsque renforcement synaptique est alt&eacute;r&eacute;e. Ensemble, nous sugg&egrave;rent que l&#39;activation de la signalisation mTOR, suivie par la surexpression de la prot&eacute;ine PSD95 et synaptogen&egrave;se, contribue &agrave; la formation de LTM invariant lorsque renforcement fonctionnel est alt&eacute;r&eacute;e.

M&eacute;canisme de formation de la m&eacute;moire &agrave; long terme lorsque le renforcement synaptique est alt&eacute;r&eacute;e.

Formazione a lungo termine di memoria (LTM) &egrave; stato collegato con rafforzamento funzionale delle sinapsi esistenti e altri processi tra cui de novo pervasivi. Tuttavia, non &egrave; chiaro se pervasivi possono contribuire alla formazione di LTM. Qui, usando mutanti carenti autophosphorylation (T286A) di &alpha;-calcio/calmodulina chinasi II, dimostrano che quando il rafforzamento funzionale &egrave; gravemente compromessa, contestuale formazione LTM &egrave; collegato con formazione indotta PSD95 up-regolamento seguito da generazione persistente delle spine multiinnervated, un tipo di sinapsi che sono caratterizzata da diversi terminali presinaptiche contattando la stessa colonna vertebrale postsinaptica. PSD95 up-regulation sia contestuale formazione LTM T286A mutanti necessaria segnalazione dalla destinazione dei mammiferi del rapamycin (mTOR). Inoltre, mostriamo che LTM contestuale resiste alla destabilizzazione in mutanti di T286A, che indica che LTM &egrave; meno flessibile quando rafforzamento sinaptico &egrave; compromessa. Presi nel loro insieme, suggeriscono che attivazione di mTOR signaling, seguita da iperespressione della proteina PSD95 e pervasivi, contribuisce alla formazione di LTM invariante quando rafforzamento funzionale &egrave; compromessa.

Meccanismo per la formazione di memoria a lungo termine quando rafforzamento sinaptico &egrave; compromessa.

Understanding the molecular and cellular process specifically regulated during fear memory consolidation and extinction is a critical step toward development of new strategies in the treatment of human fear disorders. Here we used inhibitory component of AP-1 transcription factor, JunB, in order to map brain regions where JunB-dependent transcription is regulated during consolidation and extinction of contextual fear memory. We found that contextual fear memory consolidation induced JunB expression in the medial nucleus and intercalated cells of the amygdala while extinction training induced JunB in the CA1 and CA3 areas of the dorsal hippocampus. JunB upregulation induced by contextual fear memory extinction was absent in alphaCaMKII autophosphorylation-deficient mice which have impaired contextual fear memory extinction. Thus, our data suggest that JunB expression in the medial nucleus and intercalated cells of the amygdala is involved in fear memory consolidation while alphaCaMKII-autophosphorylation-dependent JunB expression in the areas CA1 and CA3 of the dorsal hippocampus regulates fear memory extinction.

Mapping fear memory consolidation and extinction-specific expression of JunB.

The development of addiction is associated with a dysregulation of glutamatergic transmission in the brain reward circuit. α isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is one of the key proteins that regulates structural and functional plasticity of glutamatergic synapses. αCaMKII activity can be controlled by the autophosphorylation of threonine 286. The role of this autophosphorylation in the regulation of addiction-related behaviors has been proposed but is still poorly understood. Here, using αCaMKII autophosphorylation-deficient mutant mice (T286A), we show that, in comparison with wild-type animals, they are less resistant to high doses of alcohol and do not show psychostimulant response neither to alcohol injections nor during voluntary alcohol drinking. T286A mutants are also less prone to develop alcohol addiction-related behaviors including an increased motivation for alcohol, persistent alcohol seeking during withdrawal and alcohol consumption on relapse. Finally, we demonstrate that αCaMKII autophosphorylation regulates also alcohol-induced remodeling of glutamatergic synapses in the hippocampus and amygdala. In conclusion, our data suggest that αCaMKII autophosphorylation-dependent remodeling of glutamatergic synapses is a plausible mechanism for the regulation of the alcohol addiction-related behaviors.

Kasia Radwanska

Department of Molecular and Cellular Neuroscience

Nencki Institute of Experimental Biology

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Kasia Radwanska

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular and Cellular Neuroscience

Nencki Institute of Experimental Biology

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Department of Molecular and Cellular Neuroscience