Elsevier

Behavioural Brain Research

Volume 163, Issue 1, 30 August 2005, Pages 128-135
Behavioural Brain Research

Research report
Behavioral testing upregulates pCaMKII, BDNF, PSD-95 and egr-1 in hippocampus of FVB/N mice

https://doi.org/10.1016/j.bbr.2005.04.010Get rights and content

Abstract

Several protein cascades are proposed to be involved in the formation of synaptic plasticity and have been linked to neuronal information processing and storage. Although modified expression of specific proteins following behavioral testing has been shown, no systematic approach for their concomitant determination has been reported. We therefore determined hippocampal expression of signaling proteins, transcription factors and synaptosomal-associated proteins representing key elements of neuronal plasticity in mice following behavioral training.

Male FVB/N mice, 12 weeks of age, were used for behavioral testing. After completion of tests mice were sacrificed and hippocampi were dissected.

Levels of total and autophosphorylated (T286) αcalcium-calmodulin dependent kinase II (CaMKII, pCaMKII), total and phosphorylated mitogen-activated protein kinase (MAPK, pMAPK), total and phosphorylated calcium-responsive element binding (creb, pcreb), early-growth response protein 1 (egr-1), brain derived neurotrophic factor (BDNF), tyrosine kinase receptor B (trk B), drebrin and postsynaptic density-95 (PSD-95) were quantified in hippocampi of behavior trained animals (n = 7) and naïve caged controls (n = 7).

Expression of pCaMKII, BDNF, PSD-95 and egr-1 was significantly increased in the behavior-trained group. Expression of total CaMKII, total and pMAPK, total and pcreb, trk B and drebrin was comparable between groups.

Detection of significantly increased pCaMKII, BDNF, PSD-95 and egr-1 induced by behavioral training at the protein level per se is intriguing and supports the proposed importance of these molecules for neuronal information storage.

Introduction

The “Synaptic Plasticity and Memory Hypothesis” postulates that experience produces plastic changes in synaptic strength and that the persistence of these changes gives rise to neuronal information storage (nis) [46].

Although long-term potentiation/depression (LTP/LTD) is widely accepted as a concept for nis at the cellular level, it has been recognized that mechanisms underlying these cellular processes engage the genetic program of neurons and de novo synthesis of proteins [3].

“Enduring changes in synaptic efficacy may initially require phosphorylation of selected proteins, followed by changes in gene expression of the very same proteins that initially participate in the posttranslational modification” [35]. More than a hundred molecules ranging from ion channels and transcription factors to signaling molecules and cytoskeleton proteins have been implicated in nis [41], with some of them impressing as exceptionally important.

The pivotal role of CaMKII for synaptic plasticity is well known and documented (see for review [10], [48]). Through its unique regulatory properties CaMKII mediates synaptic transmission in a manifold manner. Autophosphorylation at T286 permits calcium-independent signaling, phosphorylation of N-methyl-d-aspartate receptors (NMDARs) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) alters channel conductance and cytoskeletal rearrangements promote activity-induced modulation of spine morphology. Indeed, hippocampal LTP has been shown to be essentially dependent on CaMKII [33], [44], [54]. In addition to activity-dependent phosphorylation of AMPAR subunit GluR1 at S831 by activated CaMKII, AMPARs show activity-dependent dynamic changes in their subcellular distribution. This AMPAR trafficking has attracted special attention with regards to the “silent synapse”-concept. Accumulating electrophysiological and morphological evidence indicates that synapses lacking AMPARs but containing functional NMDARs—thus postsynaptically “silent” can be “unsilenced” by the activity-induced appearance of AMPAR, thereby potentiating synaptic transmission [50]. Supportive of the idea that postsynaptic activation could be mediated through the new delivery of AMPARs to the postsynaptic membrane are reports of activity-dependent increase of AMPARs in vitro [43] and in vivo [6], [56]. To characterize the physiological role of CaMKII in vivo, several knock-out mouse models have been generated. CaMKII null mutants and mutants with a point mutation in the CaMKII gene that blocks autophosphorylation at T286 [15] are severely impaired in spatial memory tasks and display deficient LTP [13], [53] thus further confirming the crucial role of CaMKII for nis.

Another protein critically associated with AMPAR trafficking is PSD-95, a key component of the postsynaptic density, originally described as NMDAR-associated protein. As such it has been suggested that PSD-95 through its multiple protein interaction domains can mediate the association of NMDAR with different downstream signaling elements, herby regulating bi-directional synaptic plasticity. Moreover, it has been shown that activity-dependent synaptic plasticity can modulate PSD-95 association with NMDAR thus affecting the dynamic interactions between CaMKII, PSD-95 and NMDAR [14]. Shuttling of CaMKII and PSD-95 to and from NMDAR complexes is hypothesized to represent an essential molecular mechanism involved in the regulation of postsynaptic function in response to neuronal activation. While NMDARs directly bind PSD-95 [26], AMPARs are indirectly associated [9]. Only recently, PSD-95 has been found to control AMPAR incorporation during long-term potentiation and experience-driven synaptic plasticity [12]. One current model of activity-dependent synaptic delivery of AMPARs proposes that AMPARs are captured from an extrasynaptic pool and that the number of AMPAR-tethering proteins at the postsynaptic membrane is a critical determinant of AMPAR density [49]. PSD-95 has emerged as a strong candidate for anchoring of AMPARs to the postsynaptic membrane, thus potentiating synaptic transmission [49], [50].

Another major component of the memory formation cascade is mitogen-activated protein kinase (MAPK/ERK). It has become clear in the past 10 years that MAPK acts as a point of convergence for several signaling cascades implicated in information processing and storage in the brain. The multiple substrates, from cytoskeletal proteins (such as activity-regulated cytoskeleton-associated protein arc) to synaptosomal proteins (such as synapsin) and transcription factors (such as creb), predict an array of changes following MAPK activation (see for review [31]).

Creb-dependent transcription following MAPK activation has been critically associated with long-term memory formation in several forms of learning in a number of species (see for review [28]). Creb phosphorylation has been closely coupled to de novo protein synthesis and generation of dendritic spines, the primary targets of excitatory synaptic inputs associated with long-term morphological modifications seen during LTP [42]. Drebrin has been established as specific protein marker for dendritic spines [59] which are considered as “hot spots” for synaptic plasticity [22]. Although in vivo knock-down of drebrin in a rat model did not result in impaired spatial memory [23] it has been suggested that decreased drebrin is responsible for deficits in cognitive function since reduced drebrin level have been observed in brain of patients with Alzheimer's disease and Down syndrome [52].

Another transcription factor strongly associated with nis is egr-1 (zif286), an inducible transcription factor belonging to the egr family of immediate early genes.

egr-1 has been demonstrated essential for the protein synthesis-dependent late-phase LTP leading to synapse remodelling and structural reorganization of neuronal networks [11]. Moreover, several studies have shown that egr-1 expression is enhanced in experimental set-ups inducing LTP and by behavioral paradigms involving learning processes and memory formation (see for review [11]). egr-1 mutant mice presenting with impaired acquisition and severe deficits in long-term spatial memory further emphasize the relevance of egr-1 for stabilization of synaptic plasticity.

BDNF is a further key structure of plasticity-related events in the nervous system. BDNF and its preferential receptor trk B have been proven critical for induction of LTP and nis by a series of in vitro and in vivo experimental paradigms. (see for review [57]). More specifically, BDNF-induced trk B phosphorylation has been shown to activate the MAPK cascade during LTP memory formation [16]. Stimulation patterns capable of inducing LTP also increased hippocampal BNDF mRNA [7], [45] thus linking BDNF to memory events at the cellular level. Knock-out mice lacking BDNF or trk B exhibit LTP impairments [27], [38], disruption of hippocampal BDNF expression using BDNF antisense oligonucleotides resulted in severely impaired spatial learning [32]. These findings further confirm the critical role of BDNF for nis in vivo.

Although the importance of the abovementioned proteins for nis is well-documented and the induction of individual elements following behavioral training has been reported [20], [39] information on their concomitant protein expressional pattern is missing.

The objective of the present study was to determine the specific protein expressional pattern following behavioral training in mouse hippocampus, a brain region known for its pivotal role in nis. More specifically, we first intended to show that late changes in CaMKII, MAPK, creb, BDNF, trk B, PSD-95, egr-1 and drebrin protein expression related to nis can be induced by behavioral training. Second, we aimed to demonstrate that these changes are reflected by alterations at the protein level and can be revealed by Western blotting in mouse whole hippocampal tissue. Third, the importance of protein phosphorylation of CaMKII, MAPK and creb for nis was addressed by analysing levels of total CaMKII, MAPK, creb and their phosphorylated forms in hippocampus of behavior tested mice as compared to naïve caged controls. And indeed, data obtained in this study provide evidence for the concomitant upregulation of specific plasticity-related proteins following behavioral training in mouse hippocampus.

Section snippets

Behavioral training procedures

The subjects were 14 male FVB/NHim mice, aged 12 weeks at initiation of the experiments. Mice were maintained in cages made of Makrolon and filled with soft wood bedding. A standard rodent diet (Ssniff R/M-H) was available ad libitum. In the breeding colony water (acidified to pH 2.5–3.0 with HCl) was supplied by automatic valves, during experiments in water bottles. Room temperature was 22 ± 1 °C, relative humidity 50 ± 10%. Ventilation with fresh air had an air change of 15 times per hour. The

Behavioral analysis

To induce hippocampal-dependent nis mice were trained to locate a submerged platform in the Morris Water-Maze (MWM). Mice completed the training trials within (mean ± S.D.) 80.45 ± 37.21 s on the first day, 74.65 ± 40.14 s on the second day and 76.77 ± 39.28 s on the third day. Their latency to find the platform was reduced to 63.01 ± 28.42 s in the memory trial and even 50.16 ± 46.06 s in the reversal task (see Table 1). In order to ensure that animals do not fail in the MWM due to defects in basic

Discussion

The principal findings of the present study are that neuronal processes induced by behavioral training consisting of a hippocampus dependent maze task complemented by a battery of test commonly used for mouse standard phenotypic analysis are reflected by upregulation of pCaMKII, PSD-95, BDNF and egr-1 in mouse hippocampus at the protein level. It could be shown that protein expression changes following behavioral training are of such a magnitude that they can be detected by an immunochemical

Acknowledgements

We gratefully acknowledge provision of technical means for behavioral studies by Prof. Dr. Hermann Bubna-Littitz, University of Veterinary Medicine, Vienna, Austria. The technical skills of Milena Savija and Christina Widhalm in Western blot experiments and Violetta Kubesch in behavioral analysis are strongly appreciated. We are highly indebted to the Red Bull Company for generous financial assistance.

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