Distinct set of kinases induced after retrieval of spatial memory discriminate memory modulation processes in the mouse hippocampus.

Protein phosphorylation and dephosphorylation events play a key role in memory formation and various protein kinases and phosphatases have been firmly associated with memory performance. Here, we determined expression changes of protein kinases and phosphatases following retrieval of spatial memory in CD1 mice in a Morris Water Maze task, using antibody microarrays and confirmatory Western blot. Comparing changes following single and consecutive retrieval, we identified stably and differentially expressed kinases, some of which have never been implicated before in memory functions. On the basis of these findings we define a small signaling network associated with spatial memory retrieval. Moreover, we describe differential regulation and correlation of expression levels with behavioral performance of polo-like kinase 1. Together with its recently observed genetic association to autism-spectrum disorders our data suggest a role of this kinase in balancing preservation and flexibility of learned behavior.

Hippocampal levels of phosphorylated protein kinase A (phosphor-S96) are linked to spatial memory enhancement by SGS742.

Cognitive enhancement by the GABA (B) receptor antagonist SGS742 has been well-documented, but mechanisms of action are not fully elucidated. Previous work has proposed involvement of somatostatin-14 and protein kinase C in cognitive enhancement; phospho-protein kinase A (p-PKA), fyn, and phospho-fyn are known signaling systems for spatial memory. It was the aim of the study to determine hippocampal levels of these proteins following SGS742-treatment and to correlate them with the outcome from the Morris water maze (MWM), represented by the parameter "time spent in the target quadrant" during the probe trial. OF1 mice were used for the experiments and divided into four groups: intraperitoneal SGS742 and saline solution treatment, both, tested in the MWM, and two yoked controls. Six hours following the probe trial, hippocampal protein levels were determined by immunoblotting. In the MWM, time spent in the target quadrant was significantly enhanced by SGS742 treatment. p-PKA levels were significantly increased only in the SGS742-treated group tested in the MWM as compared to saline treatment. In yoked controls, no significant differences in p-PKA levels between SGS742 and saline treatment were observed. Somatostatin-14 levels were significantly increased in both SGS742-treated groups. No statistically significant changes of other protein levels were observed. We propose that GABA (B) antagonism represented by SGS742 treatment led to cognitive enhancement involving p-PKA, because yoked controls treated with SGS742 were comparable to yoked saline-treated controls. The finding that somatostatin-14 was also induced in the SGS742-treated yoked controls points to a drug side effect, and therefore the role of somatostatin-14 for cognitive enhancement remains open.

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742.

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two-dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ-TOF mass spectrometry. Quantification of proteins from four groups (NaCl-treated mice, SGS742-treated mice, SGS742-treated yoked controls, and NaCl-treated yoked controls) was carried out according to an in-gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABAB antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742-treated mice (mean+/-SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19+/-0.41 for synapsin Ia, and 1.41+/-0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl-treated controls and the use of yoked controls that were ruling out swimming- and stress-mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation.

Apodemus sylvaticus (LOXT) is a suitable mouse strain for testing spatial memory retention in the Morris water maze.

Information on the difference in cognitive function between laboratory and wild-caught mice is anecdotal and this question has not been systematically studied. Moreover, studying a wild-caught mouse strain per se may add information to the repertoire of mouse strains available. We aimed to study spatial memory in a wild mouse strain (Apodemus sylvaticus, AS) as compared to two individual laboratory mouse strains. Male AS (n=20), CD1 (n=19) and C57BL/6J mice (n=19), 12-14 weeks old, were used in the experiments. The Morris water maze (MWM) was used for determination of spatial memory and time spent in the target quadrant at time points 5 (D5) and 12 days (D12) was evaluated. During the acquisition phase latency to reach the platform and path length to reach the platform was evaluated. Following four training days on day 5 (D5), time spent in the target quadrant was highest in AS>CD1>C57BL/6J (P<0.006). On day 12 (D12), time spent in the target quadrant was significantly higher in AS than in both other strains (P<0.001). All animals learned the task and during the acquisition phase, latency to reach the platform as well as path length decreased significantly in AS. It is concluded that the AS is the most suitable strain for the evaluation of spatial memory in the MWM and is presenting with memory retention superior to laboratory mouse strains CD1 and C57BL/6J.

Strain-dependent effects of SGS742 in the mouse.

SGS742 has been reported to increase spatial memory in rodents. However, effects of SGS742 have not been systematically assessed in different strains so far and indeed strains show different cognitive abilities per se. The aim of the study was therefore to examine the effect of SGS742 in three different inbred (C57BL/6J, DBA/2, BALB/c) strains and three outbred strains (CD1, CF1, OF1). Mice were administered intra-peritonial (3-aminopropyl)n-butylphosphinic acid (SGS742; GABA (B)-receptor antagonist) and tested in the Morris water maze (MWM). Open field, elevated plus maze, neurological observational battery and rota rod were carried out to support interpretation of data. SGS742 enhanced performance in the MWM in an inbred strain, C57BL/6J and in the outbred strain OF1 in terms of learning and memory formation at the consolidation level. A series of side effects as e.g. reduced motor coordination and proprioception were noticed that, however, may not have been influencing results observed in the cognition task. In conclusion, SGS742 enhanced cognitive performance in two mouse strains and we learned that testing compounds for effects on spatial memory should be carried out in several strains or even different species in order to claim cognitive enhancement.

Hippocampal protein kinase C family members in spatial memory retrieval in the mouse.

Although a few individual members of the protein kinase C (PKC) family were studied in spatial memory no systematic approach was carried out to concomitantly determine all described PKC family members in spatial memory of the mouse. It was therefore the aim of the current study to link hippocampal PKCs to memory retrieval in the Morris water maze (MWM). CD1 mice were trained (n=9) or untrained (n=9) in the MWM, hippocampi were taken 6h following the test for memory retrieval and PKCs were determined in mouse hippocampi by immunoblotting. The trained animals learned the spatial memory task and kept memory at the probe trial. PKCs alpha and epsilon were comparable between groups while PKCs beta, delta, gamma (two forms, i.e. two bands on Western blotting), zeta (2 forms) were higher in trained mice and theta (2 forms) were lower in trained mice. PKC gamma (1 form) was significantly correlating with the time spent in the target quadrant (r=0.7933; P=0.0188). Changes of hippocampal levels of PKCs beta, delta, gamma, zeta and theta were paralleling memory retrieval of the MWM task but correlations revealed that spatial memory retrieval was only linked to one form of PKC gamma. Results are also in agreement with a recent publication showing that PKM zeta is not required for memory formation. These findings may be relevant for the interpretation of previous work and the design of future work on the protein kinase C family in spatial memory of the mouse.

Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze.

Evaluation of spatial learning and memory is mainly carried out using the Morris water maze as a single paradigm. We intended to test whether mice in the Barnes maze and Multiple T-maze would lead to comparable results and to test two individual mouse strains with different anxiety levels. C57BL/6J and CD1 male mice were used in the experiments. During the acquisition phase, learning was measured using parameters latency, path length, errors in the BM and correct decisions in MTM. Mice were trained for 4 days and probe trials were performed on days 5 and 12. Latencies reduction over the training period indicated that both strains learned all tasks. During retention phase at days 5 and 12 C57BL/6J performed the Barnes maze and Multiple T-maze task better than CD1 mice while CD1 performed better than C57BL/6J in the Morris water maze. In the BM at day 12, C57BL/6J kept the level of visits to target observed at day 5 whereas CD1 performed worse. Strain- and task-dependent differences were observed using the three mazes. Therefore, fair evaluation of spatial memory demands application of (at least) two different test systems, a water- and a land maze. Different anxiety-related behaviour as well as stress-responses in the strains used may help to interpret the findings reported and again may propose the use of at least two mouse strains when robust evaluation of spatial memory is considered.

Strain-dependent hippocampal protein levels of GABA(B)-receptor subunit 2 and NMDA-receptor subunit 1.

Mouse strains differ from one another in cognitive function as learning and memory as well as synaptic plasticity. Although molecular explanations for this heterogeneity have been proposed, there is no available information on strain-dependent GABA(B)-receptor levels in hippocampus that may explain observed differences in GABA(B)-receptor-mediated cognitive enhancement among strains. This formed the rationale to perform a study on GABA(B) and related somatostatin receptor 5 and NMDA receptors that have been reported to be involved in memory enhancement by GABA(B)-antagonism. Hippocampi of three inbred (C57BL/6J, DBA/2, BalbC) and three outbred (OF1, CD1, CF1) mouse strains were taken. Membrane fractions were prepared and subsequently levels of GABA(B)-receptor subunits 1 and 2, NMDA-receptor subunits 1 and 2a and b, as well as somatostatin receptor 5 were determined by immunoblotting. NMDA-receptor subunit 1 levels were significantly higher in C57BL/6J than in DBA/2. GABA(B)-receptor subunit 2 levels were significantly decreased in C57BL/6J as compared to OF1, CD1, CF1 and DBA/2. No statistically significant differences for other receptors were observed. Different NMDA-receptor subunit 1 and GABA(B)-receptor subunit 2 levels observed in naive mouse strains reflect strain-dependent expression of subunits and are proposed to lead to altered stoichiometry of GABA(B)-receptor complexes. Modification of receptor complex stoichiometry in the hippocampus in turn may well be responsible for cognitive differences observed in spatial memory between mouse strains.

The role of post-translational modifications for learning and memory formation.

Learning and memory depend on molecular mechanisms involving the protein machinery. Recent evidence proposes that post-translational modifications (PTMs) play a major role in these cognitive processes. PTMs including phosphorylation of serine, threonine, and tyrosine are already well-documented to play a role for synaptic plasticity of the brain, neurotransmitter release, vesicle trafficking and synaptosomal or synaptosomal-associated proteins are substrates of a series of specific protein kinases and their counterparts, protein phosphatases. But protein phosphorylation is only one out of many possible PTMs and first work shows a role of palmitoylation as well as glycosylation for proteins involved in memory formation. Recent technology may now allow reliable detection and even quantification of PTMs of proteins involved in the cognitive system. This will contribute to the understanding of mechanisms for learning and memory formation at the chemical level and has to complement determination of protein levels and indeed determination of protein expression per se generates limited information. The many other PTMs expected including protein nitrosylation and alkylation will even represent targets for pharmacological interventions but in turn increase the complexity of the system. Nevertheless, determination of the presence and the function of PTMs is mandatory and promising cognitive research at the protein chemical level.

The cognitive enhancer SGS742 does not involve major known signaling cascades in OF1 mice.

In a previous study we have shown that SGS742, a cognitive enhancer acting by GABA(B) receptor antagonism improved spatial learning in OF1 mice. The aim of the study was therefore to screen representatives of several signaling cascades known to be involved in memory formation at the protein level. NaCl-, SGS742- treatment and yoked controls for NaCl and SGS742 were studied. Six hours following testing OF1 mice in the Morris water maze, hippocampal signaling proteins calmodulin kinase II (CaMKII) (phosphorylated (P) and non-phosphorylated (NP) form), CREB (P, NP), ERK (P, NP), BDNF, Egr-1, Trk-B and neuro-cytoskeleton elements drebrin and PSD-95 were determined by immunoblotting. Protein patterns were comparable between the control and SGS742 treated group revealing that these proteins may not be involved in the mechanisms underlying cognitive enhancement by SGS742 and this should be taken into account for interpretation and design of previous and future related neurochemical studies.

Cognitive enhancement by SGS742 in OF1 mice is linked to specific hippocampal protein expression.

Cognitive enhancement by the GABA (B) antagonist SGS742 has been reported and we decided to search for proteins involved. Hippocampi from OF1 mice were from SGS742- treated animals and three control groups. Proteins were extracted and run on 2DE, and spots were quantified. Significantly different protein spots were identified by two mass spectrometry principles, nano-LC-ESI-MS/MS and by nano-LC-ESI(CID/ETD)-MS/MS. Signaling, chaperone and metabolic enzyme proteins were linked to memory enhancement.