Nuclear Translocation of Jacob in Hippocampal Neurons after Stimuli Inducing Long-Term Potentiation but Not Long-Term Depression

Background: In recent years a number of potential synapto-nuclear protein messengers have been characterized that are thought to be involved in plasticity-related gene expression, and that have the capacity of importin- mediated and activity-dependent nuclear import. However, there is a surprising paucity of data showing the nuclear import of such proteins in cellular models of learning and memory. Only recently it was found that the transcription factor cyclic AMP response element binding protein 2 (CREB2) transits to the nucleus during long-term depression (LTD), but not during long-term potentiation (LTP) of synaptic transmission in hippocampal primary neurons. Jacob is another messenger that couples NMDA-receptor-activity to nuclear gene expression. We therefore aimed to study whether Jacob accumulates in the nucleus in physiological relevant models of activity-dependent synaptic plasticity. Methodology/Principal Findings: We have analyzed the dynamics of Jacob’s nuclear import following induction of NMDA-receptor dependent LTP or LTD at Schaffer collateral-CA1 synapses in rat hippocampal slices. Using time-lapse imaging of neurons expressing a Jacob-Green-Fluorescent-Protein we found that Jacob rapidly translocates from dendrites to the nucleus already during the tetanization period of LTP, but not after induction of LTD. Immunocytochemical stainings confirmed the nuclear accumulation of endogenous Jacob in comparison to apical dendrites after induction of LTP but not LTD. Complementary findings were obtained after induction of NMDA-receptor dependent chemical LTP and LTD i

Reinforcement of rat hippocampal LTP by holeboard training

Hippocampal long-term potentiation (LTP) can be dissociated in early-LTP lasting 4–5 h and late-LTP with a duration of more than 8 h, the latter of which requires protein synthesis and heterosynaptic activity during its induction. Previous studies in vivo have shown that early-LTP in the dentate gyrus can protein synthesis-dependently be transformed (reinforced) into late-LTP by the association of arousing novel environmental stimuli. Here we show that consolidation of spatial memory also reinforces early-LTP in the dentate gyrus. Both memory consolidation and LTP-reinforcement depend on protein synthesis. Four groups of animals were trained by five, seven, eight or 10 trials, respectively, to recognize a fixed pattern of baited holes. The last trial was performed 15 min after tetanus. Errors of long-term reference memory during the last trial were significantly decreased only in the eight- and 10-trial experimental groups compared to pseudo-trained animals. In correlation to this learning effect we found a reinforcement of early-LTP only in these experimental groups compared to controls. The data suggest that the synthesis of new proteins required for spatial reference-memory formation also contributes to LTP maintenance in the hippocampal dentate gyrus

Protein kinase Mζ is essential for the induction and maintenance of dopamine-induced long-term potentiation in apical CA1 dendrites

Dopaminergic D1/D5-receptor-mediated processes are important for certain forms of memory as well as for a cellular model of memory, hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus. D1/D5-receptor function is required for the induction of the protein synthesis-dependent maintenance of CA1-LTP (L-LTP) through activation of the cAMP/PKA-pathway. In earlier studies we had reported a synergistic interaction of D1/D5-receptor function and N-methyl-D-aspartate (NMDA)-receptors for L-LTP. Furthermore, we have found the requirement of the atypical protein kinase C isoform, protein kinase Mζ (PKMζ) for conventional electrically induced L-LTP, in which PKMζ has been identified as a LTP-specific plasticity-related protein (PRP) in apical CA1-dendrites. Here, we investigated whether the dopaminergic pathway activates PKMζ. We found that application of dopamine (DA) evokes a protein synthesis-dependent LTP that requires synergistic NMDA-receptor activation and protein synthesis in apical CA1-dendrites. We identified PKMζ as a DA-induced PRP, which exerted its action at activated synaptic inputs by processes of synaptic tagging

Identification of transmitter systems and learning tag molecules involved in behavioral tagging during memory formation

Long-term memory (LTM) consolidation requires the synthesis of plasticity-related proteins (PRPs). In addition, we have shown recently that LTM formation also requires the setting of a “learning tag” able to capture those PRPs. Weak training, which results only in short-term memory, can set a tag to use PRPs derived from a temporal-spatial closely related event to promote LTM formation. Here, we studied the involvement of glutamatergic, dopaminergic, and noradrenergic inputs on the setting of an inhibitory avoidance (IA) learning tag and the synthesis of PRPs. Rats explored an open field (PRP donor) followed by weak (tag inducer) or strong (tag inducer plus PRP donor) IA training. Throughout pharmacological interventions around open-field and/or IA sessions, we found that hippocampal dopamine D1/D5- and β-adrenergic receptors are specifically required to induce PRP synthesis. Moreover, activation of the glutamatergic NMDA receptors is required for setting the learning tags, and this machinery further required α-Ca2+/calmodulin-dependent protein kinase II and PKA but not ERK1/2 activity. Together, the present findings emphasize an essential role of the induction of PRPs and learning tags for LTM formation. The existence of only the PRP or the tag was insufficient for stabilization of the mnemonic trace

NMDA dose-dependent increase in Jacob nuclear immunoreactivity of primary hippocampal neurons.

<p>Hippocampal primary neurons were maintained in 1 ml of neurobasal medium (NB). 12–16 hours prior stimulation the volume of NB medium was increased to 1.6 ml. Then 800 µl of conditioned NB medium was removed and NMDA applied. Five minutes later the NMDA-NB medium was replaced with the 800 µl conditioned NB medium and 25 minutes later the neurons were fixed. A) The diagram summarizes normalized nuclear Jacob immunofluorescence at DIV 18 after bath application of 20 µM or 50 µM NMDA. B) The bar diagram indicates the gain in the nuclear fluorescence signal after treatment with 20 µM or 50 µM NMDA in comparison to drug free conditions. Note the increased nuclear accumulation of Jacob at DIV 18. C) Representative images of Jacob nuclear immunoreactivity and DAPI staining in hippocampal primary neurons at DIV 23 under control, 20 µM and 50 µM NMDA conditions, respectively. ***p<0.001, **p<0.01. Scale bar is 10 µm.</p

Time-lapse imaging of Jacob-GFP nuclear translocation in acute hippocampal slices in response to LTP induction.

<p>A) Schematic representation of data acquisition and image analysis. B) A representative 12 bit grayscale confocal image of a neuron expressing Jacob-eGFP (grayscale range: 0: black to 4095: white). Other images represent F/F₀ images that were acquired at the indicated time points. ROIs are indicated for nucleus, proximal dendrite and background area with white circles and squares (horizontal white scale bar: 10 µm). The colored vertical bar encodes green as 0, and dark blue as -20% and red as +20%. C) The time course of normalized fluorescence intensities is depicted for nuclei and proximal dendrites acquired under application with anisomycin (Anis., 50 µM) and Anis. + AP5 (40 µM). Brackets and asterisks (**, p≤0.05; Mann-Whitney U-Test) indicate the significance intervals between various groups (symbol pairs on bracket). The inlet shows fEPSP-transients before, and 5 minutes after the first tetanization for Anis. and Anis.+AP5 experiments. Scale bars indicate 0.5 mV for vertical and 2 ms for horizontal bar. Vertical arrows show the time point of 100 Hz/1-s tetanization.</p