Coupling of extrasynaptic NMDA receptors to a CREB shut-off pathway is developmentally regulated

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Abstract

Electrical activation of hippocampal neurons can cause calcium influx through different entry sites which may specify nuclear signalling and induction of gene transcription and downstream physiological outputs. Genomic responses initiated by NMDA receptors (NMDARs) are critically dependent on whether synaptically or extrasynaptically located receptors are stimulated; calcium flux through synaptic NMDARs activates CREB whereas flux through extrasynaptic NMDARs triggers a CREB shut-off signal. Here we investigated the possibility that the coupling of extrasynaptic NMDARs to the CREB shut-off pathway is regulated during in vitro development. Cultured hippocampal neurons were analyzed after 7 or 12 days of in vitro culturing. We found that synaptic NMDAR activity induced CREB phosphorylation at day in vitro (DIV) 7 and DIV 12. In contrast, the extrasynaptic NMDAR-dependent CREB shut-off signal is developmentally regulated. At DIV 12 extrasynaptic NMDAR activation shuts down CREB and overrides the CREB-activating signal triggered by synaptic NMDAR activation. In contrast, at DIV 7 this shut off signal is absent; both synaptic and extrasynaptic NMDARs activate CREB function. Developmental changes in NMDAR signaling have been proposed to contribute to the emergence of glutamate excitotoxicity, which causes apoptosis or necrosis depending on the severity of the insult. Since CREB regulates a number of pro-survival genes, the emergence of this shut-off around DIV 7 may contribute to the increase in susceptibility of neurons to glutamate-induced neuropathology in vitro and in vivo during post-natal development.

Introduction

Neuronal calcium transients mediate many adaptive changes within the nervous system through the activation of calcium-dependent signalling pathways [1], [2], [3]. Such signals are responsible, for example, for short- and long-term synaptic plasticity, and can also promote cell survival or death. It is becoming increasingly clear from work carried out over the last 10 years that calcium influx into neurons, through either voltage-gated or ligand (neurotransmitter)-gated calcium channels, does not yield a pre-ordained response from the cell. The neuron is wired in a sophisticated way that enables it to respond differently to calcium signals with different characteristics. These critical parameters include the magnitude of the calcium signal, but also its spatial properties, its temporal properties, its oscillatory frequency (if applicable) and the channel through which the calcium initially flows into the neuron [4], [5], [6], [7], [8], [9], [10], [11].

The last parameter may at first sight appear to be counter-intuitive as one might expect the neuron to respond similarly to two similar calcium signals, regardless of the type of channel through which the calcium flowed from the extracellular medium. However, the emerging picture is that different channels associate with different signalling molecules or adapters which may therefore be specifically, or at least more efficiently, activated when calcium flows in through that particular channel. A classical example of calcium entry site-specific differences is the regulation of transcription mediated by the CRE-binding protein CREB. CREB activation involves a crucial step: phosphorylation on serine 133 [12]. The degree of CREB-dependent transcription depends considerably on the duration over which it remains phosphorylated [5], [11], [13].

Calcium flux though L-type voltage-gated calcium channels is a potent activator of CREB-mediated gene expression. In contrast, stimulation of NMDA receptors (NMDARs) using glutamate bath application only very transiently activates CREB [4], [8]. These findings seem to indicate that L-type calcium channels have a privileged role in CREB-mediated gene expression that has been the subject of much recent investigation [8], [14], [15], [16]. However, the stimulation paradigms used in these studies (i.e. KCl-induced membrane depolarisation or glutamate bath application) only remotely resemble physiological stimuli and thus provide limited insight into how synaptic activity controls nuclear signalling and transcription. Recently we have shown that calcium influx through the same receptor can have opposing effects on signalling to CREB, depending on the location of the receptor [17]. Calcium entry through synaptic NMDARs strongly promotes CREB activity and CREB-dependent gene expression. In direct contrast, activation of NMDARs triggers a dominant CREB shut-off signal that acts via inducing rapid dephosphorylation of CREB on serine 133; this blocks CREB-dependent reporter gene expression initiated by calcium influx through either L-type channels or synaptic NMDARs [17]. Thus, the observation that bath glutamate very transiently activates CREB and can cause CREB dephosphorylation is explained by the dominant action of extrasynaptic NMDAR signalling over synaptic NMDAR signalling, both of which are activated by the addition of glutamate to the culture medium [17].

The transcription factor CREB is thought to be central to certain aspects of adaptive responses by the neuron. It is believed to be important in consolidating activity-evoked changes to neuronal connectivity and strength that may underlie learning and memory [18]. It also controls the expression of a number of pro-survival genes and has been shown to be important in determining neuronal survival/death [19], [20], [21], [22], [23], [24], [25], [26].

The CREB shut-off/dephosphorylation signal evoked by the bath application of glutamate is under developmental control. Neurons cultured from p0 rats must remain in culture for more than 7 days in vitro (DIV 7) before they will respond to bath glutamate by inactivating CREB previously phosphorylated by either elevated cAMP or calcium influx through L-type calcium channels [27]. We have investigated further the developmental control of this CREB shut-off pathway in the light of our recent findings that point at extrasynaptic NMDARs as the mediator of this pathway [17]. We have also looked for any developmental variation in synaptic NMDAR signalling to CREB. We found that in DIV 7 neurons, like DIV 12 neurons, synaptic NMDAR-dependent calcium transients can be evoked that are strong and prolonged activators of CREB phosphorylation. In contrast to the findings in DIV 12 neurons, extrasynaptic NMDAR activation in DIV 7 neurons is unable to cause dephosphorylation of CREB that has been previously phosphorylated by synaptic NMDAR-dependent calcium transients. At DIV 12, the extrasynaptic NMDAR signal has become dominant and shuts off CREB even in the face of synaptic NMDAR activation.

Section snippets

Hippocampal cultures and stimulations

Hippocampal neurons were cultured as described [41] except that growth media was supplemented with B27 (Gibco/BRL). Stimulations were done after a culturing period of 7 to 12 days. Bursts of action potential firing were induced by treatment of cultured hippocampal neurons with 50 μM bicuculline. D(−)APV was from Sigma; MK-801 was from Tocris.

Imaging

Fluo-3 calcium imaging and confocal laser scanning microscopy was done as described [6]. Calcium concentrations were expressed as a function of the Fluo-3

Results

We initially investigated whether synaptically evoked calcium transients could be evoked in DIV 7 neurons. In DIV 12 hippocampal neurons this was done by blocking inhibitory GABAA receptors with bicuculline. As the hippocampal cultures used contain inhibitory interneurons (∼10%), which impose a tonic inhibition on the neuronal network, blockade of GABAA-ergic transmission causes the neurons to fire synchronous bursts of action potentials [9]. The bicuculline-induced bursts of action potentials

Discussion

Our previous study showed that in DIV 12 neurons synaptic NMDAR activation robustly activates CREB while extrasynaptic NMDAR activity de-activates it [17]. The molecular basis for this striking difference in nuclear signalling induced by synaptic and extrasynaptic NMDARs is likely due to differences in the composition of the NMDAR signalling complexes. Many proteins are known to associate with NMDARs [28] but it remains a task for the future to determine which proteins form part of the synaptic

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