Magdalena Sanhueza

The CaMKII/NMDAR complex as a molecular memory

CaMKII is a major synaptic protein that is activated during the induction of long-term potentiation (LTP) by the Ca2+ influx through NMDARs. This activation is required for LTP induction, but the role of the kinase in the maintenance of LTP is less clear. Elucidating the mechanisms of maintenance may provide insights into the molecular processes that underlie the stability of stored memories. In this brief review, we will outline the criteria for evaluating an LTP maintenance mechanism. The specific hypothesis evaluated is that LTP is maintained by the complex of activated CaMKII with the NMDAR. The evidence in support of this hypothesis is substantial, but further experiments are required, notably to determine the time course and persistence of complex after LTP induction. Additional work is also required to elucidate how the CaMKII/NMDAR complex produces the structural growth of the synapse that underlies late LTP. It has been proposed by Frey and Morris that late LTP involves the setting of a molecular tag during LTP induction, which subsequently allows the activated synapse to capture the proteins responsible for late LTP. However, the molecular processes by which this leads to the structural growth that underlies late LTP are completely unclear. Based on known binding reactions, we suggest the first molecularly specific version of tag/capture hypothesis: that the CaMKII/NMDAR complex, once formed, serves as a tag, which then leads to a binding cascade involving densin, delta-catenin, and N-cadherin (some of which are newly synthesized). Delta-catenin binds AMPA-binding protein (ABP), leading to the LTP-induced increase in AMPA channel content. The addition of postsynaptic N-cadherin, and the complementary increase on the presynaptic side, leads to a trans-synaptically coordinated increase in synapse size (and more release sites). It is suggested that synaptic strength is stored stably through the combined actions of the CaMKII/NMDAR complex and N-cadherin dimers. These N-cadherin pairs have redundant storage that could provide informational stability in a manner analogous to the base-pairing in DNA.

Intrinsic subthreshold oscillations of the membrane potential in pyramidal neurons of the olfactory amygdala

The amygdala complex is a heterogeneous group of temporal lobe brain structures involved in the processing of biologically significant sensory stimuli and in the generation of appropriate responses to them. The amygdala has also been implicated in certain forms of emotional learning and memory. While much progress has been made in understanding neural processing in the basolateral subgroup of the amygdala, physiological studies in the cortical regions of the complex, also known as olfactory amygdala, are missing. Using a rat brain slice preparation, we conducted whole‐cell recordings on pyramidal neurons of the periamygdaloid cortex and the anterior cortical nucleus, two structures receiving direct connections from the olfactory bulb. Upon depolarization by current injection through the recording electrode, a fraction of periamygdaloid cortex and most anterior cortical nucleus layer II pyramidal neurons displayed an intermittent discharge pattern, where clusters of action potentials were interspersed by periods of membrane potential subthreshold oscillations. Oscillations frequency increased with membrane potential and correlated linearly with the cluster spiking frequency. Frequency ranged from 3 to 20 Hz, considering different cells and membrane potential values (up to approximately 30 mV above resting potentials of typically approximately −70 mV). Subthreshold oscillations were preserved after pharmacological inhibition of fast excitatory and inhibitory synaptic transmission, but were abolished by application of the sodium channel blocker tetrodotoxin. We conclude that pyramidal neurons of the olfactory cortical amygdala display intrinsically generated voltage‐dependent membrane potential rhythmic fluctuations in the theta‐low beta range, requiring the activation of a sodium conductance.

Tonic and Phasic Receptor Neurons in the Vertebrate Olfactory Epithelium

Olfactory receptor neurons (ORNs) respond to odorants with characteristic patterns of action potentials that are relevant for odor coding. Prolonged odorant exposures revealed three populations of dissociated toad ORNs, which were mimicked by depolarizing currents: tonic (TN, displaying sustained firing, 49% of 102 cells), phasic (PN, exhibiting brief action potential trains, 36%) and intermediate neurons (IN, generating trains longer than PN, 15%). We studied the biophysical properties underlying the differences between TNs and PNs, the most extreme cases among ORNs. TNs and PNs possessed similar membrane capacitances (approximately 4 pF), but they differed in resting potential (-82 versus -64 mV), input resistance (4.2 versus 2.9 G(Omega)) and unspecific current, I(u) (TNs: 0 < I(u) <or= 1 pA/pF; and PNs: I(u) > 1 pA/pF). Firing behavior did not correlate with differences in voltage-gated conductances. We developed a mathematical model that accurately simulates tonic and phasic patterns. Whole cell recordings from rat ORNs in fragments (approximately 4 mm(2)) of olfactory epithelium showed that such a tissue normally contains tonic and phasic receptor neurons, suggesting that this feature is common across a wide range of vertebrates. Our findings show that the individual passive electrical properties can govern the firing patterns of ORNs.

Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Olfactory chemotransduction involves a signaling cascade. In addition to triggering transduction, odors suppress ion conductances. By stimulating with brief odorant pulses, we observed a current associated with odor-induced suppression of voltage-gated conductances and studied its time dependence. We characterized this suppression current in isolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed by odor pulses in almost every neuron, and suppression is caused by odors inducing excitation and by those inducing inhibition, indicating a nonselective phenomenon, in contrast to transduction. Suppression has a 10-fold shorter latency than transduction. Suppression was more pronounced when odors were applied to the soma than to the cilia, opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it in Drosophila photoreceptor cells, demonstrating its independence from the chemotransduction cascade. We show that odor concentrations causing suppression are similar to those triggering chemotransduction and that both suppression and transduction contribute to the odor response in isolated olfactory neurons. Furthermore, suppression affects spiking, implying a possible physiological role in olfaction.Olfactory chemotransduction involves a signaling cascade. In addition to triggering transduction, odors suppress ion conductances. By stimulating with brief odorant pulses, we observed a current associated with odor-induced suppression of voltage-gated conductances and studied its time dependence. We characterized this suppression current in isolated Caudiverbera caudiverbera olfactory neurons. All four voltage-gated currents are suppressed by odor pulses in almost every neuron, and suppression is caused by odors inducing excitation and by those inducing inhibition, indicating a nonselective phenomenon, in contrast to transduction. Suppression has a 10-fold shorter latency than transduction. Suppression was more pronounced when odors were applied to the soma than to the cilia, opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it in Drosophila photoreceptor cells, demonstrating its independence from the chemotransduction cascade. We show that odor concentrations causing suppression are similar to those triggering chemotransduction and that both suppression and transduction contribute to the odor response in isolated olfactory neurons. Furthermore, suppression affects spiking, implying a possible physiological role in olfaction.

On the mechanism of synaptic depression induced by CaMKIIN, an endogenous inhibitor of CaMKII.

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca2+ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.

Electrical resonance in the θ frequency range in olfactory amygdala neurons.

The cortical amygdala receives direct olfactory inputs and is thought to participate in processing and learning of biologically relevant olfactory cues. As for other brain structures implicated in learning, the principal neurons of the anterior cortical nucleus (ACo) exhibit intrinsic subthreshold membrane potential oscillations in the θ-frequency range. Here we show that nearly 50% of ACo layer II neurons also display electrical resonance, consisting of selective responsiveness to stimuli of a preferential frequency (2–6 Hz). Their impedance profile resembles an electrical band-pass filter with a peak at the preferred frequency, in contrast to the low-pass filter properties of other neurons. Most ACo resonant neurons displayed frequency preference along the whole subthreshold voltage range. We used pharmacological tools to identify the voltage-dependent conductances implicated in resonance. A hyperpolarization-activated cationic current depending on HCN channels underlies resonance at resting and hyperpolarized potentials; notably, this current also participates in resonance at depolarized subthreshold voltages. KV7/KCNQ K+ channels also contribute to resonant behavior at depolarized potentials, but not in all resonant cells. Moreover, resonance was strongly attenuated after blockade of voltage-dependent persistent Na+ channels, suggesting an amplifying role. Remarkably, resonant neurons presented a higher firing probability for stimuli of the preferred frequency. To fully understand the mechanisms underlying resonance in these neurons, we developed a comprehensive conductance-based model including the aforementioned and leak conductances, as well as Hodgkin and Huxley-type channels. The model reproduces the resonant impedance profile and our pharmacological results, allowing a quantitative evaluation of the contribution of each conductance to resonance. It also replicates selective spiking at the resonant frequency and allows a prediction of the temperature-dependent shift in resonance frequency. Our results provide a complete characterization of the resonant behavior of olfactory amygdala neurons and shed light on a putative mechanism for network activity coordination in the intact brain.

NMDA Receptor Subunit Composition Controls Dendritogenesis of Hippocampal Neurons through CAMKII, CREB‐P and H3K27ac

Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. While NR1NR2B receptors are predominantly expressed in immature neurons and promote plasticity, NR1NR2A receptors are mainly expressed in mature neurons and induce circuit stability. How the different subunits regulate these processes is unclear, but this is likely related to the presence of their distinct C‐terminal sequences that couple different signaling proteins. Calcium‐calmodulin‐dependent protein kinase II (CaMKII) is an interesting candidate as this protein can be activated by calcium influx through NMDARs. CaMKII triggers a series of biochemical signaling cascades, involving the phosphorylation of diverse targets. Among them, the activation of cAMP response element‐binding protein (CREB‐P) pathway triggers a plasticity‐specific transcriptional program through unknown epigenetic mechanisms. Here, we found that dendritogenesis in hippocampal neurons is impaired by several well‐characterized constructs (i.e., NR2B‐RS/QD) and peptides (i.e., tatCN21) that specifically interfere with the recruitment and interaction of CaMKII with the NR2B C‐terminal domain. Interestingly, we found that transduction of NR2AΔIN, a mutant NR2A construct with increased interaction to CaMKII, reactivates dendritogenesis in mature hippocampal neurons in vitro and in vivo. To gain insights into the signaling and epigenetic mechanisms underlying NMDAR‐mediated dendritogenesis, we used immunofluorescence staining to detect CREB‐P and acetylated lysine 27 of histone H3 (H3K27ac), an activation‐associated histone tail mark. In contrast to control mature neurons, our data shows that activation of the NMDAR/CaMKII/ERK‐P/CREB‐P signaling axis in neurons expressing NR2AΔIN is not correlated with increased nuclear H3K27ac levels.

Competition between Persistent Na+ and Muscarine-Sensitive K+ Currents Shapes Perithreshold Resonance and Spike Tuning in CA1 Pyramidal Neurons

Neurons from many brain regions display intrinsic subthreshold theta-resonance, responding preferentially to theta-frequency oscillatory stimuli. Resonance may contribute to selective communication among neurons and to orchestrate brain rhythms. CA1 pyramidal neurons receive theta activity, generating place fields. In these neurons the expression of perithreshold frequency preference is controversial, particularly in the spiking regime, with evidence favoring either non-resonant (integrator-like) or resonant behavior. Perithreshold dynamics depends on the persistent Na+ current INaP developing above −70 mV and the muscarine-sensitive K+ current IM activating above −60 mV. We conducted current and voltage clamp experiments in slices to investigate perithreshold excitability of CA1 neurons under oscillatory stimulation. Around 20% of neurons displayed perithreshold resonance that is expressed in spiking. The remaining neurons (~80%) acted as low-pass filters lacking frequency preference. Paired voltage clamp measurement of INaP and IM showed that perithreshold activation of IM is in general low while INaP is high enough to depolarize neurons toward threshold before resonance expression, explaining the most abundant non-resonant perithreshold behavior. Partial blockade of INaP by pharmacological tools or dynamic clamp changed non-resonant to resonant behavior. Furthermore, shifting IM activation toward hyperpolarized potentials by dynamic clamp also transformed non-resonant neurons into resonant ones. We propose that the relative levels of INaP and IM control perithreshold behavior of CA1 neurons constituting a gating mechanism for theta resonance in the spiking regime. Both currents are regulated by intracellular signaling and neuromodulators which may allow dynamic switching of perithreshold behavior between resonant and non-resonant.

TRP, TRPL and cacophony channels mediate Ca2+ influx and exocytosis in photoreceptors axons in Drosophila.

In Drosophila photoreceptors Ca2+-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca2+ channel cacophony is also present there. Measurements in the lamina with the Ca2+ fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca2+ increments mediated by cacophony. Additional Ca2+ influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca2+ also contributes to exocytosis, since this process was reduced after Ca2+-store depletion. Therefore, several Ca2+ pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca2+ release and the activation of TRP and TRPL coupled to Ca2+ depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.

CaMKII: A Master Functional and Structural Molecule in Synaptic Plasticity and Memory

Learning and memory relies, at least in part, on activity-dependent synaptic plasticity. A major plasticity model at glutamatergic synapses is NMDA-receptor (NMDAR)-dependent long-term potentiation (LTP). Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) is critical for LTP and several forms of learning. It is a major component of post-synaptic densities and dendritic spines. Kinase interactions with key proteins in these specializations are differentially modulated by activity and dynamically regulate holoenzyme activity. During LTP CaMKII is activated, autophosphorylated and persistently translocated to synapses through NMDAR binding. Pharmacological or genetic interference with these processes impair LTP and learning. CaMKII may cause potentiation by synaptic recruitment of AMPA-type receptors (AMPARs) through regulation of receptor binding to scaffolding proteins. Additionally, CaMKII-dependent phosphorylation increases AMPAR conductance. Interestingly, CaMKII is also involved in metaplasticity, as it can regulate the sign of synaptic modification (potentiation or depression). The advent of high-resolution optical techniques has allowed inspection of CaMKII localization and activity in spine microdomains, providing new insights on holoenzyme multifaceted involvement in activity-dependent functional and structural changes. Finally, evidence suggests a role of CaMKII interaction with NMDARs in the maintenance of synaptic strength and spine stability. Thus, CaMKII emerges as a critical and complex controller of synaptic function and information storage, playing both enzymatic and structural roles.