αCaMKII controls the establishment of cocaine's reinforcing effects in mice and humans.

Although addiction develops in a considerable number of regular cocaine users, molecular risk factors for cocaine dependence are still unknown. It was proposed that establishing drug use and memory formation might share molecular and anatomical pathways. Alpha-Ca(2+)/calmodulin-dependent protein kinase-II (αCaMKII) is a key mediator of learning and memory also involved in drug-related plasticity. The autophosphorylation of αCaMKII was shown to accelerate learning. Thus, we investigated the role of αCaMKII autophosphorylation in the time course of establishing cocaine use-related behavior in mice. We found that αCaMKII autophosphorylation-deficient αCaMKII(T286A) mice show delayed establishment of conditioned place preference, but no changes in acute behavioral activation, sensitization or conditioned hyperlocomotion to cocaine (20 mg kg(-1), intraperitoneal). In vivo microdialysis revealed that αCaMKII(T286A) mice have blunted dopamine (DA) and blocked serotonin (5-HT) responses in the nucleus accumbens (NAcc) and prefrontal cortex after acute cocaine administration (20 mg kg(-1), intraperitoneal), whereas noradrenaline responses were preserved. Under cocaine, the attenuated DA and 5-HT activation in αCaMKII(T286A) mice was followed by impaired c-Fos activation in the NAcc. To translate the rodent findings to human conditions, several CAMK2A gene polymorphisms were tested regarding their risk for a fast establishment of cocaine dependence in two independent samples of regular cocaine users from Brazil (n=688) and Switzerland (n=141). A meta-analysis across both samples confirmed that CAMK2A rs3776823 TT-allele carriers display a faster transition to severe cocaine use than C-allele carriers. Together, these data suggest that αCaMKII controls the speed for the establishment of cocaine's reinforcing effects.

Long-lasting regulation of hippocampal Bdnf gene transcription after contextual fear conditioning.

Long-term memory formation requires de novo protein synthesis and gene transcription. During contextual long-term memory formation brain-derived neurotrophic factor (BDNF) gene expression changes in conjunction with alterations of DNA methylation in the Bdnf gene. However, little is known about the molecular mechanisms underlying the maintenance and persistence of contextual long-term memory. Here, we examined the transcription of specific Bdnf exons in the hippocampus for long periods after contextual fear conditioning. We found changes in transcription lasting for at least 24 h after contextual fear conditioning, with some sex-specific effects. In addition, hypomethylation at a CpG site in CpG island 2 located at the end of Bdnf exon III sequence was detected at 0.5 h and maintained for up to 24 h after contextual fear conditioning. The identification of these long-lasting changes in transcription and DNA methylation at the Bdnf gene suggests that BDNF might have a role for storage of contextual long-term memory in the hippocampus.

Calcium/calmodulin kinase kinase beta has a male-specific role in memory formation.

The calcium/calmodulin (CaM) kinase cascade regulates gene transcription, which is required for long-term memory formation. Previous studies with Camkk2 null mutant mice have shown that in males calcium/calmodulin kinase kinase beta (CaMKKbeta) is required for spatial memory formation and for activation of the transcription factor cyclic AMP-responsive element binding protein (CREB) in the hippocampus by spatial training. Here we show that CaMKKbeta is not required for spatial memory formation in female mice as female Camkk2 null mutants were not impaired in spatial memory formation and they had the same level of hippocampal CREB phosphorylation after spatial training as female wild-type mice. Furthermore, we show that male but not female Camkk2 null mutants were impaired in long-term potentiation (LTP) at hippocampal CA1 synapses. Finally, a transcriptional analysis of male Camkk2 null mutants led to the identification of a gene, glycosyl phosphatidyl-inositol anchor attachment protein 1 (GAA1), whose hippocampal mRNA expression was up-regulated by spatial and contextual training in male but not in female wild-type mice. Taken together, we conclude that CaMKKbeta has a male-specific function in hippocampal memory formation and we have identified male-restricted transcription occurring during hippocampal memory formation.

Structure of the neuronal protein calexcitin suggests a mode of interaction in signalling pathways of learning and memory.

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Sexual dimorphisms in the effect of low-level p25 expression on synaptic plasticity and memory.

p25, a degradation product of p35, has been reported to accumulate in the forebrain of patients with Alzheimer's disease. p25 as well as p35 are activators of cyclin-dependent kinase 5 (Cdk5) although p25/Cdk5 and p35/Cdk5 complexes have distinct properties. Several mouse models with high levels of p25 expression exhibit signs of neurodegeneration. On the contrary, we have shown that low levels of p25 expression do not cause neurodegeneration and are even beneficial for particular types of learning and memory [Angelo et al., (2003) Eur J. Neurosci., 18, 423-431]. Here, we have studied the influence of low-level p25 expression in hippocampal synaptic plasticity and in learning and memory for each sex separately in two different genetic backgrounds (129B6F1 and C57BL/6). Surprisingly, we found that low-level p25 expression had different consequences in male and female mutants. In the two genetic backgrounds LTP induced by a strong stimulation of the Schaffer's collaterals (four trains, 1-s duration, 5-min interval) was severely impaired in male, but not in female, p25 mutants. Furthermore, in the two genetic backgrounds spatial learning in the Morris water maze was faster in female p25 mutants than in male transgenic mice. These results suggest that, in women, the production of p25 in Alzheimer's disease could be a compensation for some early learning and memory deficits.

Crystallization and preliminary X-ray diffraction analysis of calexcitin from Loligo pealei: a neuronal protein implicated in learning and memory.

The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 A suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 A resolution and yields data of high quality using synchrotron radiation.

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus.

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long-term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of alphaCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short-term potentiation (STP). Tetanus-induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of alphaCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mM tetraethyl-ammonium (TEA) or 5 min exposure to 41 mM potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two-fold increase in autonomous activity measured immediately after stimulation was short-lived, returning to baseline within 2-5 min after re-exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of alphaCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity of CaMKII is not required for the expression of LTP.

The contribution of autophosphorylated alpha-calcium-calmodulin kinase II to injury-induced persistent pain.

Increases in neuronal activity in response to tissue or nerve injury can lead to prolonged functional changes in the spinal cord resulting in an enhancement/sensitization of nociceptive processing. To assess the contribution of alpha-calcium-calmodulin kinase II (alpha-CaMKII) to injury-induced inflammation and pain, we evaluated nociceptive responses in mice that carry a point mutation in the alpha-CaMKII gene at position 286 (threonine to alanine). The mutated protein is unable to autophosphorylate and thus cannot function independently of calcium and calmodulin. Responses to acute noxious stimuli did not differ between alpha-CaMKII T286A mutant and wild type mice. However, the ongoing pain produced by formalin injury was significantly reduced in the mutant mice, as was formalin-evoked spinal Fos-immunoreactivity. In contrast, the decreased mechanical and thermal thresholds associated with nerve injury, Complete Freund's Adjuvant-induced inflammation or formalin-evoked tissue injury were manifest equally in wild-type and mutant mice. Double-labeling immunofluorescence studies revealed that in the mouse alpha-CaMKII is expressed in the superficial dorsal horn as well as in a population of small diameter primary afferent neurons. In summary, our results suggest that alpha-CaMKII, perhaps secondary to an N-methyl-D-aspartate-mediated calcium increase in postsynaptic dorsal horn nociresponsive neurons, is a critical contributor to the spontaneous/ongoing component of tissue-injury evoked persistent pain.

Handling and environmental enrichment do not rescue learning and memory impairments in alphaCamKII(T286A) mutant mice.

Environmental enrichment and postnatal handling have been shown to improve learning and memory in the Morris water maze, and to rescue impairments caused by genetic modification, age or genetic background. Mice with a targeted point mutation that prevents autophosphorylation at threonine-286 of the alpha-isoform of the Ca2+/calmodulin-dependent kinase II have impaired hippocampus-dependent and -independent strategy learning and memory in the water maze. We have investigated whether these impairments can be rescued with a combination of postnatal handling and environmental enrichment in a hybrid genetic background. Severe impairments were seen in acquisition and probe trials in both enriched and nonenriched mutants, indicating that enrichment did not rescue the learning and memory impairments. However, enrichment did rescue a specific performance deficit; enhanced floating behaviour, in the mutants. In summary, we have shown the lack of autophosphorylation of the alpha-isoform of the Ca2+/calmodulin-dependent kinase II prevents enrichment-induced rescues of strategy learning and memory impairments. Furthermore, we have established that there are enrichment mechanisms that are independent of this autophosphorylation.

Hippocampus-dependent learning and memory is impaired in mice lacking the Ras-guanine-nucleotide releasing factor 1 (Ras-GRF1).

Previous results have suggested that the Ras signaling pathway is involved in learning and memory. Ras is activated by nucleotide exchange factors, such as the calmodulin-activated guanine-nucleotide releasing factor 1 (Ras-GRF1). To test whether Ras-GRF1 is required for learning and memory, we inactivated the Ras-GRF1 gene in mice. These mutants performed normally in a rota-rod motor coordination task, and in two amygdala-dependent tasks (inhibitory avoidance and contextual conditioning). In contrast the mutants were impaired in three hippocampus-dependent learning tasks: contextual discrimination, the social transmission of food preferences, and the hidden-platform version of the Morris water maze. These studies indicate that Ras-GRF1 plays a role in hippocampal-dependent learning and memory.

Modulation of excitability as a learning and memory mechanism: a molecular genetic perspective.

Gene targeting has contributed substantially to the investigation of the neurobiological basis of mammalian learning and memory (L&M). These experiments start with an hypothesis as to a mechanism underlying L&M, then genes of interest are manipulated, and the impact on neuronal physiology and L&M is studied. Previous gene targeting studies have focussed mainly on the role of synaptic plasticity in L&M. Some of those reports provide evidence that processes other than, or additional to, long-term potentiation (LTP) are required for L&M. Accordingly, it is possible that altered neuronal excitability is an essential mechanism. The properties of ion channels determine neuronal excitability and so genetic alteration of ion channel properties is an appropriate method for testing whether the modulation of excitability affects L&M. K(v)beta 1.1-deficient mice were the first mutants used to study the role of altered excitability in mammalian L&M. K(v)beta 1.1 is a regulatory subunit with a restricted expression pattern in the brain, and it confers fast inactivation on otherwise noninactivating K(+) channel subunits. In hippocampal pyramidal neurones Kv beta 1.1-deficiency results in a reduced slow after-hyperpolarisation (sAHP), modulation of which is thought to contribute to L&M. The L&M phenotype of the mutants supports this sAHP hypothesis. It is expected that further gene targeting studies on excitability will lead to valuable insights into the processes of L&M.

The role of alpha-CaMKII autophosphorylation in neocortical experience-dependent plasticity.

Calcium/calmodulin kinase type II (CaMKII) is a major postsynaptic density protein. CaMKII is postulated to act as a 'molecular switch', which, when triggered by a transient rise in calcium influx, becomes active for prolonged periods because of its ability to autophosphorylate. We studied experience-dependent plasticity in the barrel cortex of mice carrying a point mutation of the alpha-CaMKII gene (T286A), which abolishes this enzyme's ability to autophosphorylate. Plasticity was prevented in adult and adolescent mice homozygous for the mutation, but was normal in heterozygotes and wild-type littermates. These results provide evidence that the molecular switch hypothesis is valid for neocortical experience-dependent plasticity.

Functional and molecular aspects of voltage-gated K+ channel beta subunits.

Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.

Molecular, cellular, and neuroanatomical substrates of place learning.

Learning and remembering the location of food resources, predators, escape routes, and immediate kin is perhaps the most essential form of higher cognitive processing in mammals. Two of the most frequently studied forms of place learning are spatial learning and contextual conditioning. Spatial learning refers to an animal's capacity to learn the location of a reward, such as the escape platform in a water maze, while contextual conditioning taps into an animal's ability to associate specific places with aversive stimuli, such as an electric shock. Recently, transgenic and gene targeting techniques have been introduced to the study of place learning. In contrast with the abundant literature on the neuroanatomical substrates of place learning in rats, very little has been done in mice. Thus, in the first part of this article, we will review our studies on the involvement of the hippocampus in both spatial learning and contextual conditioning. Having demonstrated the importance of the hippocampus to place learning, we will then focus attention on the molecular and cellular substrates of place learning. We will show that just as in rats, mouse hippocampal pyramidal cells can show place specific firing. Then, we will review our evidence that hippocampal-dependent place learning involves a number of interacting physiological mechanisms with distinct functions. We will show that in addition to long-term potentiation, the hippocampus uses a number of other mechanisms, such as short-term-plasticity and changes in spiking, to process, store, and recall information. Much of the focus of this article is on genetic studies of learning and memory (L&M). However, there is no single experiment that can unambiguously connect any cellular or molecular mechanism with L&M. Instead, several different types of studies are required to determine whether any one mechanism is involved in L&M, including (i) the development of biologically based learning models that explain the involvement of a given mechanism in L&M, (ii) lesion experiments (genetics and pharmacology), (iii) direct observations during learning, and (iv) experiments where learning is triggered by turning on the candidate mechanism. We will show how genetic techniques will be key to unraveling the molecular and cellular basis of place learning.

Abnormal hippocampal spatial representations in alphaCaMKIIT286A and CREBalphaDelta- mice.

Hippocampal "place cells" fire selectively when an animal is in a specific location. The fine-tuning and stability of place cell firing was compared in two types of mutant mice with different long-term potentiation (LTP) and place learning impairments. Place cells from both mutants showed decreased spatial selectivity. Place cell stability was also deficient in both mutants and, consistent with the severities in their LTP and spatial learning deficits, was more affected in mice with a point mutation [threonine (T) at position 286 mutated to alanine (A)] in the alpha calmodulin kinase II (alphaCaMKIIT286A) than in mice deficient for the alpha and Delta isoforms of adenosine 3'5'-monophosphate-responsive element binding proteins (CREBalphaDelta-). Thus, LTP appears to be important for the fine tuning and stabilization of place cells, and these place cell properties may be necessary for spatial learning.

Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.

The calcium-calmodulin-dependent kinase II (CaMKII) is required for hippocampal long-term potentiation (LTP) and spatial learning. In addition to its calcium-calmodulin (CaM)-dependent activity, CaMKII can undergo autophosphorylation, resulting in CaM-independent activity. A point mutation was introduced into the alphaCaMKII gene that blocked the autophosphorylation of threonine at position 286 (Thr286) of this kinase without affecting its CaM-dependent activity. The mutant mice had no N-methyl-D-aspartate receptor-dependent LTP in the hippocampal CA1 area and showed no spatial learning in the Morris water maze. Thus, the autophosphorylation of alphaCaMKII at Thr286 appears to be required for LTP and learning.

Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kvbeta1.1-deficient mice with impaired learning.

A-type K+ channels are known to regulate neuronal firing, but their role in repetitive firing and learning in mammals is not well characterized. To determine the contribution of the auxiliary K+ channel subunit Kvbeta1.1 to A-type K+ currents and to study the physiological role of A-type K+ channels in repetitive firing and learning, we deleted the Kvbeta1.1 gene in mice. The loss of Kvbeta1.1 resulted in a reduced K+ current inactivation in hippocampal CA1 pyramidal neurons. Furthermore, in the mutant neurons, frequency-dependent spike broadening and the slow afterhyperpolarization (sAHP) were reduced. This suggests that Kvbeta1.1-dependent A-type K+ channels contribute to frequency-dependent spike broadening and may regulate the sAHP by controlling Ca2+ influx during action potentials. The Kvbeta1.1-deficient mice showed normal synaptic plasticity but were impaired in the learning of a water maze test and in the social transmission of food preference task, indicating that the Kvbeta1.1 subunit contributes to certain types of learning and memory.

Gene targeting and the biology of learning and memory.

The general goal of genetic studies of learning and memory is to develop and test theories that explain the animal's behavior in neuroanatomical, neurophysiological, cellular, and molecular terms. In this review we describe the role that gene targeting and other transgenic techniques have had in the study of mammalian learning and memory. We focus especially on the hippocampus, a brain structure that is thought to be central to the processing and temporary storage of complex information. We also discuss the main issues that confront this young field, as well as our vision for its future.