The effect of autonomous alpha-CaMKII expression on sensory responses and experience-dependent plasticity in mouse barrel cortex.

The calcium/calmodulin kinase II (CaMKII) autophosphorylation site is thought to be important for plasticity, learning and memory. If autophosphorylation is prevented by a point mutation (T286A) LTP is blocked in the hippocampus and cortex. Conversely, if the point mutation mimics autophosphorylation (T286D) a range of frequencies that normally produce LTP in wild types cause LTD instead. In order to test whether the alphaCaMKII-T286D mutation increases levels of depression in vivo, we examined the effect of the alphaCaMKII-T286D transgene on plasticity induced in the barrel cortex by whisker deprivation. Surprisingly, the mutation did not affect depression or potentiation. However, in animals reared with the transgene turned on from birth, the surround receptive field responses were greater than normal. This effect may be due to the potentiating action of autophosphorylated CaMKII during early development.

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse.

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and beta-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the alpha-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, beta-galactosidase and immunochemical stainings revealed differences in GFP and beta-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense beta-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

Regional and strain-specific gene expression mapping in the adult mouse brain.

To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions.

Olfactory based spatial learning in neonatal mice and its dependence on CaMKII.

Spatial learning and memory involves the ability to encode geometric relationships between perceived cues and depends critically on the hippocampus. Visually guided spatial learning has been demonstrated in adult animals. As infant animals rely heavily on olfaction, olfactory based spatial learning was assessed in infant mice. When 12-day-old pups were displaced from their nest, they learned within a few training trials to use the spatial pattern of odor cues to move back to the nest. However, mouse pups that over-expressed Ca2+/calmodulin-dependent protein kinase (CaMKII) in hippocampal neurons were impaired in olfactory based spatial learning.

Genetic approaches to memory storage.

The ability to remember is perhaps the most significant and distinctive feature of our mental life. We are who we are largely because of what we have learned and what we remember. In turn, impairments in learning and memory can lead to disorders that range from the moderately inconvenient benign senescent forgetfulness associated with normal aging to the devastating memory losses associated with Alzheimer disease. Of the various, higher-cognitive abilities that human beings possess, such as reasoning and language, memory is the only one that can be studied effectively in simple experimental organisms that are accessible to genetic manipulation, such as snails, flies and mice. In these organisms, the effectiveness of genetic approaches in the study of memory has improved significantly in the past five years. Below we review these advances.

Cellular and molecular mechanisms of memory: the LTP connection.

Studies of the cellular and molecular mechanisms of memory formation have focused on the role of long-lasting forms of synaptic plasticity such as long-term potentiation (LTP). A combination of genetic, electrophysiological and behavioral techniques have been used to examine the possibility that LTP is a cellular mechanism of memory storage in the mammalian brain. Although a definitive answer remains elusive, it is clear that in many cases manipulations that alter LTP alter memory, and training regimens that produce memory can produce LTP-like potentiation of synaptic transmission.

Inducible and reversible gene expression with the rtTA system for the study of memory.

To obtain rapidly inducible and reversible expression of transgenes in the forebrain of the mouse, we have combined the reverse tetracycline-controlled transactivator (rtTA) system with the CaMKIIalpha promoter. We show that doxycycline induces maximal gene expression in neurons of the forebrain within 6 days and that this expression can be reversed by removal of doxycycline. Using calcineurin as a test transgene, we show that doxycycline-induced expression impairs both an intermediate form of LTP (I-LTP) in the hippocampus and the storage of spatial memory. The reversibility of the rtTA system in turn allowed us to examine the effects of the transgene on memory retrieval after normal storage had occurred. This examination suggests that retrieval requires some of the same molecular components required for storage.

Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory.

To investigate the roles phosphatases play in hippocampal-dependent memory, we studied transgenic mice overexpressing a truncated form of calcineurin. These mice have normal short-term memory but defective long-term memory evident on both a spatial task and on a visual recognition task, providing genetic evidence for the role of the rodent hippocampus in spatial and nonspatial memory. The defect in long-term memory could be fully rescued by increasing the number of training trials, suggesting that the mice have the capacity for long-term memory. We next analyzed mice overexpressing calcineurin in a regulated manner and found the memory defect is reversible and not due to a developmental abnormality. Our behavioral results suggest that calcineurin has a role in the transition from short- to long-term memory, which correlates with a novel intermediate phase of LTP.

Memory and behavior: a second generation of genetically modified mice.

The use of standard genetic techniques, such as gene targeting and transgenesis, to study cognitive function in adult animals suffers from the limitations that the gene under study is often altered in many brain regions, and that this alteration is present during the entire developmental history of the animal. Furthermore, to relate cognitive defects to neuronal mechanisms of memory, studies have relied on examining long-term potentiation - an artificially induced form of synaptic plasticity. Recent technical advances allow the expression of a genetic alteration in mice to be restricted both anatomically and temporally, making possible a more precise examination of the role of various forms of synaptic plasticity, such as long-term potentiation and long-term depression, in memory formation. Recordings from so-called 'place cells' -hippocampal cells that encode spatial location -in freely moving, genetically modified mice have further advanced our understanding of how the actual cellular representation of space is influenced by genetic alterations that affect long-term potentiation.

Rescuing impairment of long-term potentiation in fyn-deficient mice by introducing Fyn transgene.

To examine the physiological role of the Fyn tyrosine kinase in neurons, we generated transgenic mice that expressed a fyn cDNA under the control of the calcium/calmodulin-dependent protein kinase IIalpha promoter. With this promoter, we detected only low expression of Fyn in the neonatal brain. In contrast, there was strong expression of the fyn-transgene in neurons of the adult forebrain. To determine whether the impairment of long-term potentiation (LTP) observed in adult fyn-deficient mice was caused directly by the lack of Fyn in adult hippocampal neurons or indirectly by an impairment in neuronal development, we generated fyn-rescue mice by introducing the wild-type fyn-transgene into mice carrying a targeted deletion in the endogenous fyn gene. In fyn-rescue mice, Schaffer collateral LTP was restored, even though the morphological abnormalities characteristic of fyn-deficient mice were still present. These results suggest that Fyn contributes, at least in part, to the molecular mechanisms of LTP induction.

Control of memory formation through regulated expression of a CaMKII transgene.

One of the major limitations in the use of genetically modified mice for studying cognitive functions is the lack of regional and temporal control of gene function. To overcome these limitations, a forebrain-specific promoter was combined with the tetracycline transactivator system to achieve both regional and temporal control of transgene expression. Expression of an activated calcium-independent form of calcium-calmodulin-dependent kinase II (CaMKII) resulted in a loss of hippocampal long-term potentiation in response to 10-hertz stimulation and a deficit in spatial memory, a form of explicit memory. Suppression of transgene expression reversed both the physiological and the memory deficit. When the transgene was expressed at high levels in the lateral amygdala and the striatum but not other forebrain structures, there was a deficit in fear conditioning, an implicit memory task, that also was reversible. Thus, the CaMKII signaling pathway is critical for both explicit and implicit memory storage, in a manner that is independent of its potential role in development.

Subregion- and cell type-restricted gene knockout in mouse brain.

Using the phage P1-derived Cre/loxP recombination system, we have developed a method to create mice in which the deletion (knockout) of virtually any gene of interest is restricted to a subregion or a specific cell type in the brain such as the pyramidal cells of the hippocampal CA1 region. The Cre/loxP recombination-based gene deletion appears to require a certain level of Cre protein expression. The brain subregional restricted gene knockout should allow a more precise analysis of the impact of a gene mutation on animal behaviors.

Mice expressing activated CaMKII lack low frequency LTP and do not form stable place cells in the CA1 region of the hippocampus.

To relate different forms of synaptic plasticity to the formation and maintenance of place cells in the hippocampus, we have recorded place cells in freely behaving, transgenic mice that express a mutated Ca2+-independent form of CaM Kinase II. These mice have normal long-term potentiation (LTP) at 100 Hz, but they lack LTP in response to stimulation at 5-10 Hz and are impaired on spatial memory tasks. In these transgenic mice, the place cells in the CA1 region have three important differences from those of wild types: they are less common, less precise, and less stable. These findings suggest that LTP in the 5-10 Hz range may be important for the maintenance of place-field stability and that this stability may be essential for the storage of spatial memory.

The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation of mRNA in dendrites.

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The alpha subunit of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII alpha) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKII alpha provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKII alpha promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3'-untranslated region of the CaMKII alpha gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKII alpha gene, only the lacZ transcripts bearing the 3'-untranslated region are localized to dendrites. The beta-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP.

To investigate the function of the autophosphorylated form of CaMKII in synaptic plasticity, we generated transgenic mice that express a kinase that is Ca2+ independent as a result of a point mutation of Thr-286 to aspartate, which mimics autophosphorylation. Mice expressing the mutant form of the kinase show an increased level of Ca(2+)-independent CaMKII activity similar to that seen following LTP. The mice nevertheless exhibit normal LTP in response to stimulation at 100 Hz. However, at lower frequencies, in the range of 1-10 Hz, there is a systematic shift in the size and direction of the resulting synaptic change in the transgenic animals that favors LTD. The regulation of this frequency-response function by Ca(2+)-independent CaMKII activity seems to account for two previously unexplained synaptic phenomena, the relative loss of LTD in adult animals compared with juveniles and the enhanced capability for depression of facilitated synapses.

Impairment of spatial but not contextual memory in CaMKII mutant mice with a selective loss of hippocampal LTP in the range of the theta frequency.

We assessed hippocampal-dependent memory in mice with a Ca(2+)-independent form of CaMKII generated by the introduction of an aspartate at amino acid 286. The CaMKII-Asp-286 mice show normal LTP at high frequency stimulation, but in the 5-10 Hz range, they show a shift in the frequency-response curve favoring LTD. This range of frequencies is similar to the theta rhythm, which is associated with exploration in rodents. Using the Barnes maze to assess spatial memory, we found the transgenic mice could not learn to navigate to a specific location using spatial cues. In contrast, one line of transgenic mice performed normally in contextual fear conditioning, a task that is also hippocampal dependent. This dissociation between spatial and contextual memory suggests that even though both require the hippocampus, they may be mediated by different synaptic mechanisms.

Transgenic approaches to cognition.

Reverse genetic techniques, including gene 'knockouts' and transgenesis, allow defined mutations to be introduced into the mouse genome. The application of these techniques to neurobiology is beginning to provide a bridge between genes and cognition. Specifically, genetically altered mice make it possible to explore molecular mechanisms underlying implicit and explicit forms of learning, short-term and long-term memory, and emotional behaviors. The analysis of these mutant mice has begun to link specific behavioral deficits to defined changes in synaptic physiology.

Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia.

A form of learning in the marine mollusk Aplysia, long-term sensitization of the gill- and siphon-withdrawal reflex, results in the formation of new synaptic connections between the presynaptic siphon sensory neurons and their target cells. These structural changes can be mimicked, when the cells are maintained in culture, by application of serotonin, an endogenous facilitating neurotransmitter in Aplysia. A group of cell surface proteins, designated Aplysia cell adhesion molecules (apCAM's) was down-regulated in the sensory neurons in response to serotonin. The deduced amino acid sequence obtained from complementary DNA clones indicated that the apCAM's are a family of proteins that seem to arise from a single gene. The apCAM's are members of the immunoglobulin class of cell adhesion molecules and resemble two neural cell adhesion molecules, NCAM and fasciclin II. In addition to regulating newly synthesized apCAM, serotonin also altered the amount of preexisting apCAM on the cell surface of the presynaptic sensory neurons. By contrast, the apCAM on the surface of the postsynaptic motor neuron was not modulated by serotonin. This rapid, transmitter-mediated down-regulation of a cell adhesion molecule in the sensory neurons may be one of the early molecular changes in long-term synaptic facilitation.