No abstract

Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the specific LVGCC isoform involved. Both of the major LVGCC subtypes found in the brain (Ca v 1.2 and Ca v 1.3) are targets of the pharmacological manipulations used in earlier work. In this study, we used mice in which the gene for the pore-forming subunit (␣ 1D ) Ca v 1.3 was deleted (Ca v 1.3 knockout mice) to elucidate its contribution to consolidation and extinction of conditioned fear. We find that Ca v 1.3 knockout mice exhibit significant impairments in consolidation of contextual fear conditioning. However, once sufficiently overtrained, the Ca v 1.3 knockout mice exhibit rates of extinction that are identical to that observed in wild-type mice. We also find that Ca v 1.3 knockout mice perform as well as wild-type mice on the hidden platform version of the Morris water maze, suggesting that the consolidation deficit in conditioned fear observed in the Ca v 1.3 knockout mice is not likely the result of an inability to encode the context, but may reflect an inability to make the association between the context and the unconditioned stimulus.In Pavlovian fear conditioning, pairing a conditional stimulus (CS) with an aversive unconditional stimulus (US) results in a conditioned fear response. A fear response is said to be contextually conditioned when it is elicited by the context in which the US was delivered. In this case, the context serves as the CS (for recent review, see Fanselow and Poulos 2005). After contextual fear conditioning, extended exposure to the context in the absence of the US results in reduced probability and amplitude-or extinction-of the conditioned response. Both consolidation and extinction of conditioned fear have been demonstrated to be critically dependent on the amygdala (for review, see Maren 2003).Recently, many studies have investigated the molecular basis of fear conditioning and its extinction in rodents. Molecules involved in synaptic plasticity within the amygdala have been of particular interest. In light of evidence that a form of long-term potentiation (LTP) that depends on L-type voltage-gated calcium channels (LVGCCs) exists in the amygdala (Weisskopf et al. 1999), multiple groups have explored the role of LVGCCs in fear conditioning and its extinction. In rats, blockade of LVGCCs in the lateral amygdala impairs consolidation of auditory conditioned fear (Bauer et al. 2002). Systemic blockade of LVGCCs in mice, however, does not impair the acquisition, consolidation, or expression of conditioned fear, but rather its extinction (Cain et al. 2002;Suzuki et al. 2004). Demonstration that infusions into the basolateral amygdala of an LVGCC antagonist block, whereas infusions of an LVGCC agonist facilitate, extinction of conditioned fear ...

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. Observed neuropathologies associated with FXS include abnormal length, morphology, and density of dendritic spines, reported in individuals with FXS and in Fmr1 knockout (KO) mice, an animal model of FXS. To date, however, these neuropathologies have been studied in Fmr1 KO mice bred in a FVB background (a strain with genetic mutations that complicate interpretation of results) and findings have been inconsistent. Here, Golgi-Cox impregnation was used to investigate length, morphology, and density of dendritic spines on layer V pyramidal neurons in visual cortices of Fmr1 KO and wildtype (WT) mice bred in a C57BL/6 background. We report that spine abnormalities in these animals parallel abnormalities reported in humans with FXS, perhaps to a greater degree than KO mice bred in an FVB background. Specifically, Fmr1 KO mice bred in a C57BL/6 background exhibited significantly more longer dendritic spines and fewer shorter spines, as well as more spines with immature-appearing morphology and fewer with mature-appearing morphology than WT littermates. Spine length abnormalities were demonstrated to be largely independent of spine morphology abnormalities, as the length phenotype was observed in KOs even within a morphological category. Fmr1 KO mice also had a greater overall spine density than WTs. These findings provide powerful support for the essence of the dendritic spine abnormalities in the absence of FMRP, now found to be largely consistent with human data across two mouse backgrounds.

To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for remote memory consolidation, we generated conditional knockout mice in which the L-VGCC isoform Ca V 1.2 was postnatally deleted in the hippocampus and cortex. In the Morris water maze, both Ca V 1.2 conditional knockout mice (Ca V 1.2 cKO ) and control littermates displayed a marked decrease in escape latencies and performed equally well on probe trials administered during training. In distinct contrast to their performance during training, Ca V 1.2 cKO mice exhibited significant impairments in spatial memory when examined 30 d after training, suggesting that Ca V 1.2 plays a critical role in consolidation of remote spatial memories.In recent years, a wealth of experimental evidence has emerged suggesting that activity-dependent changes in neuronal gene expression are regulated in large part by calcium influx via L-VGCCs. For example, calcium influx through L-VGCCs activates calcium-dependent calmodulin kinase IV and Ras/mitogenactivated kinase, which, in turn, phosphorylate the transcription factor CREB at serine 133 (Wu et al. 2001). Once phosphorylated, CREB becomes part of an active transcriptional complex that binds to cAMP-response element DNA sequences to regulate transcription of a number of gene products. Importantly, calciummediated activation of CREB has been implicated as a key subcellular signaling cascade in a wide range of behavioral processes including long-term memory consolidation (West et al. 2002;Deisseroth et al. 2003). Given the prominent role that L-VGCCs play in the activation of these signaling cascades, we have begun to examine the impact of altering L-VGCC function on memory consolidation. Consistent with the idea that L-VGCCs are a key component of this process, we have recently demonstrated that deletion of the L-VGCC isoform Ca V 1.3 produces a deficit in consolidation of contextual fear conditioning (McKinney and Murphy 2006). Here we report here that forebrain deletion of the alternate L-VGCC isoform Ca V 1.2 specifically disrupts remote spatial memories while sparing recently acquired spatial memories.To obtain regionally restricted deletion of Ca V 1.2, mice were generated using a BAC clone containing Cacna1c (the mouse Ca V 1.2 gene) obtained by screening a murine strain SV/129J BAC library (Genome Systems Inc.) with a probe representing exon 2 of Cacna1c. DNA fragments containing portions of Cacna1c were subcloned and mapped. Three adjacent XbaI fragments (4, 0.6, and 1.9 kb) were subcloned into a modified pflox vector (Chui et al. 1997) in which the thymidine kinase (TK) cassette had been removed. The MC1-TK cassette (Thomas and Capecchi 1987) was introduced at the junction of the 3Ј homology unit and the plasmid vector. The final targeting vector (Fig. 1A) had 4 kb of 5Ј homology, a loxP site introduced into intron 1, a 0.6-kb region containing exon 2, a loxP-Neo-loxP cassette introduced into intron 2, 1.9 kb of 3Ј homology, and the MC1-TK cassette.The targeting vector was linearized and e...

Researchers and clinicians are increasingly recognizing that psychological and psychiatric disorders are often developmentally progressive, and that diagnosis often represents a point along that progression that is defined largely by our abilities to detect symptoms. As a result, strategies that guide our searches for the root causes and etiologies of these disorders are beginning to change. This review describes interactions between genetics and experience that influence the development of psychopathologies. Following a discussion of normal brain development that highlights how specific cellular processes may be targeted by genetic or environmental factors, we focus on four disorders whose origins range from genetic (fragile X syndrome) to environmental (fetal alcohol syndrome) or a mixture of both factors (depression and schizophrenia). C.H. Waddington's canalization model (slightly modified) is used as a tool to conceptualize the interactive influences of genetics and experience in the development of these psychopathologies. Although this model was originally proposed to describe the 'canalizing' role of genetics in promoting normative development, it serves here to help visualize, for example, the effects of adverse (stressful) experience in the kindling model of depression, and the multiple etiologies that may underlie the development of schizophrenia. Waddington's model is also useful in understanding the canalizing influence of experience-based therapeutic approaches, which also likely bring about 'organic' changes in the brain. Finally, in light of increased evidence for the role of experience in the development and treatment of psychopathologies, we suggest that future strategies for identifying the underlying causes of these disorders be based less on the mechanisms of action of effective pharmacological treatments, and more on increased knowledge of the brain's cellular mechanisms of plastic change.

Trains of action potentials in CA1 pyramidal neurons are followed by a prolonged calciumdependent post-burst afterhyperpolarization (AHP) that serves to limit further firing to a sustained depolarizing input. A reduction in the AHP accompanies acquisition of several types of learning and increases in the AHP are correlated with age-related cognitive impairment. The AHP develops primarily as the result of activation of outward calcium-activated potassium currents; however the precise source of calcium for activation of the AHP remains unclear. There is substantial experimental evidence suggesting that calcium influx via voltage-gated L-type calcium channels (L-VGCCs) contributes to the generation of the AHP. Two L-VGCC subtypes are predominately expressed in the hippocampus, Ca V 1.2 and Ca V 1.3, however it is not known which L-VGCC subtype is involved in generation of the AHP. This ambiguity is due in large part to the fact that at present there are no subunit-specific agonists or antagonists. Therefore, using mice in which the gene encoding Ca V 1.2 or Ca V 1.3 was deleted, we sought to determine the impact of alterations in levels of these two L-VCGG subtypes on neuronal excitability. No differences in any AHP measure were seen between neurons from Ca V 1.2 knockout mice and controls. However, the total area of the AHP was significantly smaller in neurons from Ca V 1.3 knockout mice as compared to neurons from wildtype controls. A significant reduction in the amplitude of the AHP was also seen at the 1 sec time point in neurons from Ca V 1.3 knockout mice as compared to those from controls. Reductions in both the area and 1 sec amplitude suggest the involvement of calcium influx via Ca V 1.3 in the slow AHP (sAHP). Thus, the results of our study demonstrate that deletion of Ca V 1.3, but not Ca V 1.2, significantly impacts the generation of the sAHP.

The Scn8a gene encodes the a-subunit of Na v 1.6, a neuronal voltage-gated sodium channel. Mice homozygous for mutations in the Scn8a gene exhibit motor impairments. Recently, we described a human family with a heterozygous protein truncation mutation in SCN8A. Rather than motor impairment, neuropsychological abnormalities were more common, suggesting a role for Scn8a in a more diverse range of behaviors. Here, we characterize mice heterozygous for a null mutation of Scn8a (Scn8a 1/2 mice) in a number of behavioral paradigms. We show that Scn8a 1/2 mice exhibit greater conditioned freezing in the Pavlovian fear conditioning paradigm but no apparent abnormalities in other learning and memory paradigms including the Morris water maze and conditioned taste avoidance paradigm. Furthermore, we find that Scn8a 1/2 mice exhibit more pronounced avoidance of well-lit, open environments as well as more stress-induced coping behavior. Together, these data suggest that Scn8a plays a critical role in emotional behavior in mice. Although the behavioral phenotype observed in the Scn8a 1/2 mice only partially models the abnormalities in the human family, we anticipate that the Scn8a 1/2 mice will serve as a valuable tool for understanding the biological basis of emotion and the human diseases in which abnormal emotional behavior is a primary component.

Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear using a molecular genetics approach. Previously, we used this approach to demonstrate that the Ca v 1.3 isoform mediates consolidation, but not extinction, of contextually conditioned fear. Here, we used mice in which the gene for the L-VGCC pore-forming subunit Ca v 1.2 was conditionally deleted in forebrain excitatory neurons (Ca v 1.2 cKO mice) to address the role of Ca v 1.2 in consolidation and extinction of conditioned fear. We demonstrate that Ca v 1.2 cKO mice consolidate and extinguish conditioned fear as well as control littermates. These data suggest that Ca v 1.2 is not critical for these processes and together with our previous data argue against a role for either of the brain-expressed L-VGCCs (Ca v 1.2 or Ca v 1.3) in extinction of conditioned fear. Additionally, we present data demonstrating that the L-VGCC antagonist nifedipine, which has been used in previous conditioned fear extinction studies, impairs locomotion, and induces an aversive state. We further demonstrate that this aversive state can enter into associations with conditioned stimuli that are present at the time that it is experienced, suggesting that previous studies using nifedipine were likely confounded by drug toxicity. Taken together, our genetic and pharmacological data argue against a role for Ca v 1.2 in consolidation of conditioned fear as well as a role for L-VGCCs in extinction of conditioned fear.Pavlovian fear conditioning is a popular paradigm for both the study of associative learning (Fanselow and Poulos 2005) and modeling anxiety disorders (Delgado et al. 2006;Hofmann 2007). In this paradigm, an association between a conditional stimulus (CS) and an aversive unconditional stimulus (US) is acquired through pairing the CS with the US. Learning of this association is identified by the emergence of new responses to the CS, termed conditioned fear responses. The process by which this learning is transformed into a stable long-term memory with the