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...

Neurodegenerative diseases of the central nervous system (CNS) are characterized by progressive neuronal death and neurological dysfunction, leading to increased disability and a loss of cognitive or motor functions. Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis have neurodegeneration as a primary feature. However, in other CNS diseases such as multiple sclerosis, stroke, traumatic brain injury, and spinal cord injury, neurodegeneration follows another insult, such as demyelination or ischaemia. Although there are different primary causes to these diseases, they all share a hallmark of neuroinflammation. Neuroinflammation can occur through the activation of resident immune cells such as microglia, cells of the innate and adaptive peripheral immune system, meningeal inflammation and autoantibodies directed toward components of the CNS. Despite chronic inflammation being pathogenic in these diseases, local inflammation after insult can also promote endogenous regenerative processes in the CNS, which are key to slowing disease progression. The normal aging process in the healthy brain is associated with a decline in physiological function, a steady increase in levels of neuroinflammation, brain shrinkage, and memory deficits. Likewise, aging is also a key contributor to the progression and exacerbation of neurodegenerative diseases. As there are associated co-morbidities within an aging population, pinpointing the precise relationship between aging and neurodegenerative disease progression can be a challenge. The CNS has historically been considered an isolated, “immune privileged” site, however, there is mounting evidence that adaptive immune cells are present in the CNS of both healthy individuals and diseased patients. Adaptive immune cells have also been implicated in both the degeneration and regeneration of the CNS. In this review, we will discuss the key role of the adaptive immune system in CNS degeneration and regeneration, with a focus on how aging influences this crosstalk.

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.

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

Objectives: Very low birth weight (VLBW) infants are vulnerable to nosocomial infections and subsequent morbidity; including infections caused by Staphylococcus aureus: 85% of nosocomial S. aureus infections are caused by capsular polysaccharide (CPS) types 5 and 8. Altastapht is a polyclonal investigational human immunoglobulin G (IgG) with high levels of opsonizing S. aureus CPS types 5 and 8 IgG.Methods: A Phase 2 clinical trial to assess the safety and kinetics of Altastaph in VLBW infants. Neonates in this multicenter study were randomized to receive two identical 20 ml/kg i.v. infusions of either 0.45% NaCl placebo or 1000 mg Altastaph/kg. Each infant was followed for 28 days after the second infusion or until discharge. Serum S. aureus CPS types 5 and 8 IgG levels were measured preinfusion and at various times after each infusion.Results: Of 206 neonates, 158 received both infusions. Adverse events were similar in the two treatment groups. Six subjects (3% in each group) discontinued owing to an adverse event. Geometric mean anti-type 5 IgG levels were 402 and 642 mcg/ml 1 day following infusion of the first (day 0) and Second (day 14) doses, respectively, in neonates p1000 g and slightly higher in neonates 1001 to 1500 g. Trough levels before second infusion were 188 mcg/ml. Type 8 IgG levels were similar. Geometric mean IgG levels among placebo recipients were consistently <2 and <5 mcg/ml for types 5 and 8 in both weight groups. Three episodes of S. aureus bacteremia occurred in each arm.Conclusions: Infusion of Altastaph in VLBW neonates resulted in high levels of specific S. aureus types 5 and 8 CPS IgG. The administration of this anti-staphylococcal hyperimmune globulin was well tolerated in this population.