L-type calcium currents conducted by Ca V 1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of Ca V 1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for ␤-adrenergic regulation in the fight-or-flight response. Mice expressing Ca V 1.2 channels with the distal C terminus deleted (DCT ) current in cardiomyocytes, where Ca 2ϩ enters through the channel and initiates excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release (1). Normal expression of Ca V 1.2 channels is required for cardiac contractile function and for survival beyond embryonic day 14 (2). Lack of the Ca V 1.2 channel also abolishes the development of myogenic tone and disrupts hormonal regulation of blood pressure (3). In contrast, deletion of Ca V 1.3, which also conducts L-type Ca 2ϩ currents, causes sinoatrial nodal dysfunction and cardiac arrhythmias but does not impair contractility or cause premature death (4). Overall, these gene deletion studies illustrate that L-type Ca 2ϩ currents are essential for normal cardiovascular function and for normal development.Ca V 1 channels are multisubunit complexes composed of a pore-forming ␣1 subunit and auxiliary ␤, ␣2␦, and in some cases ␥ subunits (5-7). They are a primary target for regulation by numerous hormones, protein kinases, and phosphoprotein phosphatases (5-7). In the "fight-or-flight" response, increased force of contraction is achieved largely through regulation of Ca V 1.2 channels in the heart by the sympathetic nervous system through activation of ␤-adrenergic receptors, adenylyl cyclase, and cyclic AMP-dependent protein kinase (PKA) and resulting phosphorylation of Ca V 1.2 channels (1,5,6,8). ␤-Adrenergic regulation of Ca V 1.2 channels requires A-kinase anchoring protein 15 (AKAP15), 2 which anchors the kinase to the distal C terminus of Ca V 1.2 via a modified leucine zipper (LZ) motif (9 -11).The C terminus of Ca V 1 channels undergoes proteolytic processing in vivo in skeletal and cardiac muscle (12-15). In cardiac muscle, the ␣1 subunit of Ca V 1.2 channels is present in two size forms of ϳ240 and 210 kDa, which differ by truncation of the distal C terminus (DCT) (15). This truncation leads to enhanced activity of Ca V 1.2 channels expressed in Xenopus oocytes and mammalian cell lines (16,17). Single channel conductance, modulation by ␤ and ␣2␦ subunits, and sensitivity to Ca 2ϩ channel agonists such as Bay K8644 remain unchanged (16, 17). The proteolytically cleaved DCT binds to the truncated channel and acts as potent autoinhibitor (18). Mutations of key charged residues at the interface between distal and proximal C-terminal domains of Ca V 1.2 relieves autoinhibition (18). Moreover, recent studies indicate that regulation of Ca V 1.2 channels can be reconstituted in transfect...

Regulation of Ca V 1.2 channels in cardiac myocytes by the ␤-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ␤-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for Ca V 1.2 and Ca V 1.3 channels in neurons. AKAP15 co-immunoprecipitates with Ca V 1.2 and Ca V 1.3 channels. AKAP15 has overlapping localization with Ca V 1.2 and Ca V 1.3 channels in cell bodies and proximal dendrites and is closely co-localized with Ca V 1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with Ca V 1.2 and Ca V 1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of Ca V 1.2 channels (Ca V 1.2⌬DCT) reveal that AKAP15 interacts with neuronal Ca V 1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of Ca V 1.2 channels via that interaction. Ca V 1.2⌬DCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from Ca V 1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of Ca V 1.2 and Ca V 1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal Ca V 1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.Voltage-gated calcium channels of the Ca V 1 subfamily conduct L-type calcium currents that transduce cell-surface depolarization into calcium transients and initiate excitation-contraction coupling, excitation-secretion coupling, protein phosphorylation, and gene regulation (1-5). Calcium influx via postsynaptic Ca V 1 channels supports sustained phosphorylation of cAMP response element-binding protein (CREB) 3 and CREB-dependent gene expression in hippocampal neurons (6 -13).Functional Ca V 1 channels are multimeric complexes composed of pore-forming ␣ 1 and associated ␣ 2 ␦, ␤, and in some cases, ␥ subunits (14 -19). These channels have an extended C terminus containing many protein interaction sites for regulation (5). In brain, Ca V 1 channels are composed of 70% Ca V 1.2 and 22% Ca V 1.3 with minor contributions from other Ca V 1 channels, as indicated by immunoprecipitation with specific antibodies (20). Ca V 1.2 and Ca V 1.3 channels are primarily localized in the soma and proximal dendrites (20, 21).The ␤-adrenergic pathway activates cAMP-dependent protein kinase (PKA) and increases the activity of Ca V 1 channels in skeletal and cardiac myocytes and neurons (1-5, 22, 23). PKAmediated regulatio...

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-d-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN.

Object The use of intraoperative neurophysiological monitoring (IONM) in surgical decompression surgery for myelopathy may assist the surgeon in taking corrective measures to reduce or prevent permanent neurological deficits. We evaluated the efficacy of IONM in cervical and cervicothoracic spondylotic myelopathy (CSM) cases. Methods The authors retrospectively reviewed 140 cases involving patients who underwent surgery for CSM utilizing IONM during 2011 at the University of California, San Francisco. Data on preoperative clinical variables, intraoperative changes in transcranial motor evoked potentials (MEPs), and postoperative new neurological deficits were collected. Associations between categorical variables were analyzed with the Fisher exact test. Results Of the 140 patients, 16 (11%) had significant intraoperative decreases in MEPs. In 8 of these cases, the MEP signal did not return to baseline values by the end of the operation. There were 8 (6%) postoperative deficits, of which 6 were C-5 palsies and 2 were paraparesis. Six of the patients with postoperative deficits had demonstrated persistent MEP signal change on IONM. There was a significant association between persistent MEP changes and postoperative deficits (p < 0.001). The sensitivity of intraoperative MEP monitoring was 75%, the specificity 98%, the positive predictive value 75%, and the negative predictive value 98%. Due to higher rates of false negatives, the sensitivity decreased to 60% in the subgroup of patients with vascular disease comorbidity. The sensitivity increased to 100% in elderly patients and in patients with preoperative motor deficits. The sensitivity and positive predictive value of deltoid and biceps MEP changes in predicting C-5 palsy were 67% and 67%, respectively. Conclusions The authors found a correlation between decreased intraoperative MEPs and postoperative new neurological deficits in patients with CSM. Sensitivity varies based on patient comorbidities, age, and preoperative neurological function. Monitoring of MEPs is a useful adjunct for CSM cases, and the authors have developed a checklist to standardize their responses to intraoperative MEP changes.

Patients infected with the human immunodeficiency virus (HIV), and other mammals infected with related lentiviruses, exhibit fatigue, altered sleep patterns, and abnormal circadian rhythms. A circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) temporally regulates these functions in mammals. We found that a secretary HIV transcription factor, transactivator of transcription (Tat), resets the murine circadian clock, in vitro and in vivo, at clinically relevant concentrations (EC(50) = 0.31 nM). This effect of Tat occurs only during the subjective night, when N-methyl-D-aspartate (NMDA) receptor [D-2-amino-5-phosphonovaleric acid (0.1 mM)] and nitric oxide synthase (N(G)-nitro-L-arginine methyl ester, 0.1 mM) inhibitors block Tat-induced phase shifts. Whole cell recordings of SCN neurons within the brain slice revealed that Tat did not activate NMDA receptors directly but potentiated NMDA receptor currents through the enhancement of glutamate release. Consistent with this presynaptic mechanism, inhibitors of neurotransmission block Tat-induced phase shifts, such as tetrodotoxin (1 microM), tetanus toxin (1 microM), P/Q/N type-calcium channel blockers (1 microM omega-agatoxin IVA and 1 microM omega-conotoxin GIVA) and bafilomycin A(1) (1 microM). Thus the effect of Tat on the SCN may underlie lentiviral circadian rhythm dysfunction by operating as a disease-dependent modulator of light entrainment through the enhancement of excitatory neurotransmission.

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Background Mild hypothermia is neuroprotective following cerebral ischemia but surgery involving profound hypothermia (PH, temperature <18 °C) is associated with neurological complications. Rewarming (RW) from PH injures hippocampal neurons by glutamate excitotoxicity, N-methyl-D-aspartate receptors and intracellular calcium. Because neurons are protected from hypoxia-ischemia by anesthetics that inhibit N-methyl-D-aspartic acid receptors, we tested whether anesthetics protect neurons from damage caused by PH/RW. Methods Organotypic cultures of rat hippocampus were used to model PH/RW injury, with hypothermia at 4°C followed by RW to 37°C and assessment of cell death 1 or 24 h later. Cell death and intracellular Ca2+ were assessed with fluorescent dye imaging and histology. Anesthetics were present in the culture media during PH and RW or only RW. Results Injury to hippocampal CA1, CA3, and dentate neurons following PH and RW involved cell swelling, cell rupture, and adenosine triphosphate loss; this injury was similar for 4 through 10 h of PH. Isoflurane (1 and 2%), sevoflurane (3%) and xenon (60%) reduced cell loss but propofol (3 μM) and pentobarbital (100 μM) did not. Isoflurane protection involved reduction in N-methyl-D-aspartate receptor-mediated Ca2+ influx during RW but did not involve γ-amino butyric acid receptors or KATP channels. However, cell death increased over the next day. Conclusions Anesthetic protection of neurons rewarmed from 4°C involves suppression of N-methyl-D-aspartate receptor-mediated Ca2+ overload in neurons undergoing adenosine triphosphate loss and excitotoxicity. Unlike during hypoxia/ischemia, anesthetics acting predominately on γ-amino butyric acid receptors do not protect against PH/RW. The durability of anesthetic protection against cold injury may be limited.

Clark JP, Kofuji P. Stoichiometry of N-methyl-D-aspartate receptors within the suprachiasmatic nucleus. J Neurophysiol 103: 3448 -3464, 2010. First published April 21, 2010 doi:10.1152/jn.01069.2009. The circadian pacemaker within the suprachiasmatic nucleus (SCN) confers daily rhythms to bodily functions. In nature, the circadian clock will adopt a 24-h period by synchronizing to the solar light/dark cycle. This light entrainment process is mediated, in part, at glutamatergic synapses formed between retinal ganglion afferents and SCN neurons. N-methyl-D-aspartate receptors (NMDARs) located on SCN neurons gate light-induced phase resetting. Despite their importance in circadian physiology, little is known about their functional stoichiometry. We investigated the NR2-subunit composition with whole cell recordings of SCN neurons within the murine hypothalamic brain slice using a combination of subtype-selective NMDAR antagonists and voltageclamp protocols. We found that extracellular magnesium ([Mg] o ) strongly blocks SCN NMDARs exhibiting affinities and voltage sensitivities associated with NR2A and NR2B subunits. These NMDAR currents were inhibited strongly by NR2B-selective antagonists, Ro 25-6981 (3.5 M, 55.0 Ϯ 9.0% block; mean Ϯ SE) and ifenprodil (10 M, 55.8 Ϯ 3.0% block). The current remaining showed decreased [Mg] o affinities reminiscent of NR2C and NR2D subunits but was highly sensitive to [Zn] o , a potent NR2A blocker, showing a ϳ44.2 Ϯ 1.1% maximal inhibition at saturating concentrations with an IC 50 of 7.8 Ϯ 1.1 nM. Considering the selectivity, efficacy, and potency of the drugs used in combination with [Mg] oblock characteristics of the NMDAR, our data show that both diheteromeric NR2B NMDARs and triheteromeric NR2A NMDARs (paired with an NR2C or NR2D subunits) account for the vast majority of the NMDAR current within the SCN. I N T R O D U C T I O NThe suprachiasmatic nucleus (SCN) of the hypothalamus generates a daily rhythm that is reflected in behavioral and bodily functions. The properties of this rhythm are determined in large part by the molecular machinery of neurons that form an oscillating cellular network within the SCN (Herzog 2007). In mammals, this circadian rhythm synchronizes to the environmental light-dark cycles through a direct pathway from the retina to the SCN, the retinohypothalamic tract (Johnson et al. 1988). During this light entrainment process, light depolarizes retinal ganglion cells that express the photopigment, melanopsin, which, in turn, releases glutamate from their terminals at synapses formed with neurons of the SCN Hattar et al. 2002).L-glutamate is the primary neurotransmitter of the retinohypothalamic tract and is required for the entrainment of the circadian pacemaker to the solar light-dark cycle (Hannibal 2002). Although, ionotropic glutamate receptors of the SCN, N-methyl-D-aspartate receptors (NMDARs) and amino-methyl proprionic acid/kainate receptors, participate in this process (Colwell and Menaker 1992;Ebling 1996), NMDAR activation is necessary a...