Jeffrey Vernon

[alpha]CaMKII autophosphorylation contributes to rapid learning but is not necessary for memory

Autophosphorylation of α calcium–calmodulin-dependent kinase II (αCaMKII) has been proposed to be the key event in memory storage. We tested this hypothesis with autophosphorylation-deficient mutant mice in hippocampus- and amygdala-dependent learning and memory tasks and found that the autophosphorylation of αCaMKII was required for rapid learning but was not essential for memory. We conclude that αCaMKII autophosphorylation contributes to single-trial learning but is dispensable for memory.

Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMP-hydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.

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.

Probing the presence of the ligand-binding haem in cellular nitric oxide receptors

Nitric oxide (NO) acts on receptors coupled to guanylyl cyclase (GC), leading to cGMP accumulation. The NO binding site is a haem group, oxidation or loss of which diminishes NO‐stimulated activity. Agonists reportedly engaging both these NO‐insensitive forms have emerged. Here we characterize the effect of a prototype compound (BAY 58‐2667) and use it to assess the haem status of cellular GC.

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells.

Background: The biological actions of NO depend critically on its concentration, which is very difficult to measure. Results: Recorded using the most sensitive detectors yet described, NO levels synthesized by activated brain neurons generally were in the picomolar range but varied regionally. Conclusion: NO operates physiologically at the subnanomolar concentrations that selectively target its guanylyl cyclase-linked receptors. Significance: Physiological NO levels are far lower than commonly supposed. Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36–350 nm/s, depending on area. Our measurements are 4–5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.

The Fabry disease-associated lipid Lyso-Gb3 enhances voltage-gated calcium currents in sensory neurons and causes pain

Highlights • Gb3 and Lyso-Gb3, plasma lipids accumulating in Fabry disease, cause mechanical allodynia in mice.• Lyso-Gb3 elevates intracellular calcium level in sensory neurons.• Lyso-Gb3 enhances voltage-dependent calcium currents in small-diameter DRG neurons.• Direct effects of lyso-Gb3 on sensory neurons may contribute to the pain of Fabry disease.

Mechanisms of Activity-dependent Plasticity in Cellular Nitric Oxide-cGMP Signaling

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nm peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10–20 nm). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nm being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pm provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.

Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations.

TRPs and Pain

Pain usually occurs as a result of tissue damage and has a role in healing and protection. However, in certain conditions it has no functional purpose and can become chronic and debilitating. A demand for more effective treatments to deal with this highly prevalent problem requires a better understanding of the underlying mechanisms. TRP channels are associated with numerous sensory functions across a wide range of species. Investigation into the expression patterns, electrophysiological properties and the effects of channel deletion in transgenic animal models have produced a great deal of evidence linking these channels to transduction of noxious stimuli as well as signalling within the pain system.

Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia

The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.