Discovery and development of orexin receptor antagonists as therapeutics for insomnia.

Insomnia persistently affects the quality and quantity of sleep. Currently approved treatments for insomnia primarily target γ-aminobutyric acid-A (GABA-A) receptor signalling and include benzodiazepines and GABA-A receptor modulators. These drugs are used to address this sleep disorder, but have the potential for side effects such as tolerance and dependence, making them less attractive as maintenance therapy. Forward and reverse genetic approaches in animals have implicated orexin signalling (also referred to as hypocretin signalling) in the control of vigilance and sleep/wake states. Screening for orexin receptor antagonists using in vitro and in vivo methods in animals has identified compounds that block one or other of the orexin receptors (single or dual orexin receptor antagonists [SORAs and DORAs], respectively) in animals and humans. SORAs have primarily been used as probes to further elucidate the roles of the individual orexin receptors, while a number of DORAs have progressed to clinical development as pharmaceutical candidates for insomnia. The DORA almorexant demonstrated significant improvements in a number of clinically relevant sleep parameters in animal models and in patients with insomnia but its development was halted. SB-649868 and suvorexant have demonstrated efficacy and tolerability in Phase II and III trials respectively. Furthermore, suvorexant is currently under review by the Food and Drug Administration for the treatment of insomnia. Based on the publication of recent non-clinical and clinical data, orexin receptor antagonists potentially represent a targeted, effective and well-tolerated new class of medications for insomnia.

Painful nerve injury decreases sarco-endoplasmic reticulum Ca²âº-ATPase activity in axotomized sensory neurons.

The sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a critical pathway by which sensory neurons sequester cytosolic Ca(2+) and thereby maintain intracellular Ca(2+) homeostasis. We have previously demonstrated decreased intraluminal endoplasmic reticulum Ca(2+) concentration in traumatized sensory neurons. Here we examine SERCA function in dissociated sensory neurons using Fura-2 fluorometry. Blocking SERCA with thapsigargin (1 µM) increased resting [Ca(2+)](c) and prolonged recovery (τ) from transients induced by neuronal activation (elevated bath K(+)), demonstrating SERCA contributes to control of resting [Ca(2+)](c) and recovery from transient [Ca(2+)](c) elevation. To evaluate SERCA in isolation, plasma membrane Ca(2+) ATPase was blocked with pH 8.8 bath solution and mitochondrial buffering was avoided by keeping transients small (≤ 400 nM). Neurons axotomized by spinal nerve ligation (SNL) showed a slowed rate of transient recovery compared to control neurons, representing diminished SERCA function, whereas neighboring non-axotomized neurons from SNL animals were unaffected. Injury did not affect SERCA function in large neurons. Repeated depolarization prolonged transient recovery, showing that neuronal activation inhibits SERCA function. These findings suggest that injury-induced loss of SERCA function in small sensory neurons may contribute to the generation of pain following peripheral nerve injury.

Minimal alterations in T-type calcium channel gating markedly modify physiological firing dynamics.

T-type calcium channel isoforms expressed in heterologous systems demonstrate marked differences in the biophysical properties of the resulting calcium currents. Such heterogeneity in gating behaviour not only reflects structural differences but is also observed following the regulation of channel activity by a number of ligands. However, the physiological impact of these differences in gating parameters of the T channels has never been evaluated in situ where the unique interplay between T-type calcium and other intrinsic currents is conserved, and T channel activation can be triggered by synaptic stimulation. Here, using the dynamic clamp technique, artificial T conductances were re-incorporated in thalamic neurons devoid of endogenous T currents to dissect the physiological role of the T current gating diversity on neuronal excitability. We demonstrate that the specific kinetics of the T currents in thalamocortical and nucleus reticularis thalami neurons determine the characteristic firing patterns of these neurons. We show that subtle modifications in T channel gating that are at the limit of the resolution achieved in classical biophysical studies in heterologous expression systems have profound consequences for synaptically evoked firing dynamics in native neurons. Moreover, we demonstrate that the biophysical properties of the T current in the voltage region corresponding to the foot of the activation and inactivation curves drastically condition physiologically evoked burst firing with a high degree of synaptic input specificity.

Acute alertness-promoting effects of a novel histamine subtype-3 receptor inverse agonist in healthy sleep-deprived male volunteers.

The alertness-promoting effect of MK-0249 (10 or 50 mg), a histamine subtype-3 receptor (HRH3) inverse agonist (IA), was evaluated in the stimulant reference sleep deprivation model (SRSDM) using a double-blind, double-dummy, placebo- and modafinil- (200 mg) controlled, four-period crossover design in 24 healthy young men. The two primary hypotheses were related to sleep latency (first appearance of one epoch of stage 2, 3, or 4 or REM sleep, as detected using polysomnography (PSG)) at 8:00 AM on day 2. Statistically significant increases in sleep latency were observed in association with the use of modafinil 200 mg (9.07 min; P < 0.0001), MK-0249 50 mg (5.17 min; P = 0.008), and MK-0249 10 mg (5.45 min; P = 0.005) at the maintenance of wakefulness test (MWT) at 8:00 AM. Sleep latency was higher when averaged over all MWT time points (P < 0.0001 for modafinil and for both doses of MK-0249). The alertness-promoting effect with the use of MK-0249 in the SRSDM suggests that HRH3 IAs may be effective in disorders involving excessive somnolence.

A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation.

Formation of glutamatergic synapses entails development of "silent" immature contacts into mature functional synapses. To determine how this transformation occurs, we investigated the development of neurotransmission at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, followed accumulation of synapsin I. During this period, synaptic transmission was primarily mediated by activation of NMDA receptors, suggesting that most synapses were functionally silent. However, local glutamate application to silent synapses indicated that these synapses contained functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverted functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release.

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Knocking the NT4 gene into the BDNF locus rescues BDNF deficient mice and reveals distinct NT4 and BDNF activities.

To directly compare biological activities of the neurotrophins NT4 and BDNF in vivo, we replaced the BDNF coding sequence with the NT4 sequence in mice (Bdnfnt4-ki). Mice expressing NT4 in place of BDNF were viable, in contrast with BDNF null mutants, which die shortly after birth. Although the Bdnfnt4-ki/nt4-ki and wild-type Bdnf+/+ alleles yielded similar levels of NT4 and BDNF proteins, NT4 supported more sensory neurons than BDNF and promoted functional synapse formation in cultured hippocampal neurons. Homozygous Bdnfnt4-ki/nt4-ki mice showed reduced body weight, infertility and skin lesions, suggesting unique biological activities of NT4 in vivo. The distinct activities of NT4 and BDNF may result partly from differential activation of the TrkB receptor and its down-stream signals.

Neuronal polymorphism among natural alleles of a cGMP-dependent kinase gene, foraging, in Drosophila.

Natural variation in neuronal excitability and connectivity has not been extensively studied. In Drosophila melanogaster, a naturally maintained genetic polymorphism at a cGMP-dependent protein kinase (PKG) gene, foraging (for), is associated with alternative food search strategies among the allelic variants Rover (for(R); higher PKG activity) and sitter (for(s); lower PKG activity). We examined physiological and morphological variations in nervous systems of these allelic variants isolated from natural populations. Whole-cell current clamping revealed distinct excitability patterns, with spontaneous activities and excessive evoked firing in cultured sitter, but not Rover, neurons. Voltage-clamp examination demonstrated reduced voltage-dependent K(+) currents in sitter neurons. Focal recordings from synapses at the larval neuromuscular junction demonstrated spontaneous activity and supernumerary discharges with increased transmitter release after nerve stimulation. Immunolabeling showed more diffuse motor axon terminal projections with increased ectopic nerve entry points in sitter larval muscles. The differences between the two natural alleles was enhanced in laboratory-induced mutant alleles of the for gene. The pervasive effects of the for-PKG on neuronal excitability, synaptic transmission, and nerve connectivity illustrate the magnitude of neuronal variability in Drosophila that can be attributed to a single gene. These findings establish the consequences in cellular function for natural variation in an isoform of PKG and suggest a role for natural selection in maintaining variation in neuronal properties.

Concomitant alterations of physiological and developmental plasticity in Drosophila CaM kinase II-inhibited synapses.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase) has been implicated in neural plasticity that underlies learning and memory processes. Transformed strains of Drosophila, ala1 and ala2, expressing a specific inhibitor of CaM kinase are known to be impaired in an associative conditioning behavioral paradigm. We found that these transformants had altered short-term plasticity in synaptic transmission along with abnormal nerve terminal sprouting and directionality of outgrowth. These results represent an interesting parallel with the activity-dependent regulation of synaptic physiology and morphology by the cAMP cascade in Aplysia and Drosophila. In contrast to the learning mutants dunce and rutabaga, which are defective in the cAMP cascade, inhibition of CaM kinase in ala transformants caused increased sprouting at larval neuromuscular junctions near the nerve entry point, rather than altering the higher order branch segments. In addition, synaptic facilitation and potentiation were altered in a manner different from that observed in the cAMP mutants. Furthermore, synaptic currents in ala transformants were characterized by greater variability, suggesting an important role of CaM kinase in the stability of transmission.

Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na:K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.