Nondermatophyte mould onychomycosis.

Nondermatophyte moulds (NDMs) onychomycosis is often difficult to diagnose as NDMs have been considered contaminants of nails. There are several diagnostic methods used to identify NDMs, however, repeated laboratory isolation is recommended to validate pathogenicity. With NDM and mixed infection (dermatophytes plus NDM) onychomycosis on the rise, accurate clinical diagnosis along with mycological tests is recommended. Systemic antifungal agents such as itraconazole and terbinafine (e.g. pulse regimen: 1 pulse = every day for one week, followed by no treatment for three weeks) have shown efficacy in treating onychomycosis caused by various NDMs such as Aspergillus spp., Fusarium spp., Scopulariopsis brevicaulis, and Onychocola canadensis. Studies investigating topical therapy and devices for NDM onychomycosis are limited. The emergence of antifungal resistance necessitates the incorporation of antifungal susceptibility testing into diagnosis when possible, for the management of recalcitrant infections. Case studies documented in the literature show newer azoles such as posaconazole and voriconazole as sometimes effective in treating resistant NDM onychomycosis. Treatment with broad-spectrum antifungal agents (e.g. itraconazole and efinaconazole) and other combination therapy (oral + oral and/or oral + topical) may be considerations in the management of NDM onychomycosis.

Differential regulation of synapsin phosphorylation by monocular deprivation in juveniles and adults.

The rodent visual cortex retains significant ocular dominance plasticity beyond the traditional postnatal critical period. However, the intracellular mechanisms that underlie the cortical response to monocular deprivation are predicted to be different in juveniles and adults. Here we show monocular deprivation in adult, but not juvenile rats, induced an increase in the phosphorylation of the prominent presynaptic effecter protein synapsin at two key sites known to regulate synapsin function. Monocular deprivation in adults induced an increase in synapsin phosphorylation at the PKA consensus site (site 1) and the CaMKII consensus site (site 3) in the visual cortex ipsilateral to the deprived eye, which is dominated by non-deprived eye input. The increase in synapsin phosphorylation was observed in total cortical homogenate, but not synaptoneurosomes, suggesting that the pool of synapsin targeted by monocular deprivation in adults does not co-fractionate with excitatory synapses. Phosphorylation of sites 1 and 3 stimulates the release of synaptic vesicles from a reserve pool and increases in the probability of evoked neurotransmitter release, which may contribute to the strengthening of the non-deprived input characteristic of ocular dominance plasticity in adults.

A role for the cytoplasmic polyadenylation element in NMDA receptor-regulated mRNA translation in neurons.

The ability of neurons to modify synaptic connections based on activity is essential for information processing and storage in the brain. The induction of long-lasting changes in synaptic strength requires new protein synthesis and is often mediated by NMDA-type glutamate receptors (NMDARs). We used a dark-rearing paradigm to examine mRNA translational regulation in the visual cortex after visual experience-induced synaptic plasticity. In this model system, we demonstrate that visual experience induces the translation of mRNA encoding the alpha-subunit of calcium/calmodulin-dependent kinase II in the visual cortex. Furthermore, this increase in translation is NMDAR dependent. One potential source for newly synthesized proteins is the translational activation of dormant cytoplasmic mRNAs. To examine this possibility, we developed a culture-based assay system to study translational regulation in neurons. Cultured hippocampal neurons were transfected with constructs encoding green fluorescent protein (GFP). At 6 hr after transfection, approximately 35% of the transfected neurons (as determined by in situ hybridization) expressed detectable GFP protein. Glutamate stimulation of the cultures at this time induced an increase in the number of neurons expressing GFP protein that was NMDAR dependent. Importantly, the glutamate-induced increase was only detected when the 3'-untranslated region of the GFP constructs contained intact cytoplasmic polyadenylation elements (CPEs). Together, these findings define a molecular mechanism for activity-dependent synaptic plasticity that is mediated by the NMDA receptor and requires the CPE-dependent translation of an identified mRNA.

A biophysical model of bidirectional synaptic plasticity: dependence on AMPA and NMDA receptors.

In many regions of the brain, including the mammalian cortex, the magnitude and direction of activity-dependent changes in synaptic strength depend on the frequency of presynaptic stimulation (synaptic plasticity), as well as the history of activity at those synapses (metaplasticity). We present a model of a molecular mechanism of bidirectional synaptic plasticity based on the observation that long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD) correlate with the phosphorylation/dephosphorylation of sites on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit protein GluR1. The primary assumption of the model, for which there is wide experimental support, is that postsynaptic calcium concentration and consequent activation of calcium-dependent protein kinases and phosphatases are the triggers for the induction of LTP/LTD. As calcium influx through the n-methyl-d-aspartate (NMDA) receptor plays a fundamental role in the induction of LTP/LTD, changes in the properties of NMDA receptor-mediated calcium influx will dramatically affect activity-dependent synaptic plasticity (metaplasticity). We demonstrate that experimentally observed metaplasticity can be accounted for by activity-dependent regulation of NMDA receptor subunit composition and function. Our model produces a frequency-dependent LTP/LTD curve with a sliding synaptic modification threshold similar to what has been proposed theoretically by Bienenstock, Cooper, and Munro and observed experimentally.

Regulation of synaptic strength by protein phosphatase 1.

We investigated the role of postsynaptic protein phosphatase 1 (PP1) in regulating synaptic strength by loading CA1 pyramidal cells either with peptides that disrupt PP1 binding to synaptic targeting proteins or with active PP1. The peptides blocked synaptically evoked LTD but had no effect on basal synaptic currents mediated by either AMPA or NMDA receptors. They did, however, cause an increase in synaptic strength following the induction of LTD. Similarly, PP1 had no effect on basal synaptic strength but enhanced LTD. In cultured neurons, synaptic activation of NMDA receptors increased the proportion of PP1 localized to synapses. These results suggest that PP1 does not significantly regulate basal synaptic strength. Appropriate NMDA receptor activation, however, allows PP1 to gain access to synaptic substrates and be recruited to synapses where its activity is necessary for sustaining LTD.

Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo.

Experience-dependent regulation of synaptic strength has been suggested as a physiological mechanism by which memory storage occurs in the brain. Although modifications in postsynaptic glutamate receptor levels have long been hypothesized to be a molecular basis for long-lasting regulation of synaptic strength, direct evidence obtained in the intact brain has been lacking. Here we show that in the adult brain in vivo, synaptic glutamate receptor trafficking is bidirectionally, and reversibly, modified by NMDA receptor-dependent synaptic plasticity and that changes in glutamate receptor protein levels accurately predict changes in synaptic strength. These findings support the idea that memories can be encoded by the precise experience-dependent assignment of glutamate receptors to synapses in the brain.

Bidirectional, experience-dependent regulation of N-methyl-D-aspartate receptor subunit composition in the rat visual cortex during postnatal development.

In the visual cortex, as elsewhere, N-methyl-D-aspartate receptors (NMDARs) play a critical role in triggering long-term, experience-dependent synaptic plasticity. Modifications of NMDAR subunit composition alter receptor function, and could have a large impact on the properties of synaptic plasticity. We have used immunoblot analysis to investigate the effects of age and visual experience on the expression of different NMDAR subunits in synaptoneurosomes prepared from rat visual cortices. NMDARs at birth are comprised of NR2B and NR1 subunits, and, over the first 5 postnatal weeks, there is a progressive inclusion of the NR2A subunit. Dark rearing from birth attenuates the developmental increase in NR2A. Levels of NR2A increase rapidly (in <2 hr) when dark-reared animals are exposed to light, and decrease gradually over the course of 3 to 4 days when animals are deprived of light. These data reveal that NMDAR subunit composition in the visual cortex is remarkably dynamic and bidirectionally regulated by sensory experience. We propose that NMDAR subunit regulation is a mechanism for experience-dependent modulation of synaptic plasticity in the visual cortex, and serves to maintain synaptic strength within an optimal dynamic range.

Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo.

Sensory experience is crucial in the refinement of synaptic connections in the brain during development. It has been suggested that some forms of experience-dependent synaptic plasticity in vivo are associated with changes in the complement of postsynaptic glutamate receptors, although direct evidence has been lacking. Here we show that visual experience triggers the rapid synaptic insertion of new NMDA receptors in visual cortex. The new receptors have a higher proportion of NR2A subunits and, as a consequence, different functional properties. This effect of experience requires NMDA receptor activation and protein synthesis. Thus, rapid regulation of postsynaptic glutamate receptors is one mechanism for developmental plasticity in the brain. Changes in NMDA receptor expression provide a mechanism by which brief sensory experience can regulate the properties of NMDA receptor-dependent plasticity in visual cortex.

Feeding stimulants activate an identified dopaminergic interneuron that induces the feeding motor program in Helisoma.

The neurotransmitter dopamine is shown to play a fundamental role in the generation of the feeding motor pattern and resultant feeding behavior in Helisoma. Application of exogenous dopamine triggered the fictive feeding motor pattern in the isolated CNS and triggered feeding movements in semi-intact preparations. Application of feeding stimulants to the oral cavity excited the putatively dopaminergic buccal interneuron N1a, and depolarization of interneuron N1a triggered the production of the fictive feeding motor pattern. The ability of dopamine superfusion and of interneuron N1a stimulation to activate the fictive feeding motor pattern was blocked by the dopamine antagonist sulpiride. The phase of the fictive feeding motor pattern was reset by brief hyperpolarization of interneuron N1a, demonstrating that interneuron N1a is an integral component of the buccal central pattern generator (CPG). During spontaneous fictive feeding patterns, prolonged hyperpolarizations of interneuron N1a inhibited the production of patterned activity. Exogenous dopamine maintained the fictive feeding motor pattern in the absence of interneuron N1a activity. Interneuron N1a was labeled by the formaldehyde-glutaraldehyde histochemical technique, which is indicative of the presence of dopamine in mollusks. These data suggest that interneuron N1a is an endogenous source of the neuromodulator dopamine, intrinsic to the buccal CPG, and that interneuron N1a has a prominent role in the sensory-motor integration triggering the consummatory response.

Emergence of activity-dependent, bidirectional control of microtubule-associated protein MAP2 phosphorylation during postnatal development.

Pronounced changes in neuronal morphology occur as synapses mature; however, little is known about how synaptic transmission regulates the developing neuronal cytoskeleton. The postsynaptic, microtubule-associated protein MAP2 is a target of multiple, calcium-dependent signaling pathways activated by synaptic transmission. Here we demonstrate that MAP2 phosphorylation is differentially regulated across development. In 32P-labeled hippocampal slices prepared from adult rats, depolarization stimulated a bidirectional change in the phosphorylation of immunoprecipitated MAP2. A transient increase was mediated by metabotropic glutamate receptors (mGluRs) and stimulation of mitogen-activated protein kinases (MAPKs), Ca2+/calmodulin-dependent protein kinases (CaMKs), and protein kinase C (PKC). This increase was followed by a persistent dephosphorylation mediated by NMDA receptors and activation of protein phosphatase 2B (PP2B or calcineurin). In contrast, depolarization of neonatal hippocampal slices stimulated exclusively a net increase in MAP2 phosphorylation, which was attenuated by inhibitors of MAPKs, but not CaMKs or PKC. Furthermore, although incubation in NMDA induced a time-dependent decrease in MAP2 phosphorylation in both adults and neonates, this effect was both less robust and less sensitive to calcineurin inhibitors in neonates than in adults. These data indicate that the mechanisms coupling glutamate release to MAP2 dephosphorylation are relatively lacking in the neonatal hippocampus. Highly phosphorylated MAP2 is impaired in its ability to stabilize microtubules and actin filament bundles in vitro. The neonatal propensity toward glutamate-stimulated MAP2 phosphorylation may serve to reduce cytoskeletal stability and permit dendritic arborization early in postnatal development. In mature neurons, the bidirectional control of MAP2 phosphorylation may participate in activity-dependent synaptic remodeling.

Postsynaptic mechanisms for bidirectional control of MAP2 phosphorylation by glutamate receptors.

Many activity-dependent changes in synaptic efficacy occur through elevations in postsynaptic calcium triggered by glutamate receptor activation. Here, the postsynaptic, neuron-specific microtubule-associated protein MAP2 is identified as a target of bidirectional calcium-dependent signaling pathways activated by glutamate. Glutamate produced a biphasic change in MAP2: a rapid, transient increase in phosphorylation mediated by metabotropic receptors and attenuated by inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C, followed by a persistent dephosphorylation of MAP2 mediated by NMDA receptors and activation of the calcium/calmodulin-dependent protein phosphatase 2B (calcineurin). Thus, a single transmembrane signal, glutamate, and the increased intracellular calcium it evokes can have opposing actions on a postsynaptic target phosphoprotein. The phosphorylation state of MAP2 determines its interaction with microtubules and actin filaments, suggesting that glutamatergic regulation of MAP2 phosphorylation may transduce neural activity into modifications in dendritic structure.

Plasticity in the multifunctional buccal central pattern generator of Helisoma illuminated by the identification of phase 3 interneurons.

1. The mechanism for generating diverse patterns of buccal motor neuron activity was explored in the multifunctional central pattern generator (CPG) of Helisoma. The standard pattern of motor neuron activity, which results in typical feeding behavior, consists of three distinct phases of buccal motor neuron activity. We have previously identified CPG interneurons that control the motor neuron activity during phases 1 and 2 of the standard pattern. Here we identify a pair of interneurons responsible for buccal motor neuron activity during phase 3, and examine the variability in the interactions between this third subunit and other subunits of the CPG. 2. During the production of the standard pattern, phase 3 excitation in many buccal motor neurons follows a prominent phase 2 inhibitory postsynaptic potential. Therefore phase 3 excitation was previously attributed to postinhibitory rebound (PIR) in these motor neurons. Two classes of observations indicated that PIR was insufficient to account for phase 3 activity, necessitating phase 3 interneurons. 1) A subset of identified buccal neurons is inhibited during phase 3 by discrete synaptic input. 2) Other identified buccal neurons display discrete excitation during both phases 2 and 3. 3. A bilaterally symmetrical pair of CPG interneurons, named N3a, was identified and characterized as the source of phase 3 postsynaptic potentials in motor neurons. During phase 3 of the standard motor pattern, interneuron N3a generated bursts of action potentials. Stimulation of N3a, in quiescent preparations, evoked a depolarization in motor neurons that are excited during phase 3 and a hyperpolarization in motor neurons that are inhibited during phase 3. Hyperpolarization of N3a during patterned motor activity eliminated both phase 3 excitation and inhibition. Physiological and morphological characterization of interneuron N3a is provided to invite comparisons with possible homologues in other gastropod feeding CPGs. 4. These data support a model proposed for the organization of the tripartite buccal CPG. According to the model, each of the three phases of buccal motor neuron activity is controlled by discrete subsets of pattern-generating interneurons called subunit 1 (S1), subunit 2 (S2), and subunit 3 (S3). The standard pattern of buccal motor neuron activity underlying feeding is mediated by an S1-S2-S3 sequence of CPG subunit activity. However, a number of "nonstandard" patterns of buccal motor activity were observed. In particular, S2 and S3 activity can occur independently or be linked sequentially in rhythmic patterns other than the standard feeding pattern. Simultaneous recordings of S3 interneuron N3a with effector neurons indicated that N3a can account for phase-3-like postsynaptic potentials (PSPs) in nonstandard patterns. The variety of patterns of buccal motor neuron activity indicates that each CPG subunit can be active in the absence of, or in concert with, activity in any other subunit. 5. To explore how CPG activity may be regulated to generate a particular motor pattern from the CPG's full repertoire, we applied the neuromodulator serotonin. Serotonin initiated and sustained the production of an S2-S3 pattern of activity, in part by enhancing PIR in S3 interneuron N3a after the termination of phase 2 inhibition.

An identified glutamatergic interneuron patterns feeding motor activity via both excitation and inhibition.

1. Previously we demonstrated that glutamate is an important neurotransmitter in the CNS of Helisoma. Exogenous glutamate applied to the buccal ganglia mimicked both the excitatory and inhibitory effects of subunit 2 (S2) of the tripartite central pattern generator (CPG) on S2 postsynaptic motor neurons. Here we identify buccal interneuron B2 as an S2 interneuron by utilizing a combination of electrophysiology, pharmacology, and intracellular staining. In addition, neurons that were electrophysiologically and morphologically characterized as neuron B2 demonstrated antiglutamate immunoreactivity, suggesting that neuron B2 is a source of endogenous glutamate in the buccal ganglia. 2. Depolarization of neuron B2 evoked excitatory postsynaptic potentials in motor neurons excited by S2. The excitatory effects of B2 depolarization and S2 activation were reversibly antagonized by the ionotropic glutamate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione, similar to the antagonism shown previously for application of exogenous glutamate. Depolarization of neuron B2 also evoked inhibitory postsynaptic potentials in motor neurons inhibited by S2. When such motor neurons were maintained in isolated cell culture, application of exogenous glutamate produced a direct hyperpolarization of the membrane potential. 3. The activity of neuron B2 is necessary for the production of the standard pattern of buccal motor neuron activity, which underlies functional feeding movements. The subunits of the tripartite buccal CPG must be active in the temporal sequence S1-S2-S3 to produce the standard feeding pattern. Rhythmic inhibition from neuron B2 terminated activity in S1 postsynaptic motor neurons and entrained the frequency of activity in S3 postsynaptic motor neurons. Hyperpolarization of neuron B2 disrupted the production of the standard motor pattern by eliminating S2 postsynaptic potentials in identified buccal motor neurons, thereby prolonging S1 activity and disrupting S3 bursting. 4. These data support the hypothesis that S2 neuron B2 is glutamatergic and demonstrate that glutamatergic transmission, and especially inhibition, is fundamental to the production of behaviorally critical motor neuron activity patterns in Helisoma.

Glutamate as a putative neurotransmitter in the buccal central pattern generator of Helisoma trivolvis.

1. The effects of L-glutamate superfusion over identified neurons within the buccal ganglia of Helisoma trivolvis were examined. Glutamate mirrored the effect of activity of subunit 2 (S2) of the tripartite feeding central pattern generator (CPG) on S2 postsynaptic neurons. Neurons that are excited by S2 are depolarized by glutamate, whereas neurons that are inhibited by S2 are hyperpolarized by glutamate. Glutamate also stimulated rhythmic S2 activity. 2. Different glutamate agonists could mimic specific components of the effects of glutamate on buccal neurons. Kainate produced depolarizations in neurons that receive S2 excitatory postsynaptic potentials (EPSPs) and activated rhythmic S2 activity. Quisqualate produced hyperpolarizations in neurons that receive S2 inhibitory postsynaptic potentials (IPSPs). 3. The non-N-methyl-D-aspartate glutamate receptor antagonist cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effects of S2 EPSPs and depolarizations produced by application of glutamate and kainate, but was ineffective in blocking S2 IPSPs or hyperpolarizations produced by application of glutamate and quisqualate. 4. These data support the hypothesis that glutamate is the transmitter of S2 of the feeding CPG in Helisoma, acting at CNQX-sensitive kainate-like receptors at excitatory synapses and CNQX-insensitive quisqualate-like receptors at inhibitory synapses.