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The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.

Valproic acid (VPA) is among the most teratogenic of commonly prescribed anticonvulsants, increasing the risk in humans of major malformations and impaired cognitive development. Likewise, rats exposed prenatally to VPA exhibit a variety of neuroanatomical and behavioral abnormalities. Previous work has shown that pyramidal neuron physiology in young VPA-exposed animals is marked by two strong abnormalities: an impairment in intrinsic neuronal excitability and an increase in NMDA synaptic currents. In this study, we investigated these abnormalities across postnatal development using whole-cell patch recordings from layer 2/3 neurons of medial prefrontal cortex. We found that both abnormalities were at a peak soon after birth but were gradually corrected as animals matured, to the extent that normal excitability and NMDA currents had been restored by early adolescence. The manner in which this correction happened suggested coordination between the two processes. Using computational models fitted to the physiological data, we argue that the two abnormalities trade off against each other, with the effects on network activity of the one balancing the effects of the other. This may constitute part of the nervous system's homeostatic response to teratogenic insult: an attempt to maintain stability despite a strong challenge.

Background: Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool., Methodology/principal Findings: We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2., Conclusions/significance: We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation.

Reactive oxygen species (ROS) are important regulators of intracellular signaling. We examined the expression of ROS during rat brain development and explored their role in differentiation using cortical cultures. High levels of ROS were found in newborn neurons. Neurons produced ROS, not connected with cell death, throughout embryogenesis and postnatal stages. By P20, ROS-producing cells were found only in neurogenic regions. Cells with low levels of ROS, isolated from E15 brains by FACS, differentiated into neurons, oligodendrocytes, and astrocytes in clonal cultures. Neurons produced high ROS early in culture and later differentiated into two types: large pyramidal-like neurons that fired no or only a single action potential and smaller neurons that expressed nuclear calretinin and fired repeated action potentials. Antioxidant treatment did not alter neuron number but increased the ratio of small to large neurons. These findings suggest that modulation of ROS levels influences multiple aspects of neuronal differentiation.

Neocortical neurons in vivo exist in an environment of continuous synaptic bombardment, receiving a complex barrage of excitatory and inhibitory inputs. This background activity (by depolarizing neurons, increasing membrane conductance, and introducing fluctuations) strongly alters many aspects of neuronal responsiveness. In this study, we asked how it shapes neuromodulation of postsynaptic responses. Specifically, we examined muscarinic modulation of forelimb motor cortex, a brain area in which cholinergic stimulation is known to be necessary for modifications during motor skill learning. Using a dynamic clamp system to inject simulated conductances into pyramidal neurons in motor cortical slices, we mimicked in vivo-like activity by introducing a random background of excitatory and inhibitory inputs. When muscarinic receptors were stimulated with the agonist oxotremorine-M, several previously described currents were modified, and excitability was increased. However, the presence of the background conductances strongly attenuated most muscarinic agonist effects, with the notable exception that sustained firing responses to trains of inputs were well preserved. This may be important for promoting plasticity in vivo.

A population of embryonic rat cortical cells cultured in the presence of FGF2 and having neuronal morphology expressed higher levels of reactive oxygen species (ROS) than did progenitor cells, astrocytes, and several cell lines of neuronal and non-neuronal origin. ROS were assessed using 5-(and-6)-chlormethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCF-DA), and high levels persisted in the presence of antioxidants or lowered levels of ambient oxygen. Greater than 95% of high ROS-producing cells, isolated by fluorescence-activated cell sorting, expressed the neuronal marker beta III tubulin. These cells did not incorporate BrdU or express nestin, unlike low ROS-producing cells, 99% of which exhibited both of these characteristics. Upon growth factor removal, low ROS-expressing cells differentiated into neurons and astrocytes and these neurons expressed high levels of ROS, indicating that ROS accumulation accompanies the differentiation of progenitors into neurons. ROS levels were decreased by added superoxide dismutase and catalase, suggesting that both superoxide and hydrogen peroxide contribute to the ROS signal. High ROS-expressing cells also contained higher levels of several mitochondrial respiratory chain components. Although ROS have been associated with conditions that lead to cell death, our results and recent studies on the role of ROS as regulators of signal pathways are consistent with the possibility that ROS play a role in the development of the neuronal phenotype. Moreover, the differential production of ROS provides a useful method to isolate from mixed populations cells that are highly enriched for either progenitor cells or neurons.

The psychoactive drug caffeine influences neuronal physiology; however, it is unknown whether it can dynamically alter the expression of genes that influence neurotransmission. Here, we report that caffeine stimulates transcription of the dopamine 2 receptor (D2R) gene in PC-12 cells and primary striatal cultures and increases D2R protein expression in the striatum. Physiological doses of caffeine and the specific adenosine 2A receptor antagonist 8-(3-chlorostyryl) caffeine both increased the activity of a D2R/luciferase reporter construct within 24 h, and simultaneous treatment with 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), a specific adenosine 2A receptor agonist, eliminated this effect. Tests of additional constructs revealed that specific regions of the D2R promoter (-117/-75) and 5'-untranslated region (+22/+317) were required for activation of D2R gene expression by caffeine. In primary striatal cultures, caffeine increased spontaneous firing of neurons between 12 and 80 min after treatment, whereas it increased D2R mRNA expression after only 4 h. These results indicate that regulation of D2R gene expression by caffeine occurs after the initial physiological response has subsided. In vivo, female mice treated with a dose of caffeine (50 mg/kg) showed 1.94- and 2.07-fold increases in D2R mRNA and protein expression, respectively. In contrast, male mice exhibited a 31% decrease in D2R mRNA expression and showed no changes in D2R protein expression. Collectively, these results demonstrate for the first time that caffeine alters D2R expression in neurons. They also suggest that caffeine consumption can lead to sexually dimorphic patterns of gene expression in the brain.

What is the functional significance of generating a burst of spikes, as opposed to a single spike? A dominant point of view is that bursts are needed to increase the reliability of communication between neurons. Here, we discuss the alternative, but complementary, hypothesis: bursts with specific resonant interspike frequencies are more likely to cause a postsynaptic cell to fire than are bursts with higher or lower frequencies. Such a frequency preference might occur at the level of individual synapses because of the interplay between short-term synaptic depression and facilitation, or at the postsynaptic cell level because of subthreshold membrane potential oscillations and resonance. As a result, the same burst could resonate for some synapses or cells and not resonate for others, depending on their natural resonance frequencies. This observation suggests that, in addition to increasing reliability of synaptic transmission, bursts of action potentials might provide effective mechanisms for selective communication between neurons.

Recently, variation upon a well-established hippocampal model has given rise to a new paradigm in which the strength of synaptic inputs to neocortical layer 2/3 is estimated in vitro by recording synaptically driven extracellular potentials elicited there by electrical stimulation applied to underlying layer 4. The analysis of these potentials is commonly based upon an assumption that postsynaptic spiking has played no significant role in their generation. Here, we have tested this assumption by quantifying in rats (using data obtained by cell-attached recording) the rate at which unit spikes are elicited in layer 2/3 under commonly used conditions of stimulation and recording. We found that spike responses were regularly elicited at the same latencies as field potential peaks and the rising phases of intracellularly recorded synaptic currents, and the incidence of such spiking (the fractional rate of cells spiking versus cells sampled) was sufficient to give this higher-order activity a major role in determining response amplitudes. We then analyzed layer 2/3 waveform characteristics before and after inducing long-term potentiation (LTP) by theta-burst stimulation (TBS) and found that the induction of LTP succeeded only when the initial response included a strong spike component. We further observed that LTP expression was always accompanied by a pronounced enhancement of such components. Our data suggest that, unlike in hippocampal CA1, LTP elicited by TBS in this neocortical paradigm depends upon modification of synaptically driven spike activity, through either enhanced synchronization of unitary responses, the recruitment of additional responding units, or both. This potentiation of the spike response could arise (as previously proposed) through an increase in the efficacy of synapses mediating projection from layer 4 to 2/3, but other mechanisms may also contribute, such as modification of short-range recurrent connections within layer 2/3, which are likely to play an important role in defining local-network cell ensembles.

Previous studies have shown that different pyramidal cell inputs vary in the short-term plasticity expressed when they are subjected to repetition of use. Here, we describe short-term plasticity at synapses that mediate long-range input to neocortical layer 1 and compare it with that which normally occurs in the hippocampal Schaffer collateral pathway, which also involves projection by remote inputs onto apical dendrites. We isolated tangential inputs to layer 1 in neocortical slices, stimulated these with brief 40-Hz trains, and examined postsynaptic responses by recording extracellularly from layer 1 in somatosensory, prefrontal, and visual neocortex, and intracellularly from visually identified pyramidal cell somata in layer 2/3 in somatosensory and prefrontal neocortex. Train response amplitudes were characterized by calculating paired-pulse ratios, fifth-versus-first amplitude ratios (5th/1st ratios), and a center-of-mass index "M". As expected, the hippocampal train responses facilitated strongly. In contrast, layer-1 responses displayed strong synaptic depression in all regions examined. This depression was reflected in 5th/lst ratios and M scores, but not paired-pulse ratios because it did not consistently begin until the third responses in trains. It persisted unchanged in the presence of partially blocking levels of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but was converted to strong facilitation when slices were bathed in low-Ca++ media. Intracellularly, we observed response-train depression very similar to that recorded extracellularly. These findings show that long-range inputs to neocortical layer 1 display short-term plasticity markedly different from that which normally occurs at hippocampal Schaffer collateral synapses, but similar to that which has been described previously for excitatory inputs to pyramidal cells in deeper neocortical layers.

Nicotinoids and neonicotinoids are characterized by the presence of the 3-pyridylmethylamine moiety in their structure. In the former, the amino nitrogen atom is ionized, while in the latter the corresponding nitrogen atom is not ionized but bears a partial positive charge. Both types of insecticides interact with nicotinic acetylcholine receptor (nAChR) of insect origin. The poor interaction of neonicotinoids with vertebrate nAChR was shown by its poor binding affinity to the nAChR from Torpedo electric organ and rat brain and poor activation with nAChR expressed in Xenopus oocytes. The full positive charge was essential to interact with the vertebrate nAChR, while the 3-pyridylmethylamine moiety with a partial positive charge was enough to interact with the insect nAChR. For penetration into the insect central nervous system, hydrophobicity seemed to play an important role, as indicated by the binding of the injected compounds to the housefly head nAChR. The ionization reduced hydrophobicity and limited the penetration of nicotinoids, resulting in less insecticidal activity. Among neonicotinoids, nitromethylene type compounds, though far higher in binding affinity, were less hydrophobic than the corresponding nitroimine type, and the net result was better or inferior insecticidal activity. A chlorine atom at the 6 position of the 3-pyridyl group found in commercialized neonicotinoids contributes to increased binding affinity and more importantly hydrophobicity, thus increasing insecticidal activity. N-Me-imidacloprid was found to be a propesticide of imidacloprid.

The role of the conserved carbohydrate moiety in the expression of complete acetylcholine receptor (AChR), alpha2 beta gamma delta was re-investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subunits were stably expressed and appeared to associate with other normal subunits; however, removal of carbohydrate on the alpha subunit inhibited the formation of 125I-alpha-bungarotoxin (alpha-BuTX) binding sites and functional ACh-gated ion channels. 125I-alpha-BuTX binding to AChRs was also significantly reduced by removal of the conserved carbohydrate on the gamma or delta subunits. Immunoprecipitation with monoclonal antibodies that recognize the two distinct alpha-BuTX sites on the AChR indicated that the mutant gamma subunit did not interfere with efficient formation of the alpha-BuTX binding site at the alpha/delta interface, but loss of the carbohydrate did interfere with formation of the alpha-BuTX binding site at the alpha/mutant gamma interface. A similar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated efficiently into the mature (correct tertiary conformation capable of alpha-BuTX binding) alpha beta delta or alpha beta gamma complexes, respectively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these results suggest that the formation of the two alpha-BuTX binding sites requires correct folding of the alpha gamma and alpha delta complexes, which is aided by the conserved carbohydrate on the gamma and delta subunits. Electrophysiological experiments demonstrated that functional receptors containing mutant subunits were produced, but the functional properties of the mutant receptors were differentially altered, depending on the subunit mutated. Together, our results suggest that N-glycosylation of AChR subunits ensures the correct folding of important functional domains and expression of proper functional receptors in the plasma membrane.

Aged rats often display abnormal circadian activity rhythms; the rhythm amplitude is low and entrainment to light-dark cycles is irregular. The activity rhythm of young rats can be entrained by both light and nonphotic cues, specifically food availability. In young rats, entrainment to restricted feeding cycles does not depend on intact suprachiasmatic nuclei, the presumed anatomical location of the light-entrainable oscillator. In this study, aged rats that displayed disrupted entrainment to light were assessed for their ability to entrain to restricted feeding schedules. Aged rats, young controls, and young suprachiasmatic nuclei-lesioned (SCN) rats were placed on a food restriction schedule (FR) for 14 days. Food was available for 2 h during the light phase of a 12-h light-dark cycle. Despite the absence of entrainment to light/dark cycles, both SCN-lesioned and aged groups showed entrainment to FR, with clear bouts of anticipatory activity during a period of complete food deprivation following 2 weeks of FR. The results suggest that the dysrhythmia of aged rats is a result of natural deterioration of a central circadian light-entrainable pacemaker, but that a secondary oscillator entrainable to food cycles is spared.

To examine the structural determinants for the assembly of ligand-gated receptors, we constructed mutant alpha, beta, gamma and delta subunits of the Torpedo acetylcholine receptor (AChR), lacking one of the conserved cysteine residues which forms a 13-amino acid disulfide loop in the amino terminal domain of each subunit. Mutant subunits were co-expressed with complementary wild-type subunits in Xenopus oocytes. Using subunit-specific antisera and monoclonal antibodies that recognize the two distinct alpha-bungarotoxin (alpha-BuTX) sites on the AChR, we were able to distinguish immature subunit associations from conformationally mature AChR complexes. Removal of the disulfide loop on the alpha subunit completely destroyed the formation of the two toxin-binding sites, while removal of the structure on the beta subunit had little effect. While mutant gamma and delta subunits were capable of forming associations (immature assembly) with other subunits, the formation of alpha-BTX sites between alpha and mutant gamma or mutant delta subunits was diminished. Interestingly, assembly of alpha beta gamma subunits remained efficient in the presence of mutant delta subunits, whereas assembly of alpha beta delta subunits was inefficient in the presence of mutant gamma subunits. Thus, these results indicate that the formation of the disulfide loop facilitates the conformational maturation of alpha gamma and alpha delta complexes, which may be conditional for correct subunit coupling in assembling receptors. Furthermore, it seems likely that the correct coupling between the alpha and gamma subunits is the most important step in subunit assembly.

Alzheimer disease patients exhibit irregularities in the patterns of normally circadian (daily) rhythms. Alzheimer-type pathology has been reported in the hypothalamus and in the suprachiasmatic nuclei, the putative site of the circadian oscillator. We examined the relationship between the neuropathology of Alzheimer disease, as modeled by an animal system, and circadian dysregulation by grafting genetically transformed cells that overexpress beta/A4 amyloid into the suprachiasmatic nuclei of adult rats. Grafts of beta/A4-positive cells, but not of control cells, significantly altered the pattern of activity of implanted rats. Although experimental conditions included light-dark cycles that normally tend to drive rats to 24-h rhythms, animals with grafts of beta/A4-positive cells showed abnormally high levels of activity during the light phase in addition to a disrupted circadian pattern. Periodogram analysis demonstrated significant rhythms outside of a circadian range. The body temperature rhythm of these animals was also weak 6 weeks after grafting; however, unlike activity patterns, body temperature regained a circadian period by 8 weeks after cell implantation. These data indicate that disruption of circadian activity is a behavioral measure of the consequences of beta/A4 accumulation in brain implants.

The mechanism by which the A4 (beta-amyloid) domain of the Alzheimer amyloid precursor protein (APP) is deposited in plaques is unknown, and limited information is available concerning the extent to which other APP sites are associated with plaques. To address these issues, we prepared antiserum to a peptide adjacent to the N-terminus of the APP (referred to as N1) and examined its distribution in brain relative to A4 by double-immunostaining techniques. Anti-N1 localized to both neurons and glia in control and Alzheimer patients. In the Alzheimer brain, anti-N1 detected plaques. Quantitation revealed that 85% of thioflavin-positive plaques, and 91% of A4-positive plaques were also N1 positive. Double-staining methods directly demonstrated colocalization of distant APP sites. The data suggest that suggest that proposed mechanisms for amyloid deposition during plaque formation must take into account the extracytoplasmic domain, in addition to the A4 region, rather than be confined exclusively to the A4 site.