Systematic resequencing of X-chromosome synaptic genes in autism spectrum disorder and schizophrenia.

Autism spectrum disorder (ASD) and schizophrenia (SCZ) are two common neurodevelopmental syndromes that result from the combined effects of environmental and genetic factors. We set out to test the hypothesis that rare variants in many different genes, including de novo variants, could predispose to these conditions in a fraction of cases. In addition, for both disorders, males are either more significantly or more severely affected than females, which may be explained in part by X-linked genetic factors. Therefore, we directly sequenced 111 X-linked synaptic genes in individuals with ASD (n = 142; 122 males and 20 females) or SCZ (n = 143; 95 males and 48 females). We identified >200 non-synonymous variants, with an excess of rare damaging variants, which suggest the presence of disease-causing mutations. Truncating mutations in genes encoding the calcium-related protein IL1RAPL1 (already described in Piton et al. Hum Mol Genet 2008) and the monoamine degradation enzyme monoamine oxidase B were found in ASD and SCZ, respectively. Moreover, several promising non-synonymous rare variants were identified in genes encoding proteins involved in regulation of neurite outgrowth and other various synaptic functions (MECP2, TM4SF2/TSPAN7, PPP1R3F, PSMD10, MCF2, SLITRK2, GPRASP2, and OPHN1).

Landing patterns of phloem- and wood-feeding Coleoptera on black spruce of different physiological and decay states.

We examined landing patterns of phloeophagous and xylophagous Coleoptera among trees and snags of different physiological and decay states in a pure open-canopy black spruce stand in boreal Canada to study prelanding host selection mechanisms in the absence of nonhost volatiles. Sticky traps were used to capture insects landing on high- and low-density natural snags (i.e., wood density), girdled trees, living trees, and stovepipe controls. Patterns were generally weak, with high within-group variability in species composition and landing rates. Within-group variability differed between groups, with highest variations in living trees and recent snags. Despite this evidence of frequent landing on suboptimal or inappropriate hosts, affinities were detected in most common taxa. Cerambycidae showed preferences for girdled trees. Common species of Scolytinae showed divergent preferences, because Crypturgus borealis Swaine and Dryocoetes autographus (Ratzeburg) were captured more often on high-density natural snags, Polygraphus rufipennis (Kirby) on girdled trees, and Orthotomicus latidens (LeConte) on living trees. These observed landing patterns are broadly consistent with current knowledge on the ecology of these species. Although preferences, and thus prelanding assessment of hosts based on volatiles, were detected in several species, the numerous landings observed on inappropriate hosts suggest that random landing at close range may be as common in pure stands as what was previously observed in mixed stands.

Loss of channel modulation by transmitter and protein kinase C during innervation of an identified leech neuron.

When serotonergic Retzius (R) neurons of the leech contact pressure-sensitive (P) neurons in culture, P cells selectively lose a protein kinase C-dependent cationic response to serotonin and the R cell reforms the inhibitory, chloride-dependent synapse seen in vivo. In P cells not contacted by R cells, cell-attached patches contained single cation channels sensitive to serotonin and phorbol ester with characteristic properties and high incidence (present in about one-half of the patches). P cells paired with R cells had a cation channel with similar biophysical properties and incidence, but channel activity was not stimulated by serotonin and phorbol ester. These results suggest that the early clearing of the non-synaptic (excitatory) response to serotonin is due to the loss of activation by protein kinase C (and not the number) of cation channels as a prelude to inhibitory synapse formation.

Loss of extrasynaptic channel modulation by protein kinase C underlies the selection of serotonin responses in an identified leech neuron.

Pressure-sensitive (P) neurons contacted by serotonergic Retzius (R) neurons of the leech in culture selectively reduce a protein kinase C (PKC)-dependent cation response to serotonin and are innervated by the inhibitory, Cl(-)-dependent synapse seen in vivo. We have examined whether the reduction of extrasynaptic cation channel modulation is due to changes in sensitivity of the channels to second messenger. In inside-out membrane patches from single, uncontacted P cells in culture, cation channel activity was increased by rat brain PKC and cofactors. In contrast, the activity of cation channels in patches isolated from P cells paired with R cells was unaffected by PKC. These results demonstrate the loss of extrasynaptic channel modulation by PKC during synapse formation.

Synchronization of an embryonic network of identified spinal interneurons solely by electrical coupling.

There is a need to understand the mechanisms of neural synchronization during development because correlated rhythmic activity is thought to be critical for the establishment of proper connectivity. The relative importance of chemical and electrical synapses for synchronization of electrical activity during development is unclear. We examined the activity patterns of identified spinal neurons at the onset of motor activity in zebrafish embryos. Rhythmic activity appeared early and persisted upon blocking chemical neurotransmission but was abolished by inhibitors of gap junctions. Paired recordings revealed that active spinal neurons were electrically coupled and formed a simple network of motoneurons and a subset of interneurons. Thus, the earliest spinal central pattern generator consists of synchronously active, electrically coupled neurons.

Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy.

The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.

From contact to connection: early events during synaptogenesis.

When neuronal processes first come into contact, chemical synapses can form rapidly. Many neurons synthesize synaptic machinery through intrinsic programs before cell-cell interactions. During the formation of chemical synapses, contact with appropriate targets has been found to trigger intracellular signals that induce the assembly of pre-existing synaptic machinery. We propose that 'promiscuous' neurons secrete transmitter before contacting their targets, and form over-abundant synapses, which undergo additional activity-dependent refinement; 'selective' neurons, which retain their original connectivity, require concerted retrograde and anterograde signaling to ensure their correct matching.

Motoneuron activity patterns related to the earliest behavior of the zebrafish embryo.

As a first step in the study of the developing motor circuitry of the embryonic zebrafish spinal cord, we obtained patch-clamp recordings in vivo from identified motoneurons in curarized embryos from the onset of the first motor behavior. At an early developmental stage in which embryos showed slow and repetitive spontaneous contractions of the trunk, motoneurons showed periodic depolarizations that triggered rhythmic bursts of action potentials with a frequency and duration that were consistent with those of the spontaneous contractions. The periodic depolarizations were blocked by tetrodotoxin or Cd(2+). Surprisingly, the contractions and periodic depolarizations were insensitive to general blockade of synaptic transmission (by elevated Mg(2+) and reduced Ca(2+), or by Co(2+)) and to selective blockade of the major neurotransmitter receptors of the mature spinal cord (acetylcholine, GABA(A), NMDA, AMPA/kainate, and glycine). The periodic depolarizations were suppressed by heptanol or by intracellular acidification, treatments that are known to uncouple gap junctions, indicating that electrotonic synapses could underlie the earliest motor behavior. A few hours later, most motoneurons already showed a new pattern of repetitive activity consisting of bursts of glycinergic synaptic events, but these were not necessary for the spontaneous contractions. Transecting the spinal cord at the hindbrain border did not affect the rhythmic activity patterns of the motoneurons. We suggest that spontaneous contractions of the zebrafish embryo are mediated by an early spinal circuit that is independent of the main neurotransmitter systems and descending hindbrain projections that are required for locomotion in the mature vertebrate spinal cord.

Recovery from open channel block by acetylcholine during neuromuscular transmission in zebrafish.

At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recorded in vivo have an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast ("flickering") open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as the experimentally observed slowing of the time course of mEPCs recorded at a hyperpolarized compared with a depolarized potential. Recovery from AChR block is a novel mechanism of synaptic transmission that may contribute in part at all NMJs.

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells.

Na+-ATPase of high-K+ and low-K" sheep red cells was examined with respect to the sidedness of Na+ and K+ effects, using inside-out membrane vesicles and very low ATP concentrations (less than or equal to 2 muM). With varying amounts of Na+ in the medium, i.e., at the cytoplasmic surface, Na+cyt, the activation curves show that high-K+ Na+-ATPase has a higher affinity for Na+cyt compared to low-K+. The apparent affinity for Na+cyt is also increased by increasing the ATP concentrations in high-K+ but now low-K+. With Na+cyt present, Na+-ATPase is stimulated by intravesicular Na+, i.e., Na+ at the originally external surface, Na+cyt, to a greater extent in low-K+ than high-K+. Intravesicular K+ (K+ext) activates Na+-ATPase in high-K+ but not in low-K+ vesicles and extravesicular K+ (K+cyt) inhibits low-K+ but not high-K+ Na+-ATPase. Thus, the genetic difference between high-K+ and low-K+ is expressed as differences in apparent affinities for both Na+ and K+ and these differences are evident at both cytoplasmic and external membrane surfaces.

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.

Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.

Transmitter localization and vesicle turnover at a serotoninergic synapse between identified leech neurons in culture.

An electron microscopic study has been made of chemical synapses that develop between identified nerve cells isolated from the CNS of the leech and maintained in culture. Structures resembling synapses were observed in pairs of Retzius cells and P sensory cells at which chemical transmission had been demonstrated by recording with microelectrodes. Vesicle recycling was shown by following the uptake of extracellular markers after stimulation. The membrane separation between the presynaptic Retzius cell (which is known to liberate serotonin) and the postsynaptic P cell was wider in synaptic than in extrasynaptic regions. The Retzius cell contained clusters of clear vesicles apposed to thickenings of the presynaptic membrane. These clear vesicle clusters were capped by a layer of dense core vesicles that did not contact the presynaptic membrane thickenings. Subsynaptic cisternae were found in the postsynaptic cell opposite the presynaptic membrane thickenings. Occasional slight postsynaptic membrane thickenings were seen. Extracellular material was observed within the synaptic cleft. Similar synaptic structures developed between pairs of Retzius cells in culture; even a single Retzius cell was able to form autapses upon itself. Structures resembling transmitter release sites were found in Retzius cells at a distance from any postsynaptic membranes. These are presumed to be locations for the diffuse release of transmitter. Presynaptic structures resembling release sites were never observed in P cells apposed to Retzius cells. Antibody to serotonin (5-HT) labelled with colloidal gold showed serotonin to be localized in the dense core vesicles in Retzius cells. Stimulation of pairs of Retzius and P cells by raised concentrations of K+ resulted in uptake of extracellular markers. Only Retzius cells became labelled. Ferritin was found in cisternae, in dense core vesicles, and in clear vesicles. HRP was found in cisternae and in clear vesicles. Colloidal gold was taken up by coated vesicles and was occasionally found in both clear and dense core vesicles. The uptake of extracellular markers following stimulation was blocked by high Mg++. These results show that structures develop between pairs of cells at which chemical transmission develops and that transmitter release leads to turnover of dense core and clear vesicles.

Targeted "knockdown" of channel expression in vivo with an antisense morpholino oligonucleotide.

We have examined whether antisense morpholino oligonucleotides (morpholinos) can be used as a tool to suppress or "knockdown" the expression of ion channels during development of the zebrafish. Because the acetylcholine receptor channel is well characterized in zebrafish and is abundant as skeletal muscle is found throughout the body, we sought to knock down its expression as a general test of the feasibility of this approach. A 25-mer morpholino was designed to target the 5' region of the cloned alpha-subunit and was injected into early stage blastulae in order to trap it in all developing cells. From the time of hatching (early on the third day of development) and for a few days after, a fraction of the injected embryos were immobile, i.e. were "morphant". Injection of blastulae without the morpholino or with a control morpholino containing four mispaired bases did not affect the embryos. Although the morphant embryos were generally normal in appearance, they lacked staining with alpha-bungarotoxin or an alpha-subunit-specific monoclonal antibody. In whole muscle cell recordings from morphant embryos, miniature end-plate potentials were undetectable in many of the cells and in most they had a slower, immature time course. These results are consistent with a greatly reduced, dysfunctional level of expression of acetylcholine receptors in morphant embryos. Because of their stability and specificity, morpholinos should prove useful for targeted deletion of transmitter receptors and channels in developing zebrafish and possibly in other preparations.

Cell-specific contact selects transmitter responses in an identified leech neuron.

Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.

Voltage dependencies of the fast and slow gating modes of RIIA sodium channels.

Rat brain IIA sodium channel alpha-subunits were expressed in Xenopus oocytes, and the sodium currents were measured by intracellular voltage clamping with large agarose-tipped electrodes and by excised membrane patch-clamp recording to separate and characterize the properties of the fast and slow channel gating modes. The currents showed biexponential inactivation properties with fast and slow phases that could be isolated as distinct gating modes through differences in their inactivation properties. At holding potentials more negative than -55 mV, fast mode currents inactivated within a few milliseconds of depolarization, and could be distinguished by their rapid recovery from inactivation. Single sodium channels in the fast mode opened early after depolarization and rarely showed re-openings. At holding potentials positive to -55 mV, fast mode currents were inactivated, revealing slow mode currents which had slower activation and inactivation kinetics and showed sustained single channel activity during depolarizing pulses. The steady-state voltage dependencies of fast and slow mode activation were very similar. In contrast, slow mode inactivation occurred at potentials 27 mV more positive than fast mode inactivation. The slow mode appears to be due to destabilization of a voltage-insensitive conformation of the channel. The fast gating process dominated at high current levels, perhaps due to alpha-subunit interactions.

Signalling synapse formation between identified neurons.

We have investigated the signals between identified leech neurons during the formation of specific synapses in culture. At an inhibitory serotonergic synapse between two well-studied neurons, the postsynaptic cell has an additional (extrasynaptic) excitatory response to 5-HT which may underly a form of activity-dependent modulation. Thus, the presynaptic neuron must select which 5-HT response will be activated and which will be excluded at its synapses. The selection of these responses preceded synapse formation and was specifically induced at sites of contact with the presynaptic neuron, this not being observed for other cell pairings. Aldehyde-fixed presynaptic cells were equally effective, unless pre-treated with trypsin or wheat germ agglutinin, suggesting that contact with a specific cell-surface glycoprotein induced this physiological change in 5-HT sensitivity. The mechanism underlying the selective loss of the extrasynaptic response has been examined by single channel recording. Cation channels in the postsynaptic neuron were modulated by protein kinase C (PKC) upon binding of 5-HT to a 5-HT2 receptor. However, at sites of contact with the presynaptic neuron, the channels were no longer sensitive to PKC. Furthermore, when cation channels from uncontacted neurons were inserted or 'crammed' into contacted neurons, they were rapidly rendered insensitive to PKC, demonstrating a cytoplasmic signal for the uncoupling of channel modulation. Interestingly, the cytoplasm of contacted postsynaptic neurons showed immunoreactivity for tyrosine phosphorylation: exposure of the neurons to specific inhibitors of tyrosine kinases prevented tyrosine phosphorylation, the loss of cation channel modulation and synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term storage of functional, isolated nerve endings by slow freezing and rapid thawing.

Nerve endings (synaptosomes) were isolated from homogenized rat brain corpora striata following centrifugation on discontinuous sucrose gradients. The synaptosomes (in 0.8 M sucrose) were (i) slowly frozen by placing the tube containing the suspension in a freezer at -10 degrees C for 1 h followed by (ii) swirling in a mixture of acetone and dry ice for 15 min and (iii) were stored in liquid nitrogen for up to 6 weeks. Freshly isolated synaptosomes and synaptosomes from the same preparation that were frozen for 2, 4, or 6 weeks and rapidly thawed in a water bath at 37 degrees C were re-equilibrated with a physiological salt solution and assayed for their ability to accumulate Ca and to release transmitter (dopamine) upon depolarization in high K medium. K-dependent Ca uptake gradually declined to approximately 1/3 the value observed with freshly isolated synaptosomes after 6 weeks of storage. K-stimulated dopamine release (only from intact synaptosomes) was normal over the entire period of storage. It is concluded that synaptosomes retain their physiological properties when stored frozen for a few weeks and that cold storage may be a useful technique for experiments requiring lengthy or repeated assays or accumulation of material.

Generating a phage display antibody library against an identified neuron.

The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.

In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish.

The zebrafish is a popular model for developmental studies due to its accessibility by cellular, molecular and genetic approaches. As a complement to these other methods, we have devised an exposed hindbrain/spinal cord preparation in the curarized zebrafish embryo and larva that permits intracellular labeling and patch clamp recording from individually identified sensory neurons, motoneurons and interneurons in vivo. Regular bursts of synaptic potentials and action potentials were observed under whole-cell current clamp in embryonic motoneurons and in some identified interneurons. Larval neurons showed prolonged depolarizations with synaptically driven bursts of action potentials. Frequent spontaneous synaptic potentials were observed and synaptic currents were effectively space clamped. It is thus feasible to study in vivo the properties of identifiable neurons of the developing locomotor network in the zebrafish, including their synaptic activity, firing patterns and interconnections.