Abnormal larval neuromuscular junction morphology and physiology in Drosophila Prickle isoform mutants with defective axonal transport and adult seizure behavior

Previous studies have demonstrated that mutations of the Drosophila planar cell polarity gene prickle (pk) result in altered microtubule-mediated vesicular transport in larval motor axons, as well as adult neuronal circuit hyperexcitability and epileptic behavior. It is also known that mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms differ in phenotype but display isoform counterbalancing effects in heteroallelic pkpk/pksple flies to ameliorate adult motor circuit and behavioral hyperexcitability. We have further investigated the larval neuromuscular junction (NMJ) and uncovered robust phenotypes in both pkpk and pksple alleles (heretofore referred to as pk and sple alleles, respectively), including synaptic terminal overgrowth, as well as irregular motor axon terminal excitability, poor vesicle release synchronicity, and altered efficacy of synaptic transmission. We observed significant increase in whole-cell excitatory junctional potential (EJP) in pk homozygotes, which was restored to near WT level in pk/sple heterozygotes. We further examined motor terminal excitability sustained by presynaptic Ca2+ channels, under the condition of pharmacological blockade of Na+ and K+ channel function. Such manipulation revealed extreme Ca2+ channel-dependent nerve terminal excitability in both pk and sple mutants. However, when combined in pk/sple heterozygotes, such terminal hyper-excitability was restored to nearly normal. Focal recording from individual synaptic boutons revealed asynchronous vesicle release in both pk and sple homozygotes, which nevertheless persisted in pk/sple heterozygotes without indications of isoform counter-balancing effects. Similarly, the overgrowth at NMJs was not compensated in pk/sple heterozygotes, exhibiting an extremity comparable to that in pk and sple homozygotes. Our observations uncovered differential roles of the pk and sple isoforms and their distinct interactions in the various structural and functional aspects of the larval NMJ and adult neural circuits.

Pre-synaptic inhibition of afferent feedback in the macaque spinal cord does not modulate with cycles of peripheral oscillations around 10 Hz

Spinal interneurons are partially phase-locked to physiological tremor around 10Hz. The phase of spinal interneuron activity is approximately opposite to descending drive to motoneurons, leading to partial phase cancellation and tremor reduction. Pre-synaptic inhibition of afferent feedback modulates during voluntary movements, but it is not known whether it tracks more rapid fluctuations in motor output such as during tremor. In this study, dorsal root potentials (DRPs) were recorded from the C8 and T1 roots in two macaque monkeys following intra-spinal micro-stimulation (random inter-stimulus interval 1.5-2.5 s, 30-100?A), whilst the animals performed an index finger flexion task which elicited peripheral oscillations around 10Hz. Forty one responses were identified with latency <5ms; these were narrow (mean width 0.59 ms), and likely resulted from antidromic activation of afferents following stimulation near terminals. Significant modulation during task performance occurred in 16/41 responses, reflecting terminal excitability changes generated by pre-synaptic inhibition (Wall?s excitability test). Stimuli falling during large-amplitude 8-12Hz oscillations in finger acceleration were extracted, and sub-averages of DRPs constructed for stimuli delivered at different oscillation phases. Although some apparent phase-dependent modulation was seen, this was not above the level expected by chance. We conclude that although terminal excitability reflecting pre-synaptic inhibition of afferents modulates over the timescale of a voluntary movement, it does not follow more rapid changes in motor output. This suggests that pre-synaptic inhibition is not part of the spinal systems for tremor reduction described previously, and that it plays a role in overall ? but not moment-by-moment ? regulation of feedback gain.

Presynaptic Current Changes at the Mossy Fiber–Granule Cell Synapse of Cerebellum During LTP

The involvement of presynaptic mechanisms in the expression of long-term potentiation (LTP), an enhancement of synaptic transmission suggested to take part in learning and memory in the mammalian brain, has not been fully clarified. Although evidence for enhanced vesicle cycling has been reported, it is unknown whether presynaptic terminal excitability could change as has been observed in invertebrate synapses. To address this question, we performed extracellular focal recordings in cerebellar slices. The extracellular current consisted of a pre- (P1/N1) and postsynaptic (N2/SN) component. In ∼50% of cases, N1 could be subdivided into N1a and N1b. Whereas N1a was part of the fiber volley (P1/N1a), N1b corresponded to a Ca2+-dependent component accounting for 40–50% of N1, which could be isolated from individual mossy fiber terminals visualized with fast tetramethylindocarbocyanine perchlorate (DiI). The postsynaptic response, given its timing and sensitivity to glutamate receptor antagonists [N2 was blocked by 10 μM [1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) and SN by 100 μM APV +50 μM 7-Cl-kyn], corresponded to granule cell excitation. N2 and SN could be reduced by 1) Ca2+ channel blockers, 2) decreasing the Ca2+ to Mg2+ ratio, 3) paired-pulse stimulation, and 4) adenosine receptor activation. However, only the first two manipulations, which modify Ca2+ influx, were associated with N1(or N1b) reduction. LTP was induced by θ-burst mossy fiber stimulation (8 trains of 10 impulses at 100 Hz separated by 150-ms pauses). Interestingly, during LTP, both N1 (or N1b) and N2/SN persistently increased, whereas P1 (or P1/N1a) did not change. Average changes were N1 = 38.1 ± 31.9, N2 = 49.6 ± 48.8, and SN = 59.1 ± 35.5%. The presynaptic changes were not observed when LTP was prevented by synaptic inhibition, by N-methyl-d-aspartate and metabotropic glutamate receptor blockage, or by protein kinase C blockage. Moreover, the presynaptic changes were sensitive to Ca2+channel blockers (1 mM Ni2+ and 5 μM ω-CTx-MVIIC) and occluded by K+ channel blockers (1 mM tetraethylammmonium). Thus regulation of presynaptic terminal excitability may take part in LTP expression at a central mammalian synapse.

Na+/Ca2+ Exchanger in GABAergic Presynaptic Boutons of Rat Central Neurons

Rat Meynert neurons were acutely isolated using a dissociation technique that maintains functional GABAergic presynaptic boutons. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded under voltage-clamp conditions using whole cell patch-clamp recordings. Using the frequency of mIPSCs as a measure of presynaptic terminal excitability, the existence of a Na+/Ca2+ exchanger (NCX) in these GABAergic nerve terminals was clearly demonstrated. Both the frequency and the amplitude of mIPSCs were unaffected by replacement of extracellular Na2+. However, in this Na+-free external solution, ouabain could now induce a transient increase of mIPSCs frequency, which was not inhibited by adding Cd2+ or cyclopiazonic acid but was inhibited by removing external Ca2+. This indicates that this transient potentiation was dependent on external Ca2+, but that this Ca2+influx was not via voltage-dependent Ca2+channels. KB-R7943, an inhibitor of NCX, at a concentration of 3 × 10−6 M, reduced this transient increase of mIPSCs frequency without affecting mIPSCs amplitude and the response to exogenous GABA. These results demonstrate the existence of NCX in these GABAergic nerve terminals. In zero external Na+, ouabain causes an accumulation of intraterminal Na+ and a resultant influx of Ca2+ through the reversed mode operation of NCX. However, under more physiological conditions, NCX may also operate in a forward mode and serve to maintain low intracellular [Ca2+] in nerve terminals.