Christopher Kushmerick

Mice Deficient for the Vesicular Acetylcholine Transporter Are Myasthenic and Have Deficits in Object and Social Recognition

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

Physiological Temperatures Reduce the Rate of Vesicle Pool Depletion and Short-Term Depression via an Acceleration of Vesicle Recruitment

The timing and strength of synaptic transmission is profoundly dependent on temperature. However, the temperature dependence of the multiple mechanisms that contribute to short-term synaptic plasticity is poorly understood. Here, we use voltage-clamp recordings to quantify the temperature dependence of exocytosis at the calyx of Held synapse. EPSC and miniature EPSC amplitudes were larger at physiological temperature, but quantal content during low-frequency (0.05 Hz) stimulation was constant after temperature jumps from 22–24°C to 35–37°C. The initial degree of EPSC depression during 100 Hz stimuli trains was unchanged with temperature, as were estimates of release probability and vesicle pool size. In contrast, physiological temperatures dramatically relieved depression measured after 40 stimuli at 100 Hz by increasing twofold the rate of recovery from depression. Presynaptic calyx recordings revealed that physiological temperature increased capacitance jumps resulting from 0.5 and 1 ms depolarizations by increasing Ca2+ influx. When Ca2+ entry was equalized at the two temperatures, exocytosis exhibited little temperature dependence for brief depolarizations. However, in response to longer depolarizations, raising temperature increased a slow phase of exocytosis, without affecting overall Ca2+ entry or the size of the readily releasable pool of vesicles. Higher temperatures also increased the rate of presynaptic Ca2+ current inactivation; nevertheless, the degree of steady-state EPSC depression was greatly reduced. Our results thus suggest that changes in steady-state EPSCs during stimulus trains at physiological temperature reflect larger quantal amplitudes and faster refilling of synaptic vesicle pools, leading to reduced short-term depression during prolonged high-frequency firing.

Reliability and Precision of the Mouse Calyx of Held Synapse

Traditionally, the calyx of Held synapse is viewed as a highly reliable relay in the sound localization circuit of the auditory brainstem, with every presynaptic action potential triggering a postsynaptic action potential in vivo. However, this view is at odds with slice recordings that report large short-term depression (STD). To investigate the reliability and precision of this synapse, we compared slice and in vivo recordings from medial nucleus of the trapezoid body neurons of young adult mice. We show that the extracellularly recorded complex waveform can be used to estimate both presynaptic release and postsynaptic excitability. Whereas under standard slice conditions the synapse underwent large STD, both extracellular and whole-cell recordings indicated that in vivo the size of the EPSPs was independent of recent history. The estimated quantal content was typically <20 in vivo, much lower than in the resting synapse under standard slice conditions. However, due to the large quantal size and summation of EPSPs, the safety factor of this synapse was generally still sufficiently large and postsynaptic failures were observed only infrequently in vivo. When present, failures were typically due to stochastic fluctuations in EPSP size or postsynaptic spike depression. In vivo, the calyx of Held synapse thus functions as a tonic synapse. The price it pays for its low release probability is an increase in jitter and synaptic latency and occasional postsynaptic failures.

Retroinhibition of Presynaptic Ca2+ Currents by Endocannabinoids Released via Postsynaptic mGluR Activation at a Calyx Synapse

We investigated the mechanisms by which activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) leads to inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem. In ∼50% of the MNTB neurons tested, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent and reduced paired-pulse depression, suggestive of an inhibition of glutamate release. Presynaptic voltage-clamp experiments revealed a reversible reduction of Ca2+ currents by DHPG, with no significant modification of the presynaptic action potential waveform. Likewise, in ∼50% of the tested cells, the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN) reversibly inhibited EPSCs, presynaptic Ca2+ currents, and exocytosis. For a given cell, the amount of inhibition by DHPG correlated with that by WIN. Moreover, the inhibitory action of DHPG was blocked by the CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and occluded by WIN, indicating that DHPG and WIN operate via a common pathway. The inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mm BAPTA into the postsynaptic cell, suggesting that DHPG activated postsynaptic mGluRs. Light and electron microscopy immunolabeling indicated a presynaptic expression of CB1Rs and postsynaptic localization of mGluR1a. Our data suggest that activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+ current and glutamate release.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

ABSTRACT The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

Functional and structural features of γ-zeathionins, a new class of sodium channel blockers

γ1‐ and γ2‐zeathionins (γ1‐Z and γ2‐Z) are members of a family of small and basic peptides involved in plant protection. These plant defensins exhibit remarkable structural similarity to scorpion neurotoxins and insect defensins. In the present report, we used the whole‐cell patch clamp technique to investigate the inhibition of the sodium current (I Na) by γ1‐Z and γ2‐Z in the GH3 cell line. Both γ1‐Z and γ2‐Z rapidly and reversibly inhibited I Na without changing the kinetics or voltage dependence of activation or inactivation. To our knowledge, this is the first example of a plant protein that inhibits the sodium channel. From structural comparisons with the μ‐conotoxins, a family of peptides that block the sodium channel, we detected some similar features that could provide the basis of inhibition of sodium channels by γ‐zeathionins.

Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells.

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

Presynaptic Resurgent Na+ Currents Sculpt the Action Potential Waveform and Increase Firing Reliability at a CNS Nerve Terminal

Axonal and nerve terminal action potentials often display a depolarizing after potential (DAP). However, the underlying mechanism that generates the DAP, and its impact on firing patterns, are poorly understood at axon terminals. Here, we find that at calyx of Held nerve terminals in the rat auditory brainstem the DAP is blocked by low doses of externally applied TTX or by the internal dialysis of low doses of lidocaine analog QX-314. The DAP is thus generated by a voltage-dependent Na+ conductance present after the action potential spike. Voltage-clamp recordings from the calyx terminal revealed the expression of a resurgent Na+ current (INaR), the amplitude of which increased during early postnatal development. The calyx of Held also expresses a persistent Na+ current (INaP), but measurements of calyx INaP together with computer modeling indicate that the fast deactivation time constant of INaP minimizes its contribution to the DAP. INaP is thus neither sufficient nor necessary to generate the calyx DAP, whereas INaR by itself can generate a prominent DAP. Dialysis of a small peptide fragment of the auxiliary β4 Na+ channel subunit into immature calyces (postnatal day 5–6) induced an increase in INaR and a larger DAP amplitude, and enhanced the spike-firing precision and reliability of the calyx terminal. Our results thus suggest that an increase of INaR during postnatal synaptic maturation is a critical feature that promotes precise and resilient high-frequency firing.

Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior.

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to “turn down” neuronal circuits controlling locomotion.

Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter

J. Neurochem. (2010) 113, 943–951.