Timothy A. Benke

NKCC1 transporter facilitates seizures in the developing brain

During development, activation of Cl−-permeable GABAA receptors (GABAA-R) excites neurons as a result of elevated intracellular Cl− levels and a depolarized Cl− equilibrium potential (ECl). GABA becomes inhibitory as net outward neuronal transport of Cl− develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na+-K+-2Cl− cotransporter (NKCC1) facilitates the accumulation of Cl− in neurons. The NKCC1 blocker bumetanide shifted ECl negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABAA-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl−-extruding transporter (KCC2) in human and rat cortex showed that Cl− transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

Modulation of AMPA receptor unitary conductance by synaptic activity

Activity-dependent alteration in synaptic strength is a fundamental property of the vertebrate central nervous system and is thought to underlie learning and memory. The most extensively studied model of activity-dependent synaptic plasticity is long-term potentiation (LTP) of glutamate-responsive (glutamatergic) synapses, a widespread phenomenon involving multiple mechanisms. The best characterized form of LTP occurs in the CA1 region of the hippocampus, in which LTP is initiated by transient activation of NMDA (N-methyl-D-aspartate) receptors and is expressed as a persistent increase in synaptic transmission through AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. This increase is due, at least in part, to a postsynaptic modification of AMPA-receptor function; this modification could be caused by an increase in the number of receptors, their open probability, their kinetics or their single-channel conductance. Here we show that the induction of LTP in the CA1 region of the hippocampus is often associated with an increase in single-channel conductance of AMPA receptors. This shows that elementary channel properties can be rapidly modified by synaptic activity and provides an insight into one molecular mechanism by which glutamatergic synapses can alter their strength.

Hippocampal LTD Expression Involves a Pool of AMPARs Regulated by the NSF–GluR2 Interaction

We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinsons disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

A single episode of neonatal seizures permanently alters glutamatergic synapses.

The contribution of seizures to cognitive changes remains controversial. We tested the hypothesis that a single episode of neonatal seizures (sNS) on rat postnatal day (P) 7 permanently impairs hippocampal‐dependent function in mature (P60) rats because of long‐lasting changes at the synaptic level.

Auditory Hair Cell-Afferent Fiber Synapses Are Specialized to Operate at Their Best Frequencies

Auditory afferent fiber activity is driven by high-fidelity information transfer from the sensory hair cell. Presynaptic specializations, posited to maintain fidelity, are investigated at synapses with characteristic frequencies of 120 Hz and 320 Hz. Morphological data indicate that high-frequency cells have more synapses and higher vesicle density near dense bodies (DBs). Tracking vesicular release via capacitance changes identified three overlapping kinetic components of release corresponding to morphologically identified vesicle pools. High-frequency cells released faster; however, when normalized to release site number, low-frequency cells released faster, likely due to a greater Ca2+ load per synapse. The Ca(2+)-dependence of release was nonsaturating and independent of frequency, suggesting that release, not refilling, was rate limiting. A model of release derived from vesicle equilibration between morphologically defined pools reproduced the capacitance data, supporting a critical role in vesicle trafficking for DBs. The model suggests that presynaptic specializations enable synapses to operate most efficiently at their characteristic frequencies.

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells

1 The actions of the uncompetitive N‐methyl‐d‐aspartate (NMDA) receptor antagonists, memantine (1‐amino‐3,5‐dimethyladamantane) and (+)‐MK‐801 ((+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole‐cell patch clamp technique. 2 Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) was cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3 Memantine (0.3–30 μm) blocked L‐glutamate (100 μm)‐mediated currents in a concentration‐dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at −70 mV) of 0.93±0.15 μm, 0.82±0.12 μm and 0.47±0.06 μm (mean±s.e. mean), respectively. 4 The memantine‐induced block was strongly voltage‐dependent. Alteration of the holding potential from −70 mV to +60 mV resulted in an e‐fold increase in the IC50 values per 30–33 mV change in membrane potential, for all 3 subunit combinations investigated. 5 The kinetics of the actions of memantine (30 μm) were investigated for the NR1a/2A combination, in 6 cells (13–15 determinations). At −70 mV, the block and recovery from block were both best described by two exponentials with time‐constants of 201±23 ms (81±2%) and 3.9±0.6 s and 597±94 ms (18±1%) and 18.6±2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time‐constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6 (+)‐MK‐801 was studied briefly for comparative purposes. (+)‐MK‐801 (200 nM) preferentially blocked NMDA receptor currents (at −70 mV) in NR1a/NR2A and NR1a/NR2B (82±10% and 93±2% depressions) compared to NR1a/NR2D (38±7%) transfected cells. (+)‐MK‐801 appeared to be less voltage‐dependent than memantine on all three receptor combinations. 7 In conclusion, memantine was a voltage‐dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.

Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity

BackgroundKnowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP) and its counterpart long-term depression (LTD) of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP.ResultsWe find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism.ConclusionsThese data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation.

Antagonism of NMDA receptors as a potential treatment for Down syndrome: a pilot randomized controlled trial

Down syndrome (DS) is the most common genetic cause of intellectual disability. The N-methyl-D-aspartate (NMDA) receptor uncompetitive antagonist, memantine hydrochloride (memantine), has been shown to improve learning/memory and rescue one form of hippocampus synaptic plasticity dysfunction in the best-studied mouse model of DS available, the Ts65Dn mouse. Given the status of memantine as a treatment for Alzheimers disease (AD) approved by the Food and Drug Administration, the preclinical evidence of potential efficacy in Ts65Dn mice, and the favorable safety profile of memantine, we designed a study to investigate whether the findings in the mouse model could be translated to individuals with DS. In this pilot, proof-of-principle study we hypothesized that memantine therapy would improve test scores of young adults with DS on measures of episodic and spatial memory, which are generally considered to be hippocampus dependent. Accordingly, in this randomized, double-blind, placebo-controlled trial, we compared the effect of 16-week treatment with either memantine or placebo on cognitive and adaptive functions of 40 young adults with DS using a carefully selected set of neuropsychological outcome measures. Safety and tolerability were also monitored. Although no significant differences were observed between the memantine and placebo groups on the two primary outcome measures, we found a significant improvement in the memantine group in one of the secondary measures associated with the primary hypothesis. Only infrequent and mild adverse events were noted.

The Wada test in Austrian, Dutch, German, and Swiss epilepsy centers from 2000 to 2005: a review of 1421 procedures

Twenty-six Austrian, Dutch, German, and Swiss epilepsy centers were asked to report on use of the Wada test (intracarotid amobarbital procedure, IAP) from 2000 to 2005 and to give their opinion regarding its role in the presurgical diagnosis of epilepsy. Sixteen of the 23 centers providing information had performed 1421 Wada tests, predominantly the classic bilateral procedure (73%). A slight nonsignificant decrease over time in Wada test frequency, despite slightly increasing numbers of resective procedures, could be observed. Complication rates were relatively low (1.09%; 0.36% with permanent deficit). Test protocols were similar even though no universal standard protocol exists. Clinicians rated the Wada test as having good reliability and validity for language determination, whereas they questioned its reliability and validity for memory lateralization. Several noninvasive functional imaging techniques are already in use. However, clinicians currently do not want to rely solely on noninvasive functional imaging in all patients.