Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.

Heterozygous mutation or deletion of the beta subunit of platelet-activating factor acetylhydrolase (PAFAH1B1, also known as LIS1) in humans is associated with type I lissencephaly, a severe developmental brain disorder thought to result from abnormal neuronal migration. To further understand the function of PAFAH1B1, we produced three different mutant alleles in mouse Pafah1b1. Homozygous null mice die early in embryogenesis soon after implantation. Mice with one inactive allele display cortical, hippocampal and olfactory bulb disorganization resulting from delayed neuronal migration by a cell-autonomous neuronal pathway. Mice with further reduction of Pafah1b1 activity display more severe brain disorganization as well as cerebellar defects. Our results demonstrate an essential, dosage-sensitive neuronal-specific role for Pafah1b1 in neuronal migration throughout the brain, and an essential role in early embryonic development. The phenotypes observed are distinct from those of other mouse mutants with neuronal migration defects, suggesting that Pafah1b1 participates in a novel pathway for neuronal migration.

Hippocampal abnormalities and enhanced excitability in a murine model of human lissencephaly.

Human cortical heterotopia and neuronal migration disorders result in epilepsy; however, the precise mechanisms remain elusive. Here we demonstrate severe neuronal dysplasia and heterotopia throughout the granule cell and pyramidal cell layers of mice containing a heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birth-dating analysis using bromodeoxyuridine revealed that neurons in Lis1+/- murine hippocampus are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice were stunted and possessed fewer dendritic branches, whereas dentate granule cells were hypertrophic and formed spiny basilar dendrites from which the principal axon emerged. Both somatostatin- and parvalbumin-containing inhibitory neurons were heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission were severely disrupted, revealing hyperexcitability at Schaffer collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition, the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission was less than that of wild type. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K(+)](o) model of epilepsy. In Lis1+/- hippocampus, intense interictal bursting was observed on elevation of extracellular potassium to 6.5 mM, a condition that resulted in only minimal bursting in wild type. These anatomical and physiological hippocampal defects may provide a neuronal basis for seizures associated with lissencephaly.

Synthesis and biological evaluation of 18-methoxycoronaridine congeners. Potential antiaddiction agents.

Variation of the methoxycarbonyl and C-18 substituents of the antiaddictive compound 18-methoxycoronaridine, and contraction of its isoquinuclidine ring segment, provided 15 congeners for SAR evaluation at opioid and alpha3beta4 nicotinic acetylcholine receptors. The opioid activities were relatively low, and the alpha3beta4 nicotinic acetylcholine receptor activities were found to correlate with in vivo antiaddictive activities.

Release of D,L-threo-beta-hydroxyaspartate as a false transmitter from excitatory amino acid-releasing nerve terminals.

This study examined whether preaccumulated D,L-threo-beta-hydroxyaspartate (tHA), a competitive substrate for the high-affinity excitatory amino acid (EAA) transporter, is released as a false transmitter from EAA-releasing nerve terminals. Potassium-stimulation (50 mM for 1 min) evoked significant release of the endogenous EAAs (aspartate and glutamate) from superfused neocortical minislices. Endogenous EAA release was largely calcium-dependent and was inhibited by tetanus toxin, a neurotoxin which specifically blocks vesicular exocytosis. In parallel experiments, minislices were pre-incubated with 500 microM tHA. Potassium (50 mM) evoked significant release of tHA and this release was also calcium-dependent and reduced by tetanus toxin. Pre-accumulation of tHA did not affect the release of endogenous glutamate whereas the release of endogenous aspartate was significantly attenuated. These data suggest that tHA selectively accumulates in a vesicular aspartate pool and is released upon depolarization as a false transmitter from EAA nerve terminals.

Synaptosomal and vesicular accumulation of L-glutamate, L-aspartate and D-aspartate.

We examined the vesicular accumulation of the excitatory amino-acid (EAA) neurotransmitters, L-glutamate and L-aspartate, together with the non-metabolisable EAA analogue D-aspartate. Synaptosomes derived from whole brain were incubated in various concentrations of [3H]-amino acids under conditions to facilitate vesicular turnover. Synaptosomes were then lysed in hypotonic medium and vesicles immunoprecipitated with monoclonal anti-synaptophysin antibodies coupled to sepharose beads. Using this method, saturable vesicular accumulation was observed for [3H]-L-glutamate, [3H]-L-aspartate, and [3H]-D-aspartate but not for the excitatory amino acid receptor ligands [3H]-AMPA or [3H]-kainate. Vesicular accumulation (t(1/2)=7.45 min) was markedly slower than synaptosomal accumulation (t(1/2)=1.03 min) and was substantially reduced at 4 degrees C. Maximal accumulation of [3H]-L-glutamate, [3H]-L-aspartate, and [3H]-D-aspartate was estimated to be 98, 68, and 112 pmol/mg of synaptosomal protein, respectively, and uptake affinities 1.6, 3.4, and 2.1 mM, respectively. Maximal accumulation of [3H]-L-glutamate was non-competitively inhibited by both 100 microM unlabeled L-aspartate and 100 microM D-aspartate, suggesting that all are accumulated into a common vesicular pool by different transporters.

Functional nicotinic acetylcholine receptor expression on stem and progenitor cells of the early embryonic nervous system.

Although the adult brain contains nicotinic acetylcholine (ACh) receptors vital to cortical function, little is known about the assembly of embryonic receptor subunits into functional receptors or their role in fetal brain development. We now report the first evidence of functional nicotinic ACh receptors on stem and progenitor cells of fetal mouse cerebral cortex as early as embryonic day 10.

Potassium-induced long-term potentiation in rat hippocampal slices.

We observed that a transient increase in extracellular potassium concentration (50 mM for 40 s) was sufficient to induce long-term potentiation (LTP) of synaptic transmission in area CA1 of the hippocampal slice. Potassium-induced potentiation of the Schaffer collateral/commissural synapses demonstrated several features characteristic of tetanus-induced LTP: (1) population excitatory post-synaptic potential (EPSP) amplitudes were enhanced to a similar magnitude (on average 70% above baseline) which (2) lasted for more than 20 min; (3) induction was blocked by bath application of the specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV), and (4) was attenuated by reduction of the concentration of calcium in the extracellular medium. Induction of either potassium-induced LTP or tetanus-induced LTP occluded the subsequent expression of the other. Finally, exposure to high potassium in the absence of electrical stimulation was sufficient to induce LTP. Taken together, these data indicate that brief depolarizing stimuli other than tetanus can induce LTP. Because potassium-induced LTP is not restricted to the subset of afferents examined electrophysiologically, such a method could facilitate analyses of the biochemical events underlying both the induction and expression of LTP.

Ethanol alters calcium signaling in axonal growth cones.

Calcium (Ca2+) channels are sensitive to ethanol and Ca2+ signaling is a critical regulator of axonal growth and guidance. Effects of acute and chronic exposure to ethanol (22, 43, or 87 mM) on voltage-gated Ca2+ channels (VGCCs) in whole cells, and KCl-induced Ca2+ transients in axonal growth cones, were examined using dissociated hippocampal cultures. Whole-cell patch-clamp analysis in neurons with newly-formed axons (Stage 3) revealed that rapidly inactivating, low-voltage activated (LVA) and non-inactivating, high-voltage activated (HVA) currents were both inhibited in a dose-dependent manner by acute ethanol, with relatively greater inhibition of HVA currents. When assessed by Fluo-4-AM imaging, baseline fluorescence and Ca2+ response to ethanol in Stage 3 neurons was similar compared to neurons without axons, but peak Ca2+ transient amplitudes in response to bath-applied KCl were greater in Stage 3 neurons and were decreased by acute ethanol. The amplitude of Ca2+ transients elicited specifically in axonal growth cones by focal application of KCl was also inhibited by acute exposure to moderate-to-high concentrations of ethanol (43 or 87 mM), whereas a lower concentration (22 mM) had no effect. When 43 or 87 mM ethanol was present continuously in the medium, KCl-evoked Ca2+ transient amplitudes were also reduced in growth cones. In contrast, Ca2+ transients were increased by continuous exposure to 22 mM ethanol. Visualization using a fluorescent dihydropyridine analog revealed that neurons continuously exposed to ethanol expressed increased amounts of L-type Ca2+ channels, with greater increases in axonal growth cones than cell bodies. Thus, acute ethanol reduces Ca2+ current and KCl-induced Ca2+ responses in whole cells and axonal growth cones, respectively, and chronic exposure is also generally inhibitory despite apparent up-regulation of L-type channel expression. These results are consistent with a role for altered growth cone Ca2+ signaling in abnormal neuromorphogenesis associated with fetal alcohol spectrum disorders.

AMPA receptor flip/flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate.

AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by cyclothiazide, were analyzed for modulation of deactivation and desensitization by cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3-M4 extracellular loop.

Aspartate and glutamate mediate excitatory synaptic transmission in area CA1 of the hippocampus.

We examined whether L-aspartate (ASP) and L-glutamate (GLU) both function as endogenous neurotransmitters in area CA1 of the rat hippocampus. Radioligand displacement experiments using 3H-DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA) to label AMPA/kainate receptors and 3H-cis-4-phosphonomethyl-2-piperidine carboxylic acid (3H-CGS-19755) to label NMDA receptors confirmed that GLU (Ki approximately 500 nM) but not ASP (Ki > 1 mM) has high affinity for AMPA/kainate receptors whereas GLU (Ki approximately 250 nM) and ASP (Ki approximately 1.3 microM) both have high affinity for NMDA receptors. Elevating extracellular potassium concentration (50 mM, 1 min) evoked the calcium-dependent release of both ASP (approximately 50% increase) and GLU (approximately 200% increase) from hippocampal slices and from minislices of area CA1. Reducing extracellular glucose concentration (0.2 mM) reduced GLU release, enhanced ASP release, and reduced AMPA/kainate receptor-mediated responses more than NMDA receptor-mediated responses (to 7% and 34% of control, respectively). Fiber volleys, antidromic population spikes, membrane potential, input resistance, and ATP content all were not affected by glucose reduction. Unlike low glucose, the inhibitory neuromodulator adenosine (5 microM), which reduces ASP and GLU release to a similar extent, reduced AMPA/kainate and NMDA receptor-mediated population EPSPs similarly (to 11% and 12% of control, respectively). AMPA/kainate and NMDA receptor-mediated population EPSPs were also similarly reduced by 0.4 microM TTX (to 32% and 22% of control, respectively) and similarly enhanced by 10 microM 4-aminopyridine (to 206% and 248% of control, respectively). Finally, NMDA receptor-mediated EPSCs measured by whole-cell recording decayed faster in low glucose (73 msec vs 54 msec) but not in adenosine (73 msec vs 78 msec). Together, these results confirm that ASP and GLU are both involved in excitatory synaptic transmission at the Schaffer collateral-commissural terminals in area CA1 of the rat hippocampus.

AMPA receptor heterogeneity in rat hippocampal neurons revealed by differential sensitivity to cyclothiazide.

1. The kinetics of onset of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization by glutamate, and the extent of attenuation of AMPA receptor desensitization by cyclothiazide, showed pronounced cell-to-cell variation in cultures of rat hippocampal neurons. Cultures prepared from area CA1 stratum radiatum tended to show weaker modulation by cyclothiazide than cultures prepared from the whole hippocampus. 2. Kinetic analysis of concentration jump responses to glutamate revealed multiple populations of receptors with fast (approximately 400 ms), intermediate (approximately 2-4 s), and slow (> 20 s) time constants for recovery from modulation by cyclothiazide. The amplitudes of these components varied widely between cells, suggesting the existence of at least three populations of AMPA receptor subtypes, the relative density of which varied from cell to cell. 3. The complex patterns of sensitivity to cyclothiazide seen in hippocampal neurons could be reconstituted by assembly of recombinant AMPA receptor subunits generated from cDNAs encoding the flip (i) and flop (o) splice variants of the GluR-A and GluR-B subunits. Recovery from modulation by cyclothiazide was slower for GluR-AiBi and GluR-AoBi than for GluR-AiBo and GluR-AoBo. 4. Coexpression of the flip and flop splice variants of GluR-A, in the absence of GluR-B, revealed that heteromeric AMPA receptors with intermediate sensitivity to cyclothiazide, similar to responses observed for the combinations GluR-AoBi or GluR-AiBo, could be generated independently of the presence of the GluR-B subunit. However, recovery from modulation by cyclothiazide was twofold slower for GluR-AiBi than for homomeric GluR-Ai, indicating that the GluR-A and GluR-B subunits are not functionally equivalent in controlling sensitivity to cyclothiazide. 5. These results demonstrate that AMPA receptors expressed in hippocampal neurons are assembled in a variety of subunit and splice variant combinations that might serve as a mechanism to fine-tune the kinetics of synaptic transmission.

A novel allosteric potentiator of AMPA receptors: 4--2-(phenylsulfonylamino)ethylthio--2,6-difluoro-phenoxyaceta mide.

We report that a novel sulfonylamino compound, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), selectively potentiates glutamate receptors of the AMPA subtype. PEPA (1-200 microM) dose dependently potentiated glutamate-evoked currents in Xenopus oocytes expressing AMPA (GluRA-GluRD), but not kainate (GluR6 and GluR6+KA2) or NMDA (zeta1 + epsilon1-epsilon4), receptor subunits. PEPA was effective at micromolar concentrations and, in contrast to the action of cyclothiazide, preferentially modulated AMPA receptor flop isoforms. At 200 microM, PEPA potentiated glutamate responses by 50-fold in oocytes expressing GluRCflop (EC50 approximately 50 microM) versus only threefold for GluRCflip; a similar preference for flop isoforms was observed for other AMPA receptor subunits. Dose-response analysis for GluRCflop revealed that 100 microM PEPA produced a sevenfold increase in AMPA receptor affinity for glutamate. PEPA produced considerably weaker potentiation of kainate-evoked than glutamate-evoked currents, suggesting modulation of the process of receptor desensitization. In human embryonic kidney 293 cells transfected with AMPA receptor subunits, PEPA either abolished or markedly slowed the rate of onset of desensitization and potentiated steady-state equilibrium currents evoked by glutamate with subunit (GluRC >/= GluRD > GluRA) and splice-variant (flop > flip) selectivity similar to that observed in oocytes. Our results show that PEPA is a novel, flop-preferring allosteric modulator of AMPA receptor desensitization at least 100 times more potent than aniracetam.

Growth factor-induced transcription of GluR1 increases functional AMPA receptor density in glial progenitor cells.

We analyzed the effects of two growth factors that regulate oligodendrocyte progenitor (O-2A) development on the expression of glutamate receptor (GluR) subunits in cortical O-2A cells. In the absence of growth factors, GluR1 was the AMPA subunit mRNA expressed at the lowest relative level. Basic fibroblast growth factor (bFGF) caused an increase in GluR1 and GluR3 steady-state mRNA levels. Platelet-derived growth factor (PDGF) did not modify the mRNA levels for any of the AMPA subunits but selectively potentiated the effects of bFGF on GluR1 mRNA (4.5-fold increase). The kainate-preferring subunits GluR7, KA1, and KA2 mRNAs were increased by bFGF, but these effects were not modified by cotreatment with PDGF. Nuclear run-on assays demonstrated that PDGF+bFGF selectively increased the rate of GluR1 gene transcription (2.5-fold over control). Western blot analysis showed that GluR1 protein levels were increased selectively (sixfold over control) by PDGF+bFGF. Functional expression was assessed by rapid application of AMPA to cultured cells. AMPA receptor current densities (pA/pF) were increased nearly fivefold in cells treated with PDGF+bFGF, as compared with untreated cells. Further, AMPA receptor channels in cells treated with PDGF+bFGF were more sensitive to voltage-dependent block by intracellular polyamines, as expected from the robust and selective enhancement of GluR1 expression. Our combined molecular and electrophysiological findings indicate that AMPA receptor function can be regulated by growth factor-induced changes in the rate of gene transcription.

Functional nicotinic acetylcholine receptor expression in stem and progenitor cells of the early embryonic mouse cerebral cortex.

The adult cerebral cortex contains nicotinic acetylcholine (ACh) receptors vital to cortical function. However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role in cortical development. We now report evidence of functional nicotinic acetylcholine receptor channels in fetal mouse cerebral cortex as early as embryonic day 10 (E10), when the cortex consists of dividing stem and progenitor cells. Patch-clamp electrophysiological measurements indicate that nicotine and ACh evoke sizable inward currents characteristic of nicotinic receptors, that are strongly rectifying with a reversal potential near 0 mV. Three different nicotinic agonists, ACh, nicotine, and dimethylphenylpiperazinium, evoked cytosolic Ca(2+) signals. Agonist-evoked Ca(2+) signals and electrophysiological responses were found in greater than 70% of all E10-E11 cells tested and were blocked by nicotinic receptor antagonists. The Ca(2+) response to nicotinic agonists was markedly prolonged in cells from early embryonic stages relative to later stages of development. alpha3, alpha4, and alpha7 receptor subunit proteins were detected immunocytochemically in cortical cells from E10 to birth. The incidence of each subunit declined with embryonic age, suggesting a role in early development. We discuss the possible function of nicotinic receptors in early cortical development and their role as a target for nicotine in the developmental pathologies associated with the fetal tobacco syndrome.