A new mechanism for antiepileptic drug action: vesicular entry may mediate the effects of levetiracetam. J Neurophysiol 106: 1227-1239, 2011. First published June 8, 2011 doi:10.1152/jn.00279.2011 is one of the most commonly prescribed antiepileptic drugs, but its mechanism of action is uncertain. Based on prior information that LEV binds to the vesicular protein synaptic vesicle protein 2A and reduces presynaptic neurotransmitter release, we wanted to more rigorously characterize its effect on transmitter release and explain the requirement for a prolonged incubation period for its full effect to manifest. During whole cell patch recordings from rat hippocampal pyramidal neurons in vitro, we found that LEV decreased synaptic currents in a frequency-dependent manner and reduced the readily releasable pool of vesicles. When we manipulated spontaneous activity and stimulation paradigms, we found that synaptic activity during LEV incubation alters the time at which LEV's effect appears, as well as its magnitude. We believe that synaptic activity and concomitant vesicular release allow LEV to enter recycling vesicles to reach its binding site, synaptic vesicle protein 2A. In support of this hypothesis, a vesicular "load-unload" protocol using hypertonic sucrose in the presence of LEV quickly induced LEV's effect. The effect rapidly disappeared after unloading in the absence of LEV. These findings are compatible with LEV acting at an intravesicular binding site to modulate the release of transmitter and with its most marked effect on rapidly discharging neurons. Our results identify a unique neurobiological explanation for LEV's highly selective antiepileptic effect and suggest that synaptic vesicle proteins might be appropriate targets for the development of other neuroactive drugs.epilepsy; synaptic vesicle protein 2A; synaptic vesicle release; hippocampal slice LEVETIRACETAM (LEV) IS AN antiepileptic drug (AED) with a unique mode of action and efficacy against focal and generalized seizures. The exact mechanism by which this drug reduces seizures is still under investigation. LEV does not inhibit voltage-gated Na ϩ channels (Zona et al. 2001), nor does it modulate GABA receptors (Margineanu and Klitgaard 2003), two common AED mechanisms. It is established that LEV binds to a presynaptic protein, synaptic vesicle protein 2A (SV2A), located in the membrane of synaptic vesicles (Lynch et al. 2004). The precise function of SV2A is also unknown, but studies of cultured neurons from SV2A knockout mice show a reduced postsynaptic response, due to a lower initial vesicle release probability (Custer et al. 2006) and a decrease in the total amount of neurotransmitter release during a stimulus train compared with controls (Chang and Sudhof 2009).Because SV2A plays a modulatory, but not essential, role in neurotransmission, it is unclear how a ligand binding to SV2A might alter neurotransmitter release.The cellular mechanism of action of LEV remains paradoxical. While it seems to do nothing to normal synaptic transmission, i...

Leptin is a hormone that reduces excitability in some hypothalamic neurons via leptin receptor activation of the JAK2 and PI3K intracellular signaling pathways. We hypothesized that leptin receptor activation in other neuronal subtypes would have anticonvulsant activity and that intranasal leptin delivery would be an effective route of administration. We tested leptin's anticonvulsant action in 2 rodent seizure models by directly injecting it into the cortex or by administering it intranasally. Focal seizures in rats were induced by neocortical injections of 4-aminopyridine, an inhibitor of voltage-gated K + channels. These seizures were briefer and less frequent upon coinjection of 4-aminopyridine and leptin. In mice, intranasal administration of leptin produced elevated brain and serum leptin levels and delayed the onset of chemical convulsant pentylenetetrazoleinduced generalized convulsive seizures. Leptin also reduced neuronal spiking in an in vitro seizure model. Leptin inhibited α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor-mediated synaptic transmission in mouse hippocampal slices but failed to inhibit synaptic responses in slices from leptin receptor-deficient db/db mice. JAK2 and PI3K antagonists prevented leptin inhibition of AMPAergic synaptic transmission. We conclude that leptin receptor activation and JAK2/PI3K signaling may be novel targets for anticonvulsant treatments. Intranasal leptin administration may have potential as an acute abortive treatment for convulsive seizures in emergency situations.

MCF-7 human breast cancer cells are commonly used to model tissues responsive to estrogens and antiestrogens. We examined the effects of estradiol and the antiestrogen ICI 182780 on MCF-7 cell proliferation and insulin-like growth factor binding protein 3 (IGFBP-3) gene expression. ICI 182780-induced growth inhibition was associated with increased transcription of the IG-FBP-3 gene, increased IGFBP-3 mRNA abundance, and increased IGFBP-3 protein accumulation in the conditioned medium. The growth stimulatory effect of estradiol was associated with opposite effects, and the correlation between cellular proliferation and IGFBP-3 mRNA abundance was strong (r ‫؍‬ ؊0.91). Recombinant IGFBP-3 inhibited basal and estradiol-stimulated MCF-7 cell proliferation, and an IGFBP-3 antisense oligodeoxynucleotide abolished antiestrogen-induced growth inhibition. These results provide evidence for an estradiol and antiestrogen-regulated IGFBP-3 growth inhibitory autocrine pathway in MCF-7 cells. Insulin-like growth factors I and II (IGF-I1 and IGF-II) are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cell types, including normal and transformed breast epithelial cells (1, 2). Both IGF-I and IGF-II bind with high affinity to specific IGF-binding proteins (IGFBPs), which modulate their bioactivity. At least six IGFBPs have been described (3,4). IGFBP-3 acts as a growth inhibitor in many (but not all (5)) experimental systems (reviewed in Ref. 6). Examples of data consistent with a growth inhibitory role for IGFBP-3 include the growth inhibition associated with IG-FBP-3 gene transfection (6), the increased IGFBP-3 accumulation associated with senescence-related reduction of cellular proliferation (7), the increase in IGFBP-3 production associated with retinoid-induced growth inhibition (8), and the decrease in IGFBP-3 production associated with epidermal growth factorstimulated proliferation (9). Early studies attributed the growth inhibitory action of IGFBP-3 to the reduction of IGF-I and/or IGF-II bioactivity resulting from competition for somatomedins between IGFBP-3 and the type I IGF receptor (10). However, there is recent evidence that IGFBP-3 also has growth inhibitory activity that is independent of its IGF binding properties (11)(12)(13)(14).Antiestrogens are widely used in breast cancer treatment, and it has been proposed that the inhibitory effect of these compounds on IGF-I expression contributes to their antiproliferative activity (15, 16) (reviewed in Ref. 17). Antiestrogens have significant trophic effects on the uterus (18), and there is a negative correlation between uterine weight and uterine IG-FBP-3 expression; the positive uterotrophic actions of both estradiol and the partial estrogen receptor antagonist tamoxifen are associated with suppression of uterine IGFBP-3 expression, while the pure antiestrogen ICI 182,780 (19) causes uterine involution and markedly enhances uterine IGFBP-3 expression (20). We undertook the present study to examine the possibility that the anti-proliferativ...

Summary:Purpose: The antiepileptic drug levetiracetam (LEV) is an enigma. Despite the fact that it specifically binds to the presynaptic vesicle protein, SV2A, no satisfactory mechanism of action has yet been identified. Using a combination of electrophysiological and cellular imaging techniques, we carefully tested the hypothesis that LEV directly interferes with neurotransmitter release.Methods: We measured extracellular evoked responses in the CA1 region of rat hippocampal slices after paired pulse stimulation and after application of up to 10 pulses applied at 5-80 Hz. In parallel experiments, we used quantitative 2-photon microscopy and the fluorescent vesicular marker FM1-43 to measure the effect of repetitive stimulation on presynaptic vesicle release.Results: Acute exposure to LEV (100 µM) had no effect on paired pulse synaptic responses. However, when slices were exposed to LEV for 3 h, there was a significant alteration in paired pulse responses and a more striking reduction in late synaptic potentials delivered in an 80 Hz train. LEV significantly reduced the rate of vesicle release assessed by FM1-43 destaining during 1 Hz stimulation.Conclusion: LEV is unique among currently available antiepileptics, because it directly inhibits presynaptic neurotransmitter release in a use-dependent fashion. While there are alternate explanations for this observation, it is plausible that LEV exerts its effect by binding to a protein selectively expressed in presynaptic nerve terminals.