Gamma-hydroxybutyric acid-induced absence seizures in GluR2 null mutant mice

In this electrophysiological study, we examined the susceptibility of GluR2 mutant null mice to absence seizures in comparison with wild-type controls. The prodrug of (GHB), gamma-butyrolactone (GBL) was given systemically to induce the absence seizures. We also tested the severity and duration of the seizure activity in this model. The results showed that the latency from GBL administration to onset of seizure was significantly prolonged in GluR2(-/-) mice when compared to GluR2(+/+) mice. The duration of spike-and-wave discharges (SWD) was also significantly decreased in the GluR2(-/-) mice. Ninety minutes following GBL administration, wild-type animals continued to exhibit intermittent SWD bursts while GluR2(-/-) mice had returned to baseline. These data suggest that the GluR2 subunit may be involved in the initiation and maintenance of absence seizures induced by GBL.     

Anticonvulsant action of β-kainic acid in mice. Is β-kainic acid an N-methyl-d-aspartate antagonist?

An effect of the beta-stereoisomer of kainic acid on seizures produced by intracerebroventricular injections of excitatory amino acids was tested in mice. beta-Kainic acid preferentially antagonizes myoclonic seizures induced by N-methyl-D-aspartate and quinolinate, has less pronounced anticonvulsant action against alpha-kainate, D-homocysteinesulphinate and quisqualate, and no effect on convulsions induced by L-glutamate. The anticonvulsant activity of beta-kainic acid matches that of 2-amino-7-phosphonoheptanoic and kynurenic acids, both preferential N-methyl-D-aspartate receptor antagonists, and differs considerably from the profile of anticonvulsant action of gamma-D-glutamylaminomethylsulphonic acid, a preferential kainate/quisqualate antagonist.     

Pharmacology of β-phenylethylamine-induced seizures in mice

1. 1. The endogenous trace amine β-phenylethylamine (PE) produced tonic-clonic seizures in male Swiss mice when administered in doses of 125–200 mg/kg. The number of mice exhibiting PE-induced seizures, the latency to onset of first seizure and the latency to loss of the righting reflex were dose dependent.

2. 2. Pretreatment with the benzodlazepines diazepam, chlordiazepoxide, midazolam and clonazepam significantly reduced the incidence of PE-induced seizures. Similarly, increasing brain gamma-amlnobutyric acid (GABA) levels by injection of the GABA-transamlnase inhibitors aminooxyacetic acid or gabaculine afforded significant protection against pea's convulsant effects.

3. 3. The data suggest that PE when administered at high doses may interfere either directly or Indirectly with GABAergic neurotransmission in the central nervous system. In addition, since PE induces an epileptiform spiking pattern and produces seizures in rodents It appears possible that the amine may play a role in some forms of human epilepsy.

Reduction of predator odor-induced anxiety in mice by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to an aversive, anxiety-inducing, predator (cat) odor were examined in an odor preference test. Control untreated mice displayed an anxiogenic response to the cat odor, spending a minimal amount of time in a Y-maze in the vicinity of the cat odor. Intracerebroventricular (i.c.v.) administrations of 3αHP had an anxiolytic action, resulting in significant dose-related (0.01–1.0 μg) increases in the amount of time spent in the proximity of the cat odor. These anxiolytic effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP) having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the response to cat odor indicating that the anxiolytic actions of 3αHP were unlikely to be related to any analgesic effects. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results indicate that the allylic neurosteroid 3αHP has anxiolytic actions involving interactions with the GABA_A receptor.     

Comparison of G-protein activation in the brain by μ-, δ-, and κ-opioid receptor agonists in μ-opioid receptor knockout mice

Mice lacking the μ-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for μ-, δ-, and κ-opioid receptors in μ-opioid receptor knockout mice. Selective agonists of μ- (DAMGO), δ- (DPDPE), and κ- (U-69,593) opioid receptors stimulated [³⁵S]GTPγS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [³⁵S]GTPγS binding in these regions of μ-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a μ-opioid receptor agonist in μ-opioid receptor knockout mice, and demonstrated that coupling of the κ-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the δ-opioid receptor agonist, DPDPE, was reduced in the μ-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.     

Modulation of GABA-stimulated chloride influx by β-carbolines in rat brain membrane vesicles

We have shown that GABA-stimulated chloride influx into membrane vesicles from the rat cerebral cortex can be enhanced or inhibited by β-carbolines. The β-carbolines can produce a full spectrum of responses from maximal enhancement to maximal inhibition.     

Effects of substantia nigra γ-vinyl-GABA infusions on flurothyl seizures in adult rats

There is evidence implicating the nigral γ-aminobutyric acid (GABA) system in the control seizures. Our previous studies have demonstrated that, in rat pups, intranigrally infused γ-vinyl-GABA (GVG, 5–20 μg) strongly suppresses flurothyl-induced tonic but not clonic seizures. Furthermore, nigral infusions of bicuculline or muscimol abolish the anticonvulsant effect of GVG. In this study, we report that in adult rats bilateral infusions of GVG (20 μg) into the substantia nigra pars reticulata (SNR) significantly elevated the thresholds for both clonic and tonic seizures induced by flurothyl. Lower doses (5 and 10 μg) did not significantly protect adult rats against seizures, but there was a significant effect of GVG dose. Unilateral infusion of GVG (20 μg) in the SNR did not alter the thresholds for flurothyl-induced seizures. Intranigral infusions of bicuculline following pretreatment with GVG abolished the protective effect of GVG on flurothyl-induced seizures, indicating that the anticonvulsant effect of GVG is most likely mediated by the nigral GABA_A receptor. Intranigral administration of muscimol after GVG pretreatment significantly suppressed flurothyl-induced seizures, but the combined effect of the two drugs was not as strong as that of GVG alone. The data suggest that GVG protects adult rats against flurothyl-induced seizures. In adults, however, the dose of GVG required to protect against both clonic and tonic seizures is higher than that needed in rat pup SNR.     

A method for culturing rat oviduct γ-aminobutyric acid cells

A technique for the culture of rat oviduct gamma-aminobutyric acid (GABA) cells is described. The technique involves first explaining the fimbria and preampulla, which are the oviduct divisions with the highest density of GABA cells. The explanted tissue is cultured in a serum-free medium, to propagate the outgrowing cells. Under the experimental conditions we describe, the majority of the cells maintain GABA expression, as determined by immunostaining with a GABA antiserum.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.     

Specific [H]γ-aminobutyric acid binding to vestibular membranes of the chick inner ear

To support a postulated neurotransmitter character of γ-aminobutyric acid (GABA) in the vertebrate vestibule, [³H]GABA binding was measured in a crude membrane preparation of chick inner ear ampullary cristae. In the absence of divalent cations bound [³H]GABA was displaced by unlabeled GABA, muscimol or bicuculline, but it was not displaced by (±)-baclofen. A single population of [³H]GABA binding sites with an equilibrium constant of 19.4 nM and a maximum binding capacity of 0.58 pmol/mg protein was found. These results suggest the possible existence of a synaptic GABA_A receptor in the chick inner ear membranes and sustain the neurotransmitter role of GABA in the chick vestibule.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Metabolites of the β-amyloid precursor protein generated by β-secretase localise to the trans-golgi network and late endosome in 293 cells

Deposition of β-amyloid occurs in the brains of all sufferers of Alzheimer's disease. β-amyloid is proteolytically derived from the β-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and β-secretase cleavage site of the Alzheimer's amyloid precursor protein. The β-secretase cleavage event occurs at the extreme N-terminus of the β-amyloid peptide. Our antiserum to the N-terminus of the β-amyloid peptide (NTβ4) specifically recognises β-secretase cleaved species as opposed to intact βAPP. NTβ4 specifically immunoprecipitates a 13 kDa fragment of βAPP (p13) which is potentially amyloidogenic. We have used these anti-sera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic βAPP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of βAPP fragments possessing the Asp-1 N-terminus of β-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network. © 1996 Wiley-Liss, Inc.     

Δ-Tetrahydrocannabinol-induced changes in β-adrenergic receptor binding in mouse cerebral cortex

The effects of 3 cannabinoids, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), 11-OH-Δ⁹-tetrahydrocannabinol (11-OH-Δ⁹-THC) and cannabinol (CBD) on the binding of [³H]dihydroalprenolol ([³H]DHA) to mouse brain β-adrenergic receptors were determined. In vitro, Δ⁹-THC and 11-OH-Δ⁹-THC increased the specific binding of [³H]DHA. The increased specific binding of [³H]DHA was due to an increase in receptor affinity as indicated by a decrease in the dissociation constant (K_d). CBD had no effect on binding. Chronic administration of Δ⁹-THC in vivo caused a decrease in the number of [³H]DHA binding sites with no change in K_d.