John M. Tuchek

The biochemistry of learning and memory

An overview of some of the biochemical and molecular events involved in the process of learning and memory are presented in a short review. Two invertebrate models of learning are considered: the gill-withdrawal reflex of Aplysia and avoidance learning in Drosophila melanogaster . Particular attention is paid to the biochemical mechanisms underlying both the development of long-term potentiation (LTP) and passive avoidance learning (PAL) in the young chick. The role of several biological molecules in learning and memory are considered, for example, protein kinase C (PKC), Ca++-Calmodulin kinase II (CaMKII), GAP-43, and glutamate receptors.

A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays

Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [3H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [3H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined.

Experimental Febrile Convulsions in Epileptic Chickens: The Anticonvulsant Effect of Elevated γ-Aminobutyric Acid Concentrations

Summary: The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation. In 3‐day‐old chicks homozygous for the epilepsy gene (epileptics), elevation of body temperature using microwave diathermy evoked an initial febrile seizure resembling the clonic seizures evoked in epileptic chicks by photic stimulation. After complete recovery, this was followed by a clonic‐tonic seizure. In nonepileptic heterozygote hatchmates (carriers) of the same age, only the latter seizure pattern was observed. In 16‐ to 17‐day‐old chicks of either phenotype, both seizure patterns were observed during hyperthermia. In all cases, the temperature at which seizures occurred was significantly lower in epileptic than in nonepileptic chicks, indicating a lower threshold for febrile seizures when there is an inherited predisposition to convulse. The occurrence of seizures was dependent on the body temperature and not on the rate of rise of temperature. Elevation of the brain γ‐aminobutyric acid (GABA) concentrations by administration of the GABA transaminase inhibitor γ‐vinyl GABA reduced the incidence of the initial febrile seizures and increased the latency in those birds that were not fully protected.

Myristoyltransferase and calcineurin: novel molecular therapeutic target for epilepsy.

N-myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. These myristoylated proteins in the cell have diverse biological functions such as signal transduction, cellular transformation and oncogensis. Known myristoylated proteins [Src family kinases, the catalytic subunit of cAMP-dependent protein kinase and calcineurin (CaN)] are either protein kinases or a protein phosphatases which modulate various cellular metabolic processes. Myristoylation is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The main objective of this review is to focus on the potential role of NMT and CaN in epileptic brain and its involvement in neuronal apoptosis. The findings on the interaction of NMT and CaN with various signaling molecules in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimers disease, ischemia and epilepsy.

Enhanced forebrain nitric oxide synthase activity in epileptic fowl

Nitric oxide synthase (NOS) activity was examined in forebrain, cerebellum and optic lobes of adult domestic fowl, having a hereditary primary generalized convulsive disorder. NOS was approximately 2-fold higher in only the forebrain of adult epileptic fowl compared to non-epileptic (carrier) hatchmates. A significant increase in NOS was also evident in forebrains of 1-day-old epileptic chicks. Ca(2+)-dependency experiments confirmed that these increments were principally due to type I NOS (NOS-I). Induction of convulsions by intermittent photic stimulation did not affect pre-existing forebrain NOS-I activity. The present data suggest that an enhanced NO signaling may ensue in selected regions of the brain as an adaptive response to hereditary epileptogenesis.

Expression of calcineurin and its interacting proteins in epileptic fowl.

Calcineurin (CaN), a Ca2+–calmodulin (CaM)‐dependent protein phosphatase, is important for Ca2+‐mediated signal transduction. The main objective of this study was to examine the potential role of CaN in epileptic brain and its involvement in neuronal apoptosis. We investigated CaN expression and its interaction with various signaling molecules in normal, carrier and epileptic brain tissues of chicken. Our results revealed higher Ca2+–CaM‐dependent phosphatase activity of CaN and a correspondingly strong immunoreactive band of CaN A in epileptic and carrier brain samples compared with normal brain. Furthermore, immunohistochemical analysis showed a higher level of expression of CaN in epileptic brain tissue. However, the intensity of immunoreactivity was less in carrier than epileptic brain. We observed that the interaction of CaN with m‐calpain and µ‐calpain was strong in carrier and epileptic chickens compared with that in normal birds. In addition, the interaction of CaN with Bcl‐2, caspase‐3 and p53 was greater in carrier and epileptic fowl than in normal chickens. The greater interaction of CaN with various apoptotic factors in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis.

Pharmacology of methyl- and propyl-β-carbolines in a hereditary model of epilepsy

Abstract Intravenous administration of β-carboline-3-carboxylate methyl ester (β-CCM) produced convulsions at small doses (0.03 mg kg ) in adult chickens, homozygous for the epileptic gene. Non-epileptic heterozygote hatchmates (carriers) did not undergo seizures at doses of 1 mg kg , and doses of 3–5 mg kg produced only brief myoclonic responses. The convulsant effect of β-CCM could be prevented by pretreatment with large doses of β-carboline-3-carboxylate propyl ester (β-CCP). β-Carboline-3-carboxylate methyl ester displayed a higher affinity than diazepam in displacement studies on synaptosomal membrane preparations from brains of epileptic and carrier chickens.

Anticonvulsant activities of 4‐(4′‐fluorophenoxy) benzaldehyde semicarbazone

A series of aryloxyaryl semicarbazones had been shown previously to possess significant anticonvulsant activity in the maximal electroshock screen in both rats and mice as well as in the subcutaneous pentylenetetrazol test in mice. One member of this series, namely 4‐(4′‐fluorophenoxy)benzaldehyde semicarbazone (Compound IV), was selected for detailed studies with a view to determining whether it should proceed to full‐scale preclinical evaluation; these results are reported herein. After intraperitoneal injection, Compound IV afforded protection in the Frings audiogenic mouse test. In addition, it had activity in the rat corneal kindling screen after oral administration and in the rat hippocampal kindling screen after intraperitoneal administration, but it was virtually inactive in the amygdala kindling test in rats. When administered by the intravenous route to genetically susceptible epileptic chickens, Compound IV and eight analogs prevented convulsions induced by alternating strobe lights. Compound IV did not display proconvulsant properties when examined in the timed intravenous test in mice nor did this compound cause significant effects on liver weights, microsomal protein yields, and various hepatic enzymes after oral administration to rats. Intraperitoneal injection of Compound IV afforded little or no protection to mice who had received subcutaneously convulsive doses of bicuculline, picrotoxin, or strychnine. This compound did not elevate γ‐aminobutyric acid levels or inhibit γ‐aminobutyric acid uptake. Ion‐imaging and electrophysiological experiments suggested that the mode of action of this compound could be on calcium and sodium channels. In most of these experiments, Compound IV was superior to the widely used antiepileptic drug phenytoin. Drug Dev. Res. 46:112–125, 1999.

The Relationship Between Anticonvulsant Activity and Receptor Affinity of N-methyl-D-aspartate Antagonists in Epileptic Fowl

The N-methyl-D-aspartate (NMDA) receptor antagonists [(3-(+/-)2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP), +/- 2-amino-7-phosphonoheptanoic acid (2AP7), +/- 2-amino-5-phosphonovaleric acid (2AP5), D-alpha-aminoadipic acid (alpha AA), and +/- alpha, epsilon-diaminopimelic acid (DAP)] were tested for anticonvulsant activity in epileptic chickens. There was a high correlation between anticonvulsant potencies (ED50) and the affinity for the NMDA receptor measured by displacement of L-[3H]glutamate from synaptosomal membranes. The high seizure susceptibility is not due to abnormalities in the NMDA receptor as comparison of KD, Bmax and Ki values in synaptosomal preparations from epileptic and non-epileptic chickens indicated no differences in NMDA receptor binding receptor characteristics.

Protection by GAB A Agonists, γ‐Hydroxybutyric Acid, and Valproic Acid Against Seizures Evoked in Epileptic Chicks by Hyperthermia

Summary: With microwave diathermy, febrile seizures were produced in epileptic chicks aged 2–5 days. Drugs that enhance GABAergic activity (i.e., GABA, muscimol, and progabide), as well as valproic acid and γ‐hydroxybutyric acid, produced dose‐dependent increases in latency to onset of seizures.