Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)-independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.     

Dopamine D-1 receptor-mediated inhibition of nucleus accumbens neurons from the ventral tegmental area.

1. Spike generation by stimulation of the parafascicular nucleus of thalamus was extracellularly recorded in the nucleus accumbens of chloral hydrate-anesthetized adult Wistar rats using a silver-wire microelectrode attached along a seven-barreled micropipette, each of which was filled with dopamine, SKF 38393 (D-1 agonist), bromocriptine (D-2 agonist), haloperidol, SCH 23390 (D-1 antagonist) and domperidone (D-2 antagonist). The drugs were microiontophoretically applied to the target neurons recorded. 2. Effects of dopamine receptor antagonists on the inhibition of the spike generation by conditioning stimuli applied to the ventral tegmental area preceding the test stimulus to the parafascicular nucleus and those of dopamine agonists on the test stimulus-induced spikes were examined. 3. The parafascicular nucleus stimulation-induced spikes were inhibited by dopamine as well as D-1 and D-2 agonists and by the conditioning stimulation of the ventral tegmental area. The conditioning stimulation-induced inhibition was antagonized by haloperidol and SCH 23390, but not by domperidone. 4. Activation of D-1 receptors, which make probably synaptic contact with dopaminergic nerve terminals from the ventral tegmental area, is considered to result in inhibition of the neuronal activity of the nucleus accumbens neurons receiving input from the parafascicular nucleus of the thalamus. In addition, D-2 receptors located extrajunctionally may be involved in the inhibition of the same neurons in the nucleus accumbens.     

Neuropeptide levels in the basal ganglia of aged common marmosets following prolonged treatment with MPTP

Summary Aged common marmosets were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 0.5–2.0 mg/kg/week i.p.) for 16 or 24 weeks, observed for a total of 30 weeks and then killed for measurement of biochemical pramaters in basal ganglia. The MPTP treatment induced a marked depletion in dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the caudate nucleus and putamen. In contrast, the concentrations of five neuropeptides: [Met⁵]-enkephalin, [Leu⁵]-enkephalin, cholecystokinin, substance P and neurotensin as measured by a combined HPLC/RIA method, remained unaltered in all basal ganglia regions examined. Enkephalin precursor levels, as reflected by cryptic [Met⁵]-enkephalin content, were increased in the putamen, but not in the caudate nucleus, as a consequence of MPTP administration. Cryptic [Leu⁵]-enkephalin content remained unchanged in the striatum of MPTP treated marmosets. Overall, these results suggest an increase in striatal [Met⁵]-enkephalin release following chronic MPTP treatment of aged marmosets. However, the chronic treatment of aged marmosets with MPTP does not reproduce the neuropeptide alterations characteristic of Parkinson's disease.     

Oscillations of cortical oxidative metabolism and microcirculation in the ischaemic brain.

A new in vivo model for studying brain metabolic and haemodynamic oscillatory phenomena during ischaemia is described. In this model acute or chronic occlusion of one or two carotid arteries in the rat is performed. Due to the partial ischaemia developed, oscillations in the level of intramitochondrial pyridine nucleotides (NADH) as well as flavoproteins (Fp) were recorded from the brain by monitoring the fluorescence of these respiratory chain components. The two fluorescent signals (NADH and Fp) were measured by using the time sharing or DC fluorometer/reflectometer. The changes in the reflected light at the excitation wavelengths (366 and 450 nm) were recorded simultaneously. Bilateral carotid artery occlusion induced immediate oscillations (6-9 waves per min) in the mitochondrial redox state as well as in tissue blood volume in both hemispheres. To verify the accuracy of the NADH monitoring system, including the correction technique for haemodynamic and other artifacts, we used the intracarotid artery saline bolus injection approach. The results could be summarized as follows: (1) unilateral carotid artery occlusion resulted in delayed development of oscillations, particularly in the ipsilateral hemisphere; (2) the oscillation phenomenon was reversible if recirculation restarted within 5 min. Occlusion for more than 30 min resulted in irreversible oscillations; (3) the oscillation appearances and intensities were affected by various physiological conditions. Vasoconstriction, induced by hyperoxia, stimulated the oscillations while vasodilation, induced by hypercapnia, depressed them. Anoxia, hypoxia and spreading depression (SD) abolished the oscillations. Glucose injection was not effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of rat brain slices as an in vitro model for mechanistic evaluation of neurotoxicity-studies with acrylamide

Biochemical mechanisms underlying acrylamide induced neurotoxicity were examined using an in vitro model consisting of sagittal slices of rat brain. Incubation of brain slices under oxygen in artificial cerebrospinal fluid containing acrylamide produced a dose and time dependent inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lysosomal enzymes, acid phosphatase, N-acetyl glucosaminidase and beta-glucuronidase decreased in a similar manner, while no changes were observed in the activity of Na+K+ATPase, cytochrome c oxidase and lactate dehydrogenase. Incubation of slices with two structurally related compounds, acetamide (a non-neurotoxic amide) and methylene bis-acrylamide (a weak neurotoxin), indicated that acrylamide selectively inhibited GAPDH, enolase and N-acetyl glucosaminidase at low concentration; similar doses of acetamide and methylene bis-acrylamide did not have the same effect on brain slices. Incubation with acrylamide depleted glutathione levels in slices, and the addition of glutathione to the incubation medium prevented acrylamide induced inhibition of GAPDH and lysosomal enzymes. Time dependent inhibition of lysosomal enzymes was also observed in vivo, in the brain and sciatic nerve of rats following a single dose of acrylamide. These results demonstrate that both in vitro and in vivo, lysosomal enzymes are also inhibited following acrylamide exposure. The rat brain slice model exhibits both selectivity and sensitivity towards neurotoxicants and hence, may prove to be an useful in vitro model for the mechanistic evaluation of neurotoxicity.     

Opposite effects of phospholipase A2 on [3H]AMPA binding in adult and neonatal membranes.

In the present study, we compared the effect of phospholipase A2 (PLA2) treatment of synaptic membranes from adult and neonatal rats on the characteristics of [3H]AMPA binding sites. Whereas PLA2 treatment of membranes from adult rats produces an increased affinity for [3H]AMPA binding, the same treatment in neonatal rats results in a decrease in the maximal number of binding sites. Since activation of PLA2 has been proposed to play a critical role in the formation of long-term potentiation (LTP), possibly mediated through a modification of the AMPA receptors, the results strengthen the hypothesis that PLA2-induced modification of [3H]AMPA binding sites is an important component of synaptic plasticity.     

Differential effects of specific opioid receptor agonists on rat pup isolation calls.

The possibility was investigated that specific opioid receptor types might selectively alter the production of isolation-induced ultrasonic vocalizations. Intracisternal injections of mu, delta and kappa opioid receptor agonists were administered to isolated 10-day-old rat pups. The mu receptor agonist [D-Ala2-NMe-Phe4-Gly-ol]-enkephalin (DAMGO) and delta receptor agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) both reduced the rate of isolation-induced ultrasonic calling in the absence of sedation. The kappa receptor agonist U50,488 had the opposite effect, significantly raising the rate of vocalization. Fourteen-day-old pups, with a larger delta receptor population, showed a greater sensitivity to DPDPE than was seen in the younger animals.     

Chimeric cytotoxin IL2-PE40 inhibits relapsing experimental allergic encephalomyelitis.

IL2-PE40 is a chimeric protein composed of human interleukin-2 (IL2) genetically fused to a modified form of Pseudomonas exotoxin lacking the cell recognition domain. IL2-PE40 is cytotoxic for IL2 receptor-bearing lymphocytes in culture and can inhibit activation of T cells in vivo. IL2-PE40 can significantly diminish antigen-stimulated proliferation of lymphocytes sensitized to myelin basic protein. Intraperitoneal administration of IL2-PE40 not only markedly inhibits the clinical manifestations of adoptively transferred relapsing experimental allergic encephalomyelitis but also dramatically reduces both inflammation and demyelination characteristic of the disease.