Influence of glyoxylic acid on properties of isolated mitochondria]

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.     

Target tissues affect cellular properties of cocultured leech Retzius neurons

The development of many neurons, including the Retzius (Rz) neurons of the medicinal leech, is shaped in part by interactions with other cells in the environment. To explore the nature of the interaction between growing Rz processes and potential target tissues, adult Rz neurons were cultured directly in contact with some of the tissues that normally serve as their targets in vivo. The morphology of the regenerated processes of these neurons varied depending upon the identity of the target tissues, but other cellular properties remained unchanged. In particular, although during normal development contact with peripheral targets determines the sign of Rz neurons' response to acetylcholine (ACh) applied to the soma, these cultured neurons maintained their original response to ACh even after as long as 2 weeks in culture on novel targets. Hence, some features of cultured adult Rz neurons varied depending upon the conditions, whereas other features remained fixed. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 55–68, 1998     

The effects of procaine, strychnine and penicillin on nociceptive neurons in leech segmental ganglia

Procaine, strychnine and penicillin selectively depolarized the membrane potential and prolonged the action potential recorded in the lateral but not the medial nociceptive (N) cell in the hirudinid leech Macrobdella decora. In contrast, procaine did not differentiate between medial and lateral N cells in two other hirudinid leeches Hirudo medicinalis and Haemopis marmorata. In these species, the drug equally decreased the amplitude of action potentials in both types of N cells without effecting their resting membrane properties. In the nociceptive neurons of the glossiphoniid leech Haementeria ghilianii which possesses only one type of N cell, procaine produced a depolarization and prolonged the action potential. This finding indicates that the single pair of N cells in Haementeria is of the lateral type. The results suggest that the lateral type N cell in Macrobdella and Haementeria share a unique Na+-dependent conductance which is selectively opened by the local anesthetics procaine and strychnine as well as by penicillin. This conductance is either not present or insensitive to the drugs in the homologous N cells in the two other leech species examined.     

Ultrastructural localization of [3H]dopamine in neurons of leech (Haemopis sanguisuga) abdominal ganglia

Neurons in the cortex of the segmental ganglia of Haemopis sanguisuga accumulated [3H]DOPA in vitro. Electron microscopic autoradiography revealed deposits of silver grains over clusters of electron dense granules measuring 20-35 nM in diameter. [3H]Dopamine (DA) was localized in the giant glial cells. Dopaminergic terminals were labelled with both DOPA and dopamine and the morphology is similar to other DA synapses. The leech could be a useful model for the study of synaptic events at dopaminergic terminals.     

Control of leech swimming activity by the cephalic ganglia

We investigated the role played by the cephalic nervous system in the control of swimming activity in the leech, Hirudo medicinalis, by comparing swimming activity in isolated leech nerve cords that included the head ganglia (supra- and subesophageal ganglia) with swimming activity in nerve cords from which these ganglia were removed. We found that the presence of these cephalic ganglia had an inhibitory influence on the reliability with which stimulation of peripheral (DP) nerves and intracellular stimulation of swim-initiating neurons initiated and maintained swimming activity. In addition, swimming activity recorded from both oscillator and motor neurons in preparations that included head ganglia frequently exhibited irregular bursting patterns consisting of missed, weak, or sustained bursts. Removal of the two head ganglia as well as the first segmental ganglion eliminated this irregular activity pattern. We also identified a pair of rhythmically active interneurons, SRN1, in the subesophageal ganglion that, when depolarized, could reset the swimming rhythm. Thus the cephalic ganglia and first segmental ganglion of the leech nerve cord are capable of exerting a tonic inhibitory influence as well as a modulatory effect on swimming activity in the segmental nerve cord.     

A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The SPG method

Summary A modified approach of the glyoxylic acid (GA) condensation reaction for the visualization of biogenic amines in tissue is described.Cryostat sections are used from brain or extracerebral tissue in dog, monkey, rat and mouse and exposed for 3 s to a room temperature solution containing sucrose-potassium phosphate-glyoxylic acid (SPG). The tissues are air dried and heated in an oven for 5 min. The complete processing time from fresh tissue to microscopic examination takes 18 min. Morphologically sharp and brightly fluorescent monoamine-containing neurons, pre-and terminal axons are seen against a dark parenchymal background without drug pre-treatment. The SPG method retains the high specific sensitivity for monoamines previously described in the original technique but is, in addition, more rapid and simple and is easily accessible as a research tool to investigators inexperienced in histofluorescence techniques.     

Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid.

The involvement of protein phosphatases in regulating platelet activation was studied. The major portion of the phosphorylase phosphatase activity found in platelet lysates appears to be of the type 1 variety. The identification of this enzyme was based on the finding that greater than 80% of protein phosphatase activity was inhibited by the heat-stable inhibitor protein inhibitor 2 and, while only 20% of the phosphorylase phosphatase activity in platelet extracts was inhibited by 2 nM okadaic acid, greater than 95% of the activity was inhibited in the presence of 1 microM okadaic acid. Increases in protein phosphorylations occurred and thrombin-induced release of serotonin was prevented as a result of artificially inhibiting the enzyme with okadaic acid in intact platelets. This implies either that the regulation of okadaic acid sensitive protein phosphatases is necessary for some agonist-induced effects or that okadaic acid sensitive phosphatases are required for maintaining platelets in a responsive state.     

Application of the glyoxylic acid method to Vibratome sections for the improved visualization of central catecholamine neurons

Summary The glyoxylic acid fluorescence histochemical method for the visualization of neuronal monoamines has been applied to fresh or glyoxylic acid-perfused brain tissue, sectioned with the Vibratome intrument. This technique demonstrates the central noradrenaline and dopamine neuron systems with a sensitivity and richness in details that is superior to the standard Falck-Hillarp formaldehyde method, as observed in the following three ways: First, the entire axon, including the non-terminal portions, became fluorescent; second, more extensive terminal systems were detected in certain brain regions ; third, due to the absence of diffusion, the delicate dopamine-containing fibres in e. g. the caudate nucleus and the median eminence had a distinct fluorescence. It is concluded that the glyoxylic acid method applied to Vibratome sections should be ideal for precise and detailed neuroanatomical studies on central catecholamine neuron systems.     

Fluorescent reporters of monoamine transporter distribution and function

Serotonin is a monoamine serving as a chemical messenger in diverse brain regions, as well as in blood and various other organs. We synthesized six ethylamine functionalized fluorophores as fluorescent probes for serotonin. The one with best spectral properties and aqueous solubility, 6-amino-2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, was studied in detail both in vivo and in vitro. It was shown to act as a ligand for serotonin transporter (SERT) without acute cerebral or cardiovascular toxicity or adverse effects. Fluorescent serotonin analogs can be used for direct visualization of SERT distribution and activity in live tissue.     

Differential action of tetrodotoxin on identified leech neurons

1. In leech segmental ganglia, the maximum rate of depolarization of action potentials was found to depend largely on Na in the Retzius (R) cell, the mechanosensory P, N and T cells and an identifiable neuron of unknown function, the X cell. 2. Tetrodotoxin (TTX) 15 100 mumol/l had little or no effect on R and X cells. In contrast, membrane excitation in N, P and T cells was depressed in dose- and use-dependent fashion. 3. The data imply the existence of two kinds of Na channels in normal, fully differentiated leech neurons. Correlation of such differences should lead to a better understanding of how particular neurons perform different functions.