Characterization of functional subgroups among genetically identified cholinergic neurons in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN) is a part of the reticular activating system which is composed of cholinergic, glutamatergic and GABAergic neurons. Early electrophysiological studies characterized and grouped PPN neurons based on certain functional properties (i.e., the presence or absence of the A-current, spike latency, and low threshold spikes). Although other electrophysiological characteristics of these neurons were also described (as high threshold membrane potential oscillations, great differences in spontaneous firing rate and the presence or absence of the M-current), systematic assessment of these properties and correlation of them with morphological markers are still missing. In this work, we conducted electrophysiological experiments on brain slices of genetically identified cholinergic neurons in the PPN. Electrophysiological properties were compared with rostrocaudal location of the neuronal soma and selected morphometric features obtained with post hoc reconstruction. We found that functional subgroups had different proportions in the rostral and caudal subregions of the nucleus. Neurons with A-current can be divided to early-firing and late-firing neurons, where the latter type was found exclusively in the caudal subregion. Similar to this, different parameters of high threshold membrane potential oscillations also showed characteristic rostrocaudal distribution. Furthermore, based on our data, we propose that high threshold oscillations rather emerge from neuronal somata and not from the proximal dendrites. In summary, we demonstrated the existence and spatial distribution of functional subgroups of genetically identified PPN cholinergic neurons, which are in accordance with differences found in projection and in vivo functional findings of the subregions. Being aware of functional differences of PPN subregions will help the design and analysis of experiments using genetically encoded opto- and chemogenetic markers for in vivo experiments.

Targets, receptors and effects of muscarinic neuromodulation on giant neurones of the rat dorsal cochlear nucleus.

Although cholinergic modulation of the cochlear nucleus (CN) is functionally important, neither its cellular consequences nor the types of receptors conveying it are precisely known. The aim of this work was to characterise the cholinergic effects on giant cells of the CN, using electrophysiology and quantitative polymerase chain reaction. Application of the cholinergic agonist carbachol increased the spontaneous activity of the giant cells; which was partly the consequence of the reduction in a K(+) conductance. This effect was mediated via M4 and M3 receptors. Cholinergic modulation also affected the synaptic transmission targeting the giant cells. Excitatory synaptic currents evoked by the stimulation of the superficial and deep regions of the CN were sensitive to cholinergic modulation: the amplitude of the first postsynaptic current was reduced, and the short-term depression was also altered. These changes were mediated via M3 receptors alone and via the combination of M4, M2 and M3 receptors, when the superficial and deep layers, respectively, were activated. Inhibitory synaptic currents evoked from the superficial layer showed short-term depression, but they were unaffected by carbachol. In contrast, inhibitory currents triggered by the activation of the deep parts exhibited no significant short-term depression, but they were highly sensitive to cholinergic activation, which was mediated via M3 receptors. Our results indicate that pre- and postsynaptic muscarinic receptors mediate cholinergic modulation on giant cells. The present findings shed light on the cellular mechanisms of a tonic cholinergic modulation in the CN, which may become particularly important in evoking contralateral excitatory responses under certain pathological conditions.

Divergent cardiac effects of the first and second generation hypoglycemic sulfonylurea compounds.

The effects of first and second generation hypoglycemic sulfonylureas on the incidence of ventricular ectopic beats and on the duration of transitional ventricular fibrillation in the ischemic rat heart were investigated. First generation sulfonylurea compounds (tolbutamide, carbutamide and gliclazide) in 105 preparations increased, while second generation sulfonylurea compounds (glibenclamide and glipizide) in 50 preparations decreased in a dose-dependent manner both the number of ventricular ectopic beats and the duration of transitional ventricular fibrillation during the first 30 min after ligation of the left anterior descending coronary artery. Therefore, second generation sulfonylureas should be preferred in the treatment of type 2 diabetics with ischemic heart diseases, if satisfactory metabolic control cannot be achieved by a treatment regimen and diet alone.

Effects of first and second generation sulphonylureas on cardiotoxicity of strophanthidin in rabbits.

The effects of the first generation sulphonylurea compound gliclazide and the second generation sulphonylurea compound glipizide on strophanthidin toxicity was investigated in rabbits. The sulphonylurea pretreated animals were intravenously infused with 23 mumol/kg strophanthidin until the appearance of the first ventricular ectopic beat and continued thereafter until the appearance of ventricular fibrillation. The first generation sulphonylurea gliclazide increased, while the second generation sulphonylurea glipizide decreased the strophanthidin toxicity in a dose dependent manner. It was concluded that instead of first generation sulphonylureas, second generation sulphonylureas must be preferred in cardiac glycoside treated diabetics, when sulphonylurea treatment is necessary.

Insulin induced reversibility of altered responsiveness in femoral arterial bed of diabetic dogs.

The altered reactivities of femoral arterial bed to noradrenaline, phenylephrine, adenosine and prostacyclin were compared in 18, clinically manifest but aketotic, alloxan diabetic mongrel dogs. Alloxan treatment markedly increased the vasoconstrictor responses to noradrenaline and phenylephrine, as well as the adenosine-induced vasodilation in the femoral vasculature. These changes were prevented or normalized, respectively, in the early or late insulin-treated alloxan diabetic animals. In the case of noradrenaline not only a normalization but also an explicit overcompensation could be observed by insulin treatment. The altered reactivity to prostacyclin could not be influenced by insulin therapy. These results indicate a significant difference in the effect of insulin treatment on the altered diabetic vascular responsiveness to catecholamines and adenosine or to prostacyclin.

Relationship between vascular adrenergic receptors and prostaglandin biosyntheses in canine diabetic coronary arteries.

Before the onset of histologically detectable alterations of diabetic arteries, a considerable decrease of vasodilation ability develops. The role of an altered prostaglandin biosynthesis in this phenomenon was investigated in connection to the altered vascular adrenergic mechanisms. The effect of phenylephrine on prostacyclin production of isolated coronary arterial rings (100 mumol/l) as well as on conductivity of the coronary arterial bed (7.5-15-30-60 pmol. kg-1.min-1) were compared in 12 metabolically healthy and 12 alloxan-diabetic (560 mumol/kg) dogs. Furthermore, the effect of phentolamine (5 mumol/l) on the prostacyclin and thromboxane productions of the isolated vessels (coronary, femoral and basilar arteries) was investigated by radioimmunoassay. Although the basal prostacyclin amounts synthesized by healthy and diabetic coronary vessels were not different (5.1 +/- 1.6 and 4.9 +/- 1.4 pg/mg vessel/30 min), similarly to femoral and basilar arteries, the diabetic arterial rings produced significantly (p less than 0.05) more thromboxane than the control rings. The alpha-adrenergic blockade by phentolamine did not influence the prostacyclin production in the healthy arteries, but considerably (p less than 0.05) increased it in the diabetic coronary arteries. Phentolamine normalised the thromboxane synthesis in the diabetic group (p less than 0.01) and enhanced (p less than 0.05) it in the metabolically healthy group. Phenylephrine was ineffective (98 +/- 6%) on the prostacyclin production in vitro versus the stimulated (150 +/- 22%) prostacyclin synthesis detected in the metabolically healthy group; and in vivo induced a more significant (p less than 0.05) decrease in the coronary conductivity in diabetic than in control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of diabetic state on coronary vasoregulation in myocardial hypoxia.

It is a matter of common knowledge that under ischaemic or hypoxic conditions the oxygen demand of the myocardium could only be satisfied by enhancement of collateral blood flow. It is demonstrated that in a diabetic state an elevation of the myocardial blood flow could not develop due to lack of proper vasodilation in the coronary arterial bed.

Effects of hypoxia and adrenergic stimulation induced alterations in PGI2 synthesis by diabetic coronary arteries.

Before the onset of histologically detectable alterations in diabetic arteries, a considerable decrease in vasodilatory potential is seen. While analyzing this phenomenon, the role of altered PGI2 synthesis in rings of coronary arteries from metabolically healthy and alloxan-diabetic dogs was measured by radioimmunoassay during baseline, under the influence of phenylephrine (100 mumol/L), and during hypoxia with or without the presence of the alpha adrenergic blocker phentolamine (5 mumol/L). Basal levels of PGI2 synthetized by healthy and diabetic coronaries were no different (7.9 +/- 2.1 and 6.4 +/- 1.4 pg/mg vessel). Phenylephrine potentiated PGI2 synthesis in controls (150 +/- 22%), while it proved to be ineffective in the diabetic animals (98 +/- 6%). Under hypoxic conditions, PGI2 production of healthy coronaries (152 +/- 24%) increased, while that in the diabetic ones (82 +/- 7%) decreased (p less than 0.01). In the presence of phentolamine no difference could be detected between the two groups. Given all these data, the decreased ability of the diabetic coronaries to vasodilate develops due to diminished PGI2 production, presumably controlled by adrenergic mechanisms. Furthermore, the more severe outcome of ischaemic heart disease in diabetes mellitus might be explained by the lack of an enhanced coronary PGI2 synthesis under hypoxic conditions.

Prostaglandins and altered diabetic vasoregulation.

In previous studies an enhanced tendency to vasconstriction could be demonstrated in special regions, first of all in the coronary arterial bed of the diabetic vasculature. Analysing the role of prostaglandins in this phenomenon, the present work demonstrates that in vivo the dose-dependent decrease in the conductivity of the coronary arterial bed induced by PGF2 alpha (3, 6, 12, 24, 48 nmol/kg) administration in alloxan- (560 mumol/kg) diabetic dogs (n = 6) proved to be more expressed after indomethacin (10 mumol/kg) treatment compared with metabolically healthy (n = 6) animals. In the presence of indomethacin (3 mumol/l) PGF2 alpha (1, 3, 10, 30 mumol/l) evoked also considerably higher vasoconstriction in the isolated coronary rings of the diabetic dogs in comparison to the metabolically healthy state. The data are referring to the contribution of an altered prostaglandin biosynthesis in the diabetic coronaries to their increased tendency to vasoconstriction induced by PGF2 alpha.

The effect of various hypoglycaemic sulphonylureas on the cardiotoxicity of glycosides and arrhythmogenic activity due to myocardial ischaemia.

The effects of different sulphonylureas on the electrical cardiac activity were studied in 145 rabbits and in 103 rats as well as in 278 digitalis-treated, non-smoker non-insulin-dependent diabetics on the same therapy at least during the previous three months. In rabbits and rats glibenclamide (0.0032-100 mumol. kg-1) decreased, while tolbutamide and carbutamide (0.008-1000 mumol. kg-1) increased strophantidin toxicity and myocardial ischaemia induced transitory ventricular fibrillation dose-dependently. The differences between the dose-response curve of glibenclamide and those of tolbutamide or carbutamide were significant. In digitalized non-insulin-dependent diabetics, multifocal ectopic ventricular beats could be observed in none among the 80 glibenclamide-treated diabetics, while in 12 cases of the 71 tolbutamide and in 10 cases of the 61 carbutamide treated diabetics. Two of the 66 non-insulin-dependent diabetics receiving only diet and 7 of the 278 age and sex matched, non-smoker, metabolically healthy patients had multifocal ectopic ventricular beats. No significant difference could be found between the therapeutical groups. It was concluded that instead of tolbutamide, glibenclamide must be preferred in digitalis-treated diabetics, when metabolic control is not satisfactorily achieved by diet and regime alone.

Effects of computer controlled submaximal glucose utilization on myocardial energy potential and on survival time during acute strophantin intoxication in dogs.

We checked the effect of a 4 hourly computer controlled submaximal glucose utilization (CCSGU) of 12.01 +/- 1.19 mg/kg min under normoglycaemic conditions and of a simultaneous diminishing of myocardial NEFA supply on trigger mechanism of ventricular fibrillation during acute strophantin intoxication (4 micrograms/kg min) in 17 mongrel dogs. Dogs treated with CCSGU (protective group, n = 8) showed a nearly 30% (p less than 0.01) major survival time (47.6 +/- 3.6 min) before ventricular fibrillation occurred in comparison to a control group (33.1 +/- 3.7 min, n = 9). CCSGU induced a 90% higher left ventricular hydraulic work (5.18 +/- 0.57 Nm/g heart weight) during strophantin infusion compared to controls (2.75 +/- 0.47). No significant myocardial NEFA extraction was evident in the protective group. During CCSGU myocardial oxygen extraction was on a lower level in rest (15.8 +/- 1.0%, p less than 0.01) as well as during strophantin infusion (11.3 +/- 2.6%, p less than 0.01) compared to controls. In dogs treated with CCSGU nearly equal myocardial levels of HEP and lactate were found compared to controls in spite of a major survival time and higher left ventricular hydraulic work. A higher myocardial glycogen content was observed in the protective group (45.1 +/- 6.7 mumol/g w.w.) in comparison to controls (28.1 +/- 2.9, p less than 0.05). Our results prove that CCSGU using the device system GLUCON induces a shift in substrate utilization from NEFA to glucose, decreases myocardial oxygen extraction, increases myocardial glycogen content, enlarges heart work and protects against strophantin induced ventricular fibrillation.

Effect of acute hypoxia on cardiac function in alloxan-diabetic dogs.

The aim of the present study was to examine the left ventricular and haemodynamic parameters during acute hypoxia induced by carbon monoxide intoxication and/or after norepinephrine administration in metabolically healthy and alloxan-diabetic dogs. In metabolically healthy animals after carbon monoxide intoxication followed by norepinephrine administration myocardial oedema occurred with a concomitant elevation of diastolic stiffness in the left ventricular wall, resulting in impaired cardiac performance. In the alloxan-diabetic animals no myocardial oedema developed and therefore no further increase of the originally elevated left ventricular diastolic stiffness could be observed. Close correlations were demonstrable between the enhanced water content and diastolic stiffness of the left ventricular wall in metabolically healthy animals as well as between the myocardial contractile force and cardiac output index in both control and diabetic dogs. In contrast to the controls, following norepinephrine administration per se decrease of cardiac performance could be observed in the alloxan-diabetic animals. Based on these results, it can be concluded that an increase of left ventricular diastolic stiffness impairs the cardiac performance in acute hypoxia following norepinephrine administration both in control and in diabetic animals, even if different reasons are responsible for it.