Effect of corticotropin releasing hormone and corticotropin releasing hormone antagonist on biosynthesis of gonadotropin relasing hormone and gonadotropin relasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin.

This study aimed to determine the mechanisms governing Gonadotropin releasing hormone (GnRH) biosynthesis and luteinising hormone (LH) secretion in follicular-phase sheep after infusion of corticotropin releasing hormone (CRH) and/or CRH antagonist corticotropin releasing hormone nist (CRH-A) into the third cerebral ventricle. The study included two experimental approaches: first, we investigated the effect of CRH or CRH-A (α-helical CRH 9-41) on GnRH and GnRH receptor (GnRHR) biosynthesis in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VMH), stalk/median eminence (SME), and on GnRHR in the anterior pituitary (AP) using an enzyme-linked immunosorbent assay (ELISA); second, we used real-time PCR to analyse the influence of CRH and CRH-A on the levels of kisspeptin (Kiss1) mRNA in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by radioimmunoassay (RIA) and ELISA methods for measurement of LH and cortisol levels in the blood, respectively. Our results show that administration of CRH significantly decreased GnRH biosynthesis in the POA/hypothalamus. CRH also decreased GnRHR abundance in the hypothalamus and in the AP, but increased it in the POA. Furthermore, administration of CRH decreased plasma LH concentration and levels of Kiss1 mRNA in the POA and VMH/ARC as well as Kiss1r mRNA in these structures and in the SME. Significant increase in plasma cortisol concentration in the group treated with CRH was also observed. For CRH-A, all analysed effects were opposite to those induced by CRH. The study demonstrates that intracerebroventricular (i.c.v.) infusion of both CRH and CRH-A affects the GnRH/GnRHR biosynthesis and LH secretion in follicular-phase sheep conceivably via either central and peripheral mechanisms including Kiss1 neurons activity and cortisol signals. It has also been suggested that CRH and CRH-A infusion probably had effects directly at the AP.

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states.

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal's physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

Propofol modulates phasic and tonic GABAergic currents in spinal ventral horn interneurones.

BACKGROUND: Surgical interventions like skin incisions trigger withdrawal reflexes which require motor neurones and local circuit interneurones in the spinal ventral horn. This region plays a key role in mediating immobilizing properties of the GABAergic anaesthetic propofol. However, it is unclear how propofol modulates GABA(A) receptors in the spinal ventral horn and whether tonic or phasic inhibition is involved. METHODS: Organotypic spinal cord tissue slices were prepared from mice. Whole-cell recordings were performed for quantifying effects of propofol on GABA(A) receptor-mediated phasic transmission and tonic conductance. RESULTS: Propofol increased GABAergic phasic transmission by a prolongation of the decay time constant in a concentration-dependent manner. The amount of the charge transferred per inhibitory post-synaptic current, described by the area under the curve, was significantly augmented by 1 µM propofol (P<0.01). A GABA(A) receptor-mediated tonic current was not induced by 1 µM propofol but at a concentration of 5 µM (P<0.05). CONCLUSIONS: Propofol depresses ventral horn interneurones predominantly by phasic rather than by tonic GABA(A) receptor-mediated inhibition. However, the present results suggest that the involvement of a tonic inhibition might contribute to the efficacy of propofol to depress nociceptive reflexes at high concentrations of the anaesthetic.

4-bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor-mediated tonic conductance.

BACKGROUND AND PURPOSE: Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition. EXPERIMENTAL APPROACH: The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings. KEY RESULTS: Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 µM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected. CONCLUSIONS AND IMPLICATIONS: The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

ALX 1393 inhibits spontaneous network activity by inducing glycinergic tonic currents in the spinal ventral horn.

BACKGROUND: Strychnine-sensitive glycine receptors are activated by glycine and facilitate chloride influx into neurons. Glycinergic transmission might be either mediated by synaptic or extrasynaptic glycine receptors. While phasic neurotransmission is provided by a synaptic pathway, activation of extrasynaptic glycine receptors induces tonic inhibition. The glycine transporter 2 (GlyT2) regulates the uptake of glycine into presynaptic boutons. It is not determined yet, whether inhibition of GlyT2 by ALX 1393 can produce inhibition of spinal motoric networks and, whether phasic or tonic glycinergic inhibition is mostly enhanced. METHODS: We investigated the effect of ALX 1393 on spontaneous action potential firing activity by extracellular recordings in the ventral horn area of organotypic spinal cultures. Additionally, using the whole-cell patch-clamp technique, we defined the influence of GlyT2 inhibition on tonic and phasic glycinergic transmission in commissural interneurons of the ventral horn. RESULTS: GlyT2 inhibition by ALX 1393 potently reduced neuronal action potential activity in a concentration-dependent manner (n=211). The half maximal effect of ALX 1393 was observed at 100 ± 31 nM. Moreover, 88.3 ± 2.6% of the action potential activity was suppressed at 1 µM. Whole-cell patch-clamp recordings unveiled that ALX 1393 (200 nM) induced a tonic current (-45.7 ± 11.6 pA, n=5) that was significantly reversed by application of the competitive glycine receptor antagonist strychnine. Contrastingly, phasic glycinergic transmission was not augmented by GlyT2 inhibition (charge transferred per time period for control conditions: 1.1 ± 0.1 pC, n=7, for ALX 1393: 0.9 ± 0.2 pC, n=7, p>0.05). CONCLUSION: GlyT2 inhibition induced glycinergic tonic currents, which might be the underlying mechanism for the observed suppression of spontaneous action potential activity by ALX 1393 in the spinal ventral horn. Silencing neuronal action potential activity by blocking GlyT2 might be a novel principle to inhibit locomotor circuits in the ventral horn area and to induce muscle relaxation.

Suppressive effect of electromagnetic field on analgesic activity of tramadol in rats.

The electromagnetic fields (EMFs) have been shown to alter animal and human behavior, such as directional orientation, learning, pain perception (nociception or analgesia) and anxiety-related behaviors. The aim of this study was to evaluate the influence of electromagnetic fields of high-frequency microwaves on pain perception and anti-nociceptive activity of tramadol (TRAM) - analgetic effective in the treatment of moderate to severe acute and chronic pain states. Electromagnetic fields exposures of a)1500 MHz frequency and b) modulated, 1800 MHz (which is identical to that generated by mobile phones) were applied. Paw withdrawal latency (PWL) to thermal stimulus was measured in vehicle or tramadol (TRAM) treated animals before and after 30, 60 and 90 minutes from injections. The differences in the level of pain (PWL) between control group and rats exposed to EMF alone in three measurements, were not observed. Tramadol alone significantly increased PWLs to thermal stimulus in comparison to vehicle results at 30 (p < 0.001) and 60 minutes (p < 0.05) after drug injection. EMF exposure of both frequencies transiently suppressed analgesic effect of tramadol, significantly reducing paw withdrawal latency in animals treated with this drug at 30 minutes from the drug injection.

Organotypic cultures as tools for testing neuroactive drugs - link between in-vitro and in-vivo experiments.

The development of neuroactive drugs is a time consuming procedure. Candidate drugs must be run through a battery of tests, including receptor studies and behavioural tests on animals. As a rule, numerous substances with promising properties as assessed in receptor studies must be eliminated from the development pipeline in advanced test phases because of unforeseen problems like intolerable side-effects or unsatisfactory performance in the whole organism. Clearly, test systems of intermediate complexity would alleviate this inefficiency. In this review, we propose cultured organotypic brain slices as model systems that could bridge the 'interpolation gap' between receptors and the brain, with a focus on the development of new general anaesthetics with lesser side effects. General anaesthesia is based on the modulation of neurotransmitter receptors and other conductances located on neurons in diverse brain regions, including cerebral cortex and spinal cord. It is well known that different components of general anaesthesia, e.g. hypnosis and immobility, are produced by the depression of neuronal activity in distinct brain regions. The ventral horn of the spinal cord is an important structure for the induction of immobility. Thus, the potentially immobilizing effects of a newly designed drug can be estimated from its depressant effect on neuronal network activity in cultured spinal slices. A drug's sedative and hypnotic potential can be examined in cortical cultures. Combined with genetically engineered mice, this approach can point to receptor subtypes most relevant to the drug's intended net effect and in return can help in the design of more selective drugs. In conclusion, the use of organotypic cultures permits predictions of neuroactive properties of newly designed drugs on an intermediate level, and should therefore open up avenues for a more creative and economic drug development process.

Depression of spinal network activity by thiopental: shift from phasic to tonic GABA(A) receptor-mediated inhibition.

Interneuronal networks in the spinal ventral horn are plausible substrates for mediating anesthetic-induced immobility. Here, we investigated how their activity is affected by clinically relevant concentrations of thiopental, a barbiturate in clinical use. In cultured spinal cord slices from mice, thiopental reduced action potential activity with an EC(50) of 16.6+/-2.4microM. Recordings of GABA(A) and glycine receptor-mediated inhibitory currents indicated that the effect was largely mediated by GABA(A) receptors and that glycine receptors were not relevant targets. Specifically, 20microM thiopental prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) more than twofold. Although this prolongation of decay time increased the inhibitory charge per sIPSC the concomitant strong reduction of sIPSC frequency resulted in less inhibitory current entering the neurons via this route. However, 20microM thiopental also induced a tonic current of 30+/-10pA, mediated by GABA(A) receptors; 50microM thiopental nearly abolished sIPSC activity but augmented tonic currents to 69+/-14pA. Furthermore, at this concentration, activity-depressing mechanisms independent of GABA(A) receptors came into play. The results suggest that in the spinal ventral horn thiopental acts mostly, but not exclusively, via GABA(A) receptors. With increasing concentrations of the drug, inhibition via sIPSCs is limited by negative feedback on interneuronal firing whereas action potential-independent GABAergic inhibition due to tonic currents gains progressively in impact.

Inhibitory ligand-gated ion channels as substrates for general anesthetic actions.

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

Effects of isoflurane and enflurane on GABAA and glycine receptors contribute equally to depressant actions on spinal ventral horn neurones in rats.

BACKGROUND: Volatile anaesthetics are widely used agents in clinical anaesthesia, although their mechanism of action is poorly understood. In particular, the dominant molecular mechanisms by which volatile anaesthetics depress spinal neurones and thereby mediate spinal effects such as immobility have recently become a matter of dispute. As GABAA and glycine receptors are potential candidates we investigated the impact of both receptor systems in mediating the depressant effects of isoflurane and enflurane on spinal neurones in rats. METHODS: The effects of isoflurane and enflurane on spontaneous action potential firing were investigated by extracellular voltage recordings from ventral horn interneurones in cultured spinal cord tissue slices obtained from embryonic rats (E 14-15). RESULTS: Isoflurane and enflurane reduced spontaneous action potential firing. Concentrations causing half-maximal effects (isoflurane: 0.17 mM; enflurane: 0.50 mM) were less than EC50-immobility (isoflurane: 0.32 mM; enflurane: 0.62 mM). Effects of isoflurane were mediated by 39% by glycine receptors and 36% by GABAA receptors. The effects of enflurane were mediated 26% by GABAA receptors and 29% by glycine receptors. CONCLUSION: These results demonstrate that the effects of isoflurane and enflurane on GABAA and glycine receptors contribute almost equally to their depressant actions on spinal ventral horn neurones in rats. The fraction of inhibition mediated by both receptor systems differs between specific volatile anaesthetics. Our data argue against the theory that a dominant molecular mechanism accounts for spinal effects of volatile anaesthetics.

[The GABA(A) receptor family: possibilities for the development of better anesthetics]. / Die GABA(A)-Rezeptor-Familie: Möglichkeiten für die Entwicklung besserer Anästhetika.

Clinically used anesthetics show amnestic, sedative, hypnotic and immobilizing properties. On a molecular level these drugs affect several receptors in the cell membrane of neurons. By using genetically engineered mice a linkage can now be made between actions on certain receptors and clinically desired and undesired effects. Experiments show that a certain GABA(A) receptor subtype mediates hypnosis and immobility, whereas another subtype is involved in side-effects like sedation and hypothermia. These findings form the basis for the development of new drugs, acting highly specific and with fewer side-effects.

Psychosomatic reactions to a stressful environment and an attempt at pharmacological modification.

BACKGROUND: Extreme environmental conditions significantly influence the functioning of the human organism and trigger distinct stress reactions. In our study we attempted to create an experimental model of complex stress conditions. MATERIAL AND METHODS: Healthy male volunteers were isolated, deprived of food and sleep, and exposed to extreme temperatures for 5 consecutive days. Physical fitness and selected somatic parameters and biochemical stress markers were measured in the tested subjects. In addition, changes in behavior and mental status were assessed by means of a set of psychological tests. Finally, the effects of pharmacological modification (administration of clobazam and tramadol) on psychosomatic stress reactions were tested. CONCLUSIONS: The results indicate that our experimental stress conditions slightly altered the mental functions of the subjects, increased their anxiety level, hampered their physical efficiency, and led to weight loss. The administration of the drugs beneficially influenced the subjects' memory and physical efficiency.

How do general anaesthetics work?

Almost a century ago, Meyer and Overton discovered a linear relationship between the potency of anaesthetic agents to induce general anaesthesia and their ability to accumulate in olive oil. Similar correlations between anaesthetic potency and lipid solubility were later reported from investigations on various experimental model systems. However, exceptions to the Meyer-Overton correlation exist in all these systems, indicating that lipid solubility is an important, but not the sole determinant of anaesthetic action. In the mammalian central nervous system, most general anaesthetics act at multiple molecular sites. It seems likely that not all of these effects are involved in anaesthesia. GABAA- and NMDA-receptor/ion channels have already been identified as relevant targets. However, further mechanisms, such as a blockade of Na+ channels and an activation of K+ channels, also come into play. A comparison of different anaesthetics seems to show that each compound has its own spectrum of molecular actions and thus shows specific, fingerprint-like effects on different levels of neuronal activity. This may explain why there is no known compound that specifically antagonises general anaesthesia. General anaesthesia is a multidimensional phenomenon. Unconsciousness, amnesia, analgesia, loss of sensory processing and the depression of spinal motor reflexes are important components. It was not realised until very recently that different molecular mechanisms might underlie these different components. These findings challenge traditional views, such as the assumption that one anaesthetic can be freely replaced by another.

[Neural mechanisms of anesthesia]. / Neuronale Mechanismen der Narkose.

Positron emission tomography studies on volunteers showed that, at concentrations inducing the loss of consciousness, propofol, halothane and isoflurane reduce glucose metabolism of neocortical neurones by 20-50%. To find out whether these effects are caused by direct anaesthetic actions on cortical structures, experiments were carried out on isolated neocortical brain slices. In these investigations an excellent correlation was observed between anaesthetic concentrations causing a half-maximal depression of action potential firing in neocortical brain slices and anaesthetic blood concentrations monitored during awaking from anaesthesia in humans. Furthermore, it could be shown that, at concentrations approximately one half the MAC-value, isoflurane decreases the frequency of auditory evoked 30-40 Hz oscillations in the neocortex by 50%. Similar quantitative effects were observed on spontaneously occurring high frequency rhythms in neocortical brain slices. However, not all aspects of cerebral anaesthetic actions can be explained by direct effects on cortical neurones. The EEG synchronisation and the amplitude reduction of mid latency auditory evoked potentials are probably related to the inhibition of thalamic neurones. Halothane, isoflurane, enflurane and propofol reduced action potential firing of cortical neurones by enhancing GABAA receptor-mediated synaptic inhibition. This molecular mechanism seems also to be involved in depressing painful stimuli-induced motor responses. Nevertheless, there must be a difference between relevant anaesthetic mechanisms on the cerebral and spinal level. This follows from the observation that the relation between the concentration causing the loss of consciousness and the concentration that depresses movements considerably varies among different anaesthetic agents.

Different actions of general anesthetics on the firing patterns of neocortical neurons mediated by the GABA(A) receptor.

BACKGROUND: In cultured slice preparations of rat neocortical tissue, clinically relevant concentrations of volatile anesthetics mainly decreased action potential firing of neurons by enhancing gamma-aminobutyric acid (GABA(A)) receptor-mediated synaptic inhibition. The author's aim was to determine if other anesthetic agents are similarly effective in this model system and act via the same molecular mechanism. METHODS: The actions of various general anesthetics on the firing patterns of neocortical neurons were investigated by extracellular single-unit recordings. RESULTS: Pentobarbital, propofol, ketamine, and ethanol inhibited spontaneous action potential firing in a concentration-dependent manner. The estimated median effective concentration (EC50) values were close to or below the EC50 values for general anesthesia. Bath application of the GABA(A) antagonist bicuculline (100 microM) decreased the effectiveness of propofol, ethanol, halothane, isoflurane, enflurane, and diazepam by more than 90%, indicating that these agents acted predominantly via the GABA(A) receptor. The depressant effects of pentobarbital and ketamine were not significantly reduced by bicuculline treatment. Drugs acting mainly via the GABA(A) receptor altered the firing patterns of neocortical cells in different manners. Diazepam reduced the discharge rates by decreasing the number of action potentials per burst, leaving the burst rate unaffected. In contrast, muscimol, GABA, propofol, and volatile anesthetics decreased the burst rate. CONCLUSIONS: Although several anesthetic agents acted nearly exclusively via the GABA(A) receptor, they changed the discharge patterns of cortical neurons in different ways. This finding is explained by GABA-mimetic or benzodiazepine-like molecular interactions.

NMDA receptor-mediated changes of spontaneous activity patterns in thalamocortical slice cultures.

Spontaneous activity is a hallmark of the thalamocortical system in vivo. Up until now, in vitro preparations of this system have been shown to be spontaneously active only when inhibition was reduced or N-methyl-D-aspartate (NMDA) receptor-mediated currents were facilitated via low extracellular magnesium levels. This study investigated the dependence of spontaneous thalamocortical activity patterns on NMDA receptor function via variation of extracellular magnesium levels (0-1 mM) and by the application of the specific NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (AP5) in the absence of magnesium. We used cocultures of rat neocortical and thalamic slices which have been shown to develop reciprocal synaptic connections similar to those in vivo. Multi-site extracellular recordings revealed that the cultures were spontaneously active at all concentrations of magnesium and AP5, albeit with a high variability among cultures. Activity consisted of burst-like events which were largely synchronized within as well as among the neural tissues, and thalamic background activity during periods of neocortical quiescence. Each tissue was capable of triggering activity in the other, indicating that both thalamocortical and corticothalamic synaptic connections were functional. With increasing magnesium concentration, activity rates declined in both tissues and the site of origin of the synchronous, burst-like events shifted from neocortex to thalamus. AP5 in magnesium-free perfusion solution had qualitatively similar effects. We conclude that thalamic activity is not as dependent on the facilitation of NMDA receptor-mediated currents as neocortical activity and consequently, that the thalamus is the pacemaker of thalamocortical synchronized activity in physiological in vitro conditions.

Ultradian rhythms in Desmodium.

The leaves of Desmodium gyrans (L.F.) DC show circadian movements in the terminal and ultradian movements of the lateral leaflets. The movements are due to swelling and shrinking of motor cells in special organs. The anatomy of these pulvini is described for the lateral leaflets. Data from electrophysiological recordings using microelectrodes inserted into the lateral pulvini, together with treatments that affect the proton pumps and ion channels, have been used to develop a physiological model of the ultradian leaflet movement. It explains the oscillations in the motor cells as being due to a change between a pump state and depolarization. During the pump state, ions are taken up, causing water influx and swelling of the motor cells. Depolarization causes loss of ions and water efflux (the motor cells shrink). The roles of calcium and the phosphatidyl inositol signal chain are discussed on the basis of experiments using chemical agents that affect these processes. Since calcium oscillations are known to occur in organisms in both time and space, an attempt has been made to simulate the situation in Desmodium pulvini by a model of specially coupled oscillators. Effects of different other treatments of the lateral pulvini are discussed. Oscillations in the minute range seem to be more common and some might be related to turgor regulation and ion uptake comparable to the situation in Desmodium. The ultradian control of the lateral pulvini and the circadian control of the terminal pulvini are apparently based on different mechanisms.