Molecular actions of sex hormones in the brain and their potential treatment use in anxiety disorders.

Anxiety disorders are one of the most prevalent mood disorders that can lead to impaired quality of life. Current treatment of anxiety disorders has various adverse effects, safety concerns, or restricted efficacy; therefore, novel therapeutic targets need to be studied. Sex steroid hormones (SSHs) play a crucial role in the formation of brain structures, including regions of the limbic system and prefrontal cortex during perinatal development. In the brain, SSHs have activational and organizational effects mediated by either intracellular or transmembrane G-protein coupled receptors. During perinatal developmental periods, the physiological concentrations of SSHs lead to the normal development of the brain; however, the early hormonal dysregulation could result in various anxiety diorders later in life. Sex differences in the prevalence of anxiety disorders suggest that SSHs might be implicated in their development. In this review, we discuss preclinical and clinical studies regarding the role of dysregulated SSHs signaling during early brain development that modifies the risk for anxiety disorders in a sex-specific manner in adulthood. Moreover, our aim is to summarize potential molecular mechanisms by which the SSHs may affect anxiety disorders in preclinical research. Finally, the potential effects of SSHs in the treatment of anxiety disorders are discussed.

On the role of sex steroids in biological functions by classical and non-classical pathways. An update.

The sex steroid hormones (SSHs) play several roles in regulation of various processes in the cardiovascular, immune, muscular and neural systems. SSHs affect prenatal and postnatal development of various brain structures, including regions associated with important physiological, behavioral, cognitive, and emotional functions. This action can be mediated by either intracellular or transmembrane receptors. While the classical mechanisms of SSHs action are relatively well examined, the physiological importance of non-classical mechanism of SSHs action through membrane-associated and transmembrane receptors in the brain remains unclear. The most recent summary describing the role of SSHs in different body systems is lacking. Therefore, the aim of this review is to discuss classical and non-classical signaling pathways of testosterone and estradiol action via their receptors at functional, cellular, tissue level and to describe the effects on various body systems and behavior. Particular emphasis will be on brain regions including the hippocampus, hypothalamus, frontal cortex and cerebellum.

Lack of M4 muscarinic receptors in the striatum, thalamus and intergeniculate leaflet alters the biological rhythm of locomotor activity in mice.

The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR -/- mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

Variability in the Drug Response of M4 Muscarinic Receptor Knockout Mice During Day and Night Time.

Mice are nocturnal animals. Surprisingly, the majority of physiological/pharmacological studies are performed in the morning, i.e., in the non-active phase of their diurnal cycle. We have shown recently that female (not male) mice lacking the M4 muscarinic receptors (MR, M4KO) did not differ substantially in locomotor activity from their wild-type counterparts (C57Bl/6Tac) during the inactive period. Increased locomotion has been shown in the active phase of their diurnal cycle. We compared the effects of scopolamine, oxotremorine, and cocaine on locomotor response, hypothermia and spontaneous behavior in the open field arena in the morning (9:00 AM) and in the evening (9:00 PM) in WT and in C57Bl/6NTac mice lacking the M4 MR. Furthermore, we also studied morning vs. evening densities of muscarinic, GABAA, D1-like, D2-like, NMDA and kainate receptors using autoradiography in the motor, somatosensory and visual cortex and in the striatum, thalamus, hippocampus, pons, and medulla oblongata. At 9:00 AM, scopolamine induced an increase in motor activity in WT and in M4KO, yet no significant increase was observed at 9:00 PM. Oxotremorine induced hypothermic effects in both WT and M4KO. Hypothermic effects were more evident in WT than in M4KO. Hypothermia in both cases was more pronounced at 9:00 AM than at 9:00 PM. Cocaine increased motor activity when compared to saline. There was no difference in behavior in the open field between WT and M4KO when tested at 9:00 AM; however, at 9:00 PM, activity of M4KO was doubled in comparison to that of WT. Both WT and KO animals spent less time climbing in their active phase. Autoradiography revealed no significant morning vs. evening difference. Altogether, our results indicate the necessity of comparing morning vs. evening drug effects.

Ascorbic acid and alpha-tocopherol protect age-dependently from hypoxia-induced changes of cortical excitability in developing rats.

OBJECTIVES: The effects of ascorbic acid and α-tocopherol pre-treatment on hypoxia induced changes in brain cortex excitability were tested in immature rats exposed chronically to simulated altitude of 7 000 m. METHODS: Rat pups were kept together with their mothers for 8 hours a day in hypobaric chamber since the day of the birth till the postnatal day 11 or 17. Each day immediately before placing to hypobaric chamber pups were pretreated intraperitoneally either with ascorbic acid (100 mg/kg) or α-tocopherol (1 500 mg/kg). Cortical afterdischarges were elicited by repeated stimulation of the right sensorimotor cortex. The duration of evoked cortical afterdischarges was analyzed. RESULTS: Duration of cortical afterdischarges progressively declines with age. Hypoxia prolonged the duration of afterdischarges in 12-, 18- and 25-day-old animals. Pretratment with ascorbic acid or α-tocopherol shorted afterdischarges duration in youngest experimental group when compared with animals exposed to hypoxia only. CONCLUSION: Hypoxia significantly affects the brain cortex excitability by prolonging afterdischarges duration. This effect differs with age. Antioxidant pre-treatment brought about shorter duration of cortical afterdischarges only in the youngest experimental group. The antioxidant effect is therefore age dependent.

Impact of chronic ethanol intake of rat mothers on the seizure susceptibility of their immature male offspring.

The aim of present study was to examine the impact of prenatal ethanol exposure on seizure susceptibility of the offspring. Pregnant Wistar rats were compelled to drink either 10% or 20% ethanol solution, as the only drinking fluid since conception up to the weaning of their offspring at the age of 28 days. Pregnant and nursing rats of the control group drank water. Electrophysiological experiments (repeated electrical stimulation and analysis of cortical afterdischarges duration) were than performed on their immature offspring. Rat pups were tested on postnatal day 18, 25, and 35. Shortening of afterdischarges duration was observed in 18-day-old animals (mothers drank 20% ethanol) when compared with age matched controls and failure of post-ictal depression phenomenon was found in 25- and 35-day-old animals. Our findings signalize that ethanol exposure during pregnancy influences seizure susceptibility by acting on excitatory/inhibitory brain systems and this effect is dose- and age-dependent.

Beta3 adrenoceptors substitute the role of M(2) muscarinic receptor in coping with cold stress in the heart: evidence from M(2)KO mice.

We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

Nicotine reduces mortality of developing rats exposed to high-altitude hypoxia and partially suppresses the duration of cortical epileptic afterdischarges.

Nicotine has been repeatedly reported as substance possessing neuroprotective properties. This study focused on the possible beneficial effects of nicotine against the high-altitude hypoxia (9000 m for one hour). 15 min prior to hypoxia exposition rats (12- and 35-day-old) were treated with nicotine. Next day electrodes have been implanted and the effects of nicotine and hypoxia (or both factors) on duration of afterdischarges (ADs) were tested. Administration of nicotine declined the hypoxia-induced mortality in 35-day-old animals. Nicotine pretreatment had no effect on ADs duration in 12-day-old pups, therefore brought about suppression of ADs in 35-day-old animals. Taken together, our data show that nicotine exhibits an anticonvulsant effect that is age-dependent. The mechanisms of nicotine neuroprotective properties include probably the influence of calcium homeostasis, increase synthesis of variety of growth factors, inhibition of the caspase cascades and antioxidant capability of nicotine.

Methylprednisolone reduces axonal impairment in the experimental model of brain oedema.

OBJECTIVES: In our earlier paper we demonstrated that opening of the blood-brain barrier with an osmotic insult induces brain oedema which represents a factor triggering axonal impairment accompanied with myelin disintegration. The aim of the present study was to find whether methylprednisolone can reduce such axonal impairment in our model of brain oedema. METHODS: Brain oedema was induced by osmotic blood-brain barrier opening with 20% mannitol applied selectively into the internal carotid. Axonal changes were recognized as signs of myelin disintegration (oedematous vesicles, varicosity, myelin fragmentation) at histological sections stained with Black Gold in hippocampal areas CA1 and CA3 and in the dentate gyrus at time intervals of 30 minutes (acute group) or one week (chronic group) after the blood-brain barrier opening. At the same time intervals methylprednisolone was applied in two different ways - into peritoneal cavity or into the right carotid artery. RESULTS: Impairment of the axonal integrity (changes of the myelin sheet integrity) was identified in all areas studied in both experimental groups. Whereas in the control group axons were of the uniform diameter, in the experimental groups various forms of myelin disintegration were observed. Methylprednisolone reduced the degree of myelin disintegration in both time intervals with the highest effect in the acute group with the intracarotic administration. CONCLUSION: Methylprednisolone can effectively reduce myelin changes accompanying brain oedema induced by blood-brain barrier opening with an osmotic insult.

Neuroprotective effect of nicotine against kainic acid excitotoxicity is associated with alpha-bungarotoxin insensitive receptors subtype of nAChRs.

OBJECTIVES: Our previous study showed that administration of nicotine is capable to protect the neurons of hippocampus against the kainic acid induced damage. Here we tested the hypotheses that multiple nicotine administration would prevent the effects of kainic acid on neuronal nicotinic receptor subtypes densities (α-bungarotoxin sensitive and α-bungarotoxin insentive) and on hippocampal cell degeneration. METHODS: Radioligand binding study was used to detect the particular nAChR subtypes densities. Two histochemical methods (bis-benzimide staining and Fluoro-Jade B dye) were used to detect and evaluate neuronal degeneration. RESULTS: Our study shows that: a) kainic acid single administration increased the number of α-bungarotoxin insentive nicotinic receptors, b) nicotine was able to prevent such changes, c) repeated nicotine administration is capable to attenuate the damage of CA1 and CA3 areas of the hippocampus. No effect on α-bungarotoxin sentive nicotinic receptors was observed. Our data therefore reveal the importance of α-bungarotoxin insentive nicotinic receptors in the response to kainite and the ability of nicotine to prevent such changes both in the cell degeneration and in number of receptors. CONCLUSION: Nicotine administration influences α-bungarotoxin insensitive receptors and repeated administration is capable to protect against toxicity caused by kainic acid in hippocampal area.

Time-dependent axonal impairment in experimental model of brain oedema.

OBJECTIVES: Clinically very serious condition of ischaemia and brain injury which are often associated with brain oedema is frequently accompanied by the impairment of the structural integrity of axons. We wondered whether the brain oedema (without ischemia brain injury) can induce structural axonal impairment. METHODS: Brain oedema was induced by osmotic blood-brain barrier opening with 20% mannitol applied selectively into the internal carotid. Axonal changes were recognized as signs of myelin disintegration (oedematous vesicles, varicosity, myelin fragmentation) at histological sections stained with Black Gold in hippocampal areas CA1 and CA3 and in the dentate gyrus and cerebral cortex at time intervals of one hour, one day, three days and one week after the oedema induction. RESULTS: Impairment of the structural integrity was identified in myelin sheets in all areas studied in all experimental groups. Whereas in the control group axon were of the uniform diameter, in the experimental groups various forms of myelin disintegration were observed. The progression of myelin damage depended on the time elapsed after the oedema induction. CONCLUSION: Opening the blood-brain barrier with an osmotic insult induces brain oedema which represents a factor triggering axonal impairment accompanied with myelin changes. The development of axonal changes initiated by brain oedema only (without ischemia brain injury) is a novel observation.

Melatonin modulates hypoxia-induced changes of rat brain excitability.

The aim of our study was to test the hypothesis, if melatonin pre-treatment (in dose of 100 mg/kg) can influence the changes of brain function after short-term hypoxia exposition (simulated altitude 9000 m) in young immature rats. Experiments were performed on freely moving 12-, 25- and 35-day-old male Wistar rats. One hour prior to hypoxia exposition, animals were pre-treated with melatonin and 24 hours after hypoxia cortical afterdischarges (ADs) were elicited by repeated stimulation of the right sensorimotor cortex. The duration of evoked ADs and shape of evoked graphoelements was monitored. Short-term exposure to hypoxic conditions resulted in significantly shorter ADs duration in 12-day-old rats after stimulations (except the 2nd one stimulation) compared to control group. Administration of melatonin prolonged the duration of ADs after all stimulations except the 1st one. Analysis of the duration ADs revealed no significant changes, either after the exposition to hypobaric hypoxia or after melatonin administration in 25- and 35-day-old animals. Effects and mechanisms of melatonin action on the brain seizure susceptibility and the possible beneficial role of that treatment in hypoxic brain damage are discussed.

Nicotine effects on rat seizures susceptibility and hippocampal neuronal degeneration.

OBJECTIVES: Nicotine is a widely used drug of abuse exerting number of effects on the central nervous system. The study was aimed at the effects of nicotine in various doses on the excitability of cerebral cortex and - by using the methods of histochemistry - to identify possible signs of neuronal degeneration after nicotine administration. METHODS: Cortical afterdischarges were elicited by repeated stimulation of the right sensorimotor cortex. The duration of evoked ADs was monitored in animals treated with nicotine (0.75 or 1.0mg/kg) and in animals treated with saline. Methods of histochemistry (Fluoro-Jade B and bis-benzimide) were used to detect possible neuronal degeneration in hippocampus or in cerebral cortex. RESULTS: No Fluoro-Jade B positive cells were found in hippocampi of control animals, or animals treated with nicotine. ADs length was significantly influenced by administration of nicotine. CONCLUSION: Nicotine in 0.75 or 1.0 mg/kg dose leads to the decrease in ADs duration, influences the seizure susceptibility, and doesn't cause any detectable neuronal damage.

[Kainic acid and neurobiology]. / Kainát a neurobiologie.

Kainic acid, the analog of excitatory amino acid L-glutamate, interacts with specific receptors in the central nervous system. During last 25 years it has become a tool for studying many human brain disorders, for example human temporal lobe epilepsy, Huntington's chorea etc. Systemic administration of kainic acid results in neuronal death in experimental animals. The mechanism, by which kainic acid produces neuronal damage is still unclear, as well as physiological function of kainate receptors remain to be elucidate. This review attempts to survey the major achievements reached in the studies, which were publicized throw the last three decades.