The effect of long-term morphine application on clock genes expression in the rat brain

Cirkadiánní a opioidní systém jsou systémy podílející se na udržování homeostázy v organismu. Narušením cirkadiánního systému dochází k rozladění správného načasování fyziologických procesů, což může mít za následek vznik nebo zhoršení již existujících patologických stavů, včetně závislosti. Jedním z faktorů, které mohou ovlivňovat přesné nastavení cirkadiánního systému je i užívání a zneužívání opioidů. Vzájemný vztah cirkadiánního a opioidního systému je málo prozkoumán. Za tímto účelem byl v této práci sledován vliv morfinu a metadonu na cirkadiánní systém potkana v dospělosti i v průběhu vývoje. Cílem disertační práce bylo sledovat účinek akutního podání morfinu na expresi hodinových genů v suprachiasmatických jádrech (SCN) dospělých potkanů, zkoumat účinek dlouhodobé aplikace morfinu nebo metadonu a jejich vysazení na expresi hodinových genů v SCN a na aktivitu enzymu AA-NAT v epifýze dospělých potkanů. Správný vývoj cirkadiánních hodin významně přispívá k udržení zdraví v dospělosti a zajišťuje dobrou adaptabilitu organismu na změny vnějšího prostředí. Protože se dosud žádná studie nezaměřila na zkoumání účinků podávání opioidů v raných fázích vývoje na zrání cirkadiánních hodin v SCN nebo v periferních orgánech, bylo dalším cílem zkoumat účinek dlouhodobé aplikace morfinu nebo metadonu...The circadian and opioid systems are systems involved in maintaining homeostasis in the body. Disruption of the circadian system disturbs the proper timing of physiological processes, which can result in the development or exacerbation of pre-existing pathological conditions, including addiction. One of the factors that can influence the precise synchronization of the circadian system is the use and abuse of opioids. The interrelationship between the circadian and opioid systems is poorly studied. To this end, the present study investigated the effect of morphine and methadone on the rat circadian system in adulthood and during development. The aim of this dissertation was to observe the effect of acute morphine administration on the expression of clock genes in the suprachiasmatic nuclei (SCN) of adult rats, and to investigate the effect of long-term morphine or methadone administration and withdrawal on the expression of clock genes in the SCN and on the activity of the enzyme AA-NAT in the pineal gland of adult rats. Proper development of the circadian clock contributes significantly to the maintenance of health in adulthood and ensures good adaptability of the organism to changes in the external environment. No study to date has focused on examining the effects of opioid administration during...Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

Neonatal Clonazepam Administration Induces Long-Lasting Changes in Glutamate Receptors

γ-aminobutyric acid (GABA) pathways play an important role in neuronal circuitry formation during early postnatal development. Our previous studies revealed an increased risk for adverse neurodevelopmental consequences in animals exposed to benzodiazepines, which enhance GABA inhibition via GABAA receptors. We reported that administration of the benzodiazepine clonazepam (CZP) during postnatal days 7–11 resulted in permanent behavioral alterations. However, the mechanisms underlying these changes are unknown. We hypothesized that early CZP exposure modifies development of glutamatergic receptors and their composition due to the tight developmental link between GABAergic functions and maturation of glutamatergic signaling. These changes may alter excitatory synapses, as well as neuronal connectivity and function of the neural network. We used quantitative real-time PCR and quantitative autoradiography to examine changes in NMDA and AMPA receptor composition and binding in response to CZP (1 mg/kg/day) administration for five consecutive days, beginning on P7. Brains were collected 48 h, 1 week, or 60 days after treatment cessation, and mRNA subunit expression was assessed in the hippocampus and sensorimotor cortex. A separate group of animals was used to determine binding to NMDA in different brain regions. Patterns of CZP-induced alterations in subunit mRNA expression were dependent on brain structure, interval after CZP cessation, and receptor subunit type. In the hippocampus, upregulation of GluN1, GluN3, and GluR2 subunit mRNA was observed at the 48-h interval, and GluN2A and GluR1 mRNA expression levels were higher 1 week after CZP cessation compared to controls, while GluN2B was downregulated. CZP exposure increased GluN3 and GluR2 subunit mRNA expression levels in the sensorimotor cortex 48 h after treatment cessation. GluA3 was higher 1 week after the CZP exposure, and GluN2A and GluA4 mRNA were significantly upregulated 2 months later. Expression of other subunits was not significantly different from that of the controls. NMDA receptor binding increased 1 week after the end of exposure in most hippocampal and cortical areas, including the sensorimotor cortex at the 48-h interval. CZP exposure decreased NMDA receptor binding in most evaluated hippocampal and cortical areas 2 months after the end of administration. Overall, early CZP exposure likely results in long-term glutamatergic receptor modulation that may affect synaptic development and function, potentially causing behavioral impairment

Suprachiasmatic nuclei as a daily clock and calendar

The suprachiasmatic nuclei (SCN) harbor the master circadian pacemaker in mammals which is responsible for control and coordination of circadian rhythms throughout the body. They are a paired structure in the hypothalamus, located just above the optic chiasm, consisting of approximately 20 000 neurons. Due to their specific properties, the SCN have a unique position within the circadian system. They are connected with retina and, therefore, they can directly receive information about changes in external light/dark cycle. The individual SCN neurons are independent autonomous circadian oscillators which are inter-connected in a communication network. This network allows the individual SCN oscillators to synchronize among each other and thus to increase the precision and robustness of the oscillations. This work is focused to summarize the knowledge on the structure and function of the SCN at the level of single cells, subpopulations of cells and the whole SCN. The specific goal of this work is a summary of the factors that determine their central role within the circadian system

The effect of long-term morphine application on clock genes expression in the rat brain

The circadian and opioid systems are systems involved in maintaining homeostasis in the body. Disruption of the circadian system disturbs the proper timing of physiological processes, which can result in the development or exacerbation of pre-existing pathological conditions, including addiction. One of the factors that can influence the precise synchronization of the circadian system is the use and abuse of opioids. The interrelationship between the circadian and opioid systems is poorly studied. To this end, the present study investigated the effect of morphine and methadone on the rat circadian system in adulthood and during development. The aim of this dissertation was to observe the effect of acute morphine administration on the expression of clock genes in the suprachiasmatic nuclei (SCN) of adult rats, and to investigate the effect of long-term morphine or methadone administration and withdrawal on the expression of clock genes in the SCN and on the activity of the enzyme AA-NAT in the pineal gland of adult rats. Proper development of the circadian clock contributes significantly to the maintenance of health in adulthood and ensures good adaptability of the organism to changes in the external environment. No study to date has focused on examining the effects of opioid administration during..

Circadian system of SHR rats

The suprachiasmatic nuclei (SCN) harbor the master circadian pacemaker in mammals which is responsible for control and coordination of circadian rhythms throughout the body and ensure optimal functioning of the organism in variable external conditions. The SCN is a heterogeneous structure in its morphology and function. The present thesis focuses on comparison of the SCN and its properties in an animal model of pathological hypertension, i.e. in spontaneously hypertensive rat (SHR), and nonpathological normotensive Wistar rat. To determine selected morphological and functional markers in the SCN between both rat strains, in situ hybridization and immunohistochemistry methods were used. To compare functional properties of the SCN of SHR and Wistar rat, the effect of light pulses on the expression of c-fos and Per1 genes as well as on locomotor activity were studied in both strains. Our results suggest morphological differences in the number of neurons and in the expression of Avp a Vip in the SCN with an apparent tendencies to higher levels of the studied peptides in SHR compared to Wistar rat. Our data further indicate variation in functioning of the SCN in SHR and Wistar rat on the basis of different responses to light pulses administered in the first part of the subjective night, namely in the..

Neonatal Clonazepam Administration Induced Long-Lasting Changes in GABAA and GABAB Receptors

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7–11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment

The day/night difference in the circadian clock's response to acute lipopolysaccharide and the rhythmic Stat3 expression in the rat suprachiasmatic nucleus.

The circadian clock in the suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behaviour and is an important part of the mammalian homeostatic system. Previously, we have shown that systemic inflammatory stimulation with lipopolysaccharide (LPS) induced the daytime-dependent phosphorylation of STAT3 in the SCN. Here, we demonstrate the LPS-induced Stat3 mRNA expression in the SCN and show also the circadian rhythm in Stat3 expression in the SCN, with high levels during the day. Moreover, we examined the effects of LPS (1mg/kg), applied either during the day or the night, on the rhythm in locomotor activity of male Wistar rats. We observed that recovery of normal locomotor activity patterns took longer when the animals were injected during the night. The clock genes Per1, Per2 and Nr1d1, and phosphorylation of kinases ERK1/2 and GSK3β are sensitive to external cues and function as the molecular entry for external signals into the circadian clockwork. We also studied the immediate changes in these clock genes expressions and the phosphorylation of ERK1/2 and GSK3β in the suprachiasmatic nucleus in response to daytime or night-time inflammatory stimulation. We revealed mild and transient changes with respect to the controls. Our data stress the role of STAT3 in the circadian clock response to the LPS and provide further evidence of the interaction between the circadian clock and immune system