Lack of CRH Affects the Behavior but Does Not Affect the Formation of Short-Term Memory.

Corticotropin-releasing hormone (CRH) is involved in modification of synaptic transmission and affects spatial discrimination learning, i.e., affects the formation of memory in long-term aspect. Therefore, we have focused on CRH effect on short-term memory. We have used stress task avoidance (maze containing three zones: entrance, aversive, and neutral) and compared the behavior and short-term memory in wild-type mice and mice lacking CRH (CRH KO) experiencing one 120-min session of restraint stress. As control, non-stressed animals were used. As expected, the animals that experienced the stress situation tend to spend less time in the zone in which the restraint chamber was present. The animals spent more time in the neutral zone. There were significant differences in number of freezing bouts in the aversive and entrance zones in CRH KO animals. CRH KO control animals entered the neutral zone much more faster than WT control and spent more time immobile in the neutral zone than WT control. These data give evidence that lacking of CRH itself improves the ability of mice to escape away from potentially dangerous area (i.e., those in which the scent of stressed animal is present).

Brain region-specific effects of immobilization stress on cholinesterases in mice.

Brain acetylcholinesterase (AChE) variant AChER expression increases with acute stress, and this persists for an extended period, although the timing, strain and laterality differences, have not been explored previously. Acute stress transiently increases acetylcholine release, which in turn may increase activity of cholinesterases. Also the AChE gene contains a glucocorticoid response element (GRE), and stress-inducible AChE transcription and activity changes are linked to increased glucocorticoid levels. Corticotropin-releasing hormone knockout (CRH-KO) mice have basal glucocorticoid levels similar to wild type (WT) mice, but much lower levels during stress. Hence we hypothesized that CRH is important for the cholinesterase stress responses, including butyrylcholinesterase (BChE). We used immobilization stress, acute (30 or 120 min) and repeated (120 min daily × 7) in 48 male mice (24 WT and 24 CRH-KO) and determined AChER, AChE and BChE mRNA expression and AChE and BChE activities in left and right brain areas (as cholinergic signaling shows laterality). Immobilization decreased BChE mRNA expression (right amygdala, to 0.5, 0.3 and 0.4, × control respectively) and AChER mRNA expression (to 0.5, 0.4 and 0.4, × control respectively). AChE mRNA expression increased (1.3, 1.4 and 1.8-fold, respectively) in the left striatum (Str). The AChE activity increased in left Str (after 30 min, 1.2-fold), decreased in right parietal cortex with repeated stress (to 0.5 × control). BChE activity decreased after 30 min in the right CA3 region (to 0.4 × control) but increased (3.8-fold) after 120 min in the left CA3 region. The pattern of changes in CRH-KO differed from that in WT mice.

The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice.

We hypothesized that muscarinic receptors (MRs) in the heart have a role in stress responses and thus investigated changes in MR signaling (gene expression, number of receptors, adenylyl cyclase (AC), phospholipase C (PLC), protein kinase A and C (PKA and PKC) and nitric oxide synthase [NOS]) in the left ventricle, together with telemetric measurement of heart rate (HR) in mice (wild type [WT] and M2 knockout [KO]) during and after one (1R) or seven sessions (7R) of restraint stress (seven mice per group). Stress decreased M2 MR mRNA and cell surface MR in the left ventricle in WT mice. In KO mice, 1R, but not 7R, decreased surface MR. Similarly, AC activity was decreased in WT mice after 1R and 7R, whereas in KO mice, there was no change. PLC activity was also decreased after 1R in WT and KO mice. This is in accord with the concept that cAMP is a key player in HR regulation. No change was found with stress in NOS activity. Amount of AC and PKA protein was not changed, but was altered for PKC isoenzymes (PKCα, ß, γ, η and ϵ (increased) in KO mice, and PKCι (increased) in WT mice). KO mice were more susceptible to stress as shown by inability to compensate HR during 120 min following repeated stress. The results imply that not only M2 but also M3 are involved in stress signaling and in allostasis. We conclude that for a normal stress response, the expression of M2 MR to mediate vagal responses is essential.

Heart ventricles specific stress-induced changes in ß-adrenoceptors and muscarinic receptors.

The left and right ventricles fulfill different role in heart function. Here we compare chamber specific changes in local catecholamine concentrations; gene expression and the receptor protein amount of all three ß-adrenoceptors (ß-AR) in rat right heart ventricles exposed to acute (1 session) and repeated (7 sessions) immobilization stress (IMMO) vs. previously observed changes in left ventricles. Density of muscarinic receptors as main cardio-inhibitive receptors was also measured. In the right ventricles, noradrenaline and adrenaline were increased. No ß1-AR changes were observed, in spite of the increased sympathetic activity. On the other hand, we have found a decrease of ß2-AR gene expression (reduction to 30%) after 7 IMMO and protein (to 59%) after 1 IMMO. ß3-AR gene expression was increased after 7 IMMO. Muscarinic receptor density was not changed. When comparing correlation in left and right ventricles, there was strong correlation between adrenaline and ß2-AR gene expression, protein and ß3-AR gene expression in the left ventricles while only correlation between adrenaline and ß2-AR mRNA and protein in the right ventricles was found. Our results show that maintenance of cardiac homeostasis under stress conditions are to a great extent achieved by a balance between different receptors and also by a balanced receptor changes in left vs. right ventricles. Taken together, decrease of cardio-stimulating ß2-AR represents a new important mechanism by which ß2-AR contributes to the heart physiology.

Corticotropin-releasing hormone affects short immobilization stress-induced changes in lung cytosolic and membrane glucocorticoid binding sites.

Glucocorticoids act via glucocorticoid receptors (GR), typically localized in the cytosol (cGR). Rapid action is probably mediated via membrane receptors (mGR). In corticotropin-releasing hormone knockouts (CRH-KO), basal plasma glucocorticoid levels do differ from wild type levels (WT), but are approximately ten times lower during exposure to immobilization stress (IMMO) in comparison to WT. We tested the following hypotheses: (1) the mice lung tissue GR basal numbers would not be changed in CRH-KO (because of similar glucocorticoid levels), (2) the number of GR would be changed in WT but not in KO during short (30, 90, and 120 min) IMMO (because of higher increase of glucocorticoid levels in WT). The basal levels of cGR were not changed in CRH-KO (compared to WT), while mGR were significantly lower (62 %) in CRH-KO. In WT, there was the only decrease (to 32 %) in cGR after 120 min when we also found an increase in mGR in WT (to 201 %). In CRH-KO, IMMO caused gradual decrease in cGR (to 52 % after 30 min, to 46 % after 90 min, and to 32 % after 120 min). In CRH-KO, the only increase in mGR appeared already at 30 min of IMMO. These data suggest, on the contrary to our hypotheses, that CRH-KO are more susceptible to GR changes in early phases of stress.

Near-complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase.

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.

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.

Dopamine and Dopamine-Related Ligands Can Bind Not Only to Dopamine Receptors.

The dopaminergic system is one of the most important neurotransmitter systems in the central nervous system (CNS). It acts mainly by activation of the D1-like receptor family at the target cell. Additionally, fine-tuning of the signal is achieved via pre-synaptic modulation by the D2-like receptor family. Some dopamine drugs (both agonists and antagonists) bind in addition to DRs also to α2-ARs and 5-HT receptors. Unfortunately, these compounds are often considered subtype(s) specific. Thus, it is important to consider the presence of these receptor subtypes in specific CNS areas as the function virtually elicited by one receptor type could be an effect of other-or the co-effect of multiple receptors. However, there are enough molecules with adequate specificity. In this review, we want to give an overview of the most common off-targets for established dopamine receptor ligands. To give an overall picture, we included a discussion on subtype selectivity. Molecules used as antipsychotic drugs are reviewed too. Therefore, we will summarize reported affinities and give an outline of molecules sufficiently specific for one or more subtypes (i.e., for subfamily), the presence of DR, α2-ARs, and 5-HT receptors in CNS areas, which could help avoid ambiguous results.

Multitargeting nature of muscarinic orthosteric agonists and antagonists.

Muscarinic receptors (mAChRs) are typical members of the G protein-coupled receptor (GPCR) family and exist in five subtypes from M1 to M5. Muscarinic receptor subtypes do not sufficiently differ in affinity to orthosteric antagonists or agonists; therefore, the analysis of receptor subtypes is complicated, and misinterpretations can occur. Usually, when researchers mainly specialized in CNS and peripheral functions aim to study mAChR involvement in behavior, learning, spinal locomotor networks, biological rhythms, cardiovascular physiology, bronchoconstriction, gastrointestinal tract functions, schizophrenia, and Parkinson's disease, they use orthosteric ligands and they do not use allosteric ligands. Moreover, they usually rely on manufacturers' claims that could be misleading. This review aimed to call the attention of researchers not deeply focused on mAChR pharmacology to this fact. Importantly, limited selective binding is not only a property of mAChRs but is a general attribute of most neurotransmitter receptors. In this review, we want to give an overview of the most common off-targets for established mAChR ligands. In this context, an important point is a mention the tremendous knowledge gap on off-targets for novel compounds compared to very well-established ligands. Therefore, we will summarize reported affinities and give an outline of strategies to investigate the subtype's function, thereby avoiding ambiguous results. Despite that, the multitargeting nature of drugs acting also on mAChR could be an advantage when treating such diseases as schizophrenia. Antipsychotics are a perfect example of a multitargeting advantage in treatment. A promising strategy is the use of allosteric ligands, although some of these ligands have also been shown to exhibit limited selectivity. Another new direction in the development of muscarinic selective ligands is functionally selective and biased agonists. The possible selective ligands, usually allosteric, will also be listed. To overcome the limited selectivity of orthosteric ligands, the recommended process is to carefully examine the presence of respective subtypes in specific tissues via knockout studies, carefully apply "specific" agonists/antagonists at appropriate concentrations and then calculate the probability of a specific subtype involvement in specific functions. This could help interested researchers aiming to study the central nervous system functions mediated by the muscarinic receptor.

Mechanisms of G protein-coupled receptors regulation.

Within the last two decades of studies in the ever-expanding field of GPCR signaling, challenging insights were adopted. Growing evidence now asists the shift from classical linear model of signaling towards a considerably complex network of signaling pathways with many shared proteins and cross-talks. Considering the extensive and intriguing network of pathways activated by these receptors, it is apparent that multi-level system of regulation must exist to rigorously modulate the amplitude, duration and spatial aspects of the GPCR signaling. This review summarizes the principal mechanisms of GPCR regulation and gives the overview of recent advances in this field of research.

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.

Two Players in the Field: Hierarchical Model of Interaction between the Dopamine and Acetylcholine Signaling Systems in the Striatum.

Tight interactions exist between dopamine and acetylcholine signaling in the striatum. Dopaminergic neurons express muscarinic and nicotinic receptors, and cholinergic interneurons express dopamine receptors. All neurons in the striatum are pacemakers. An increase in dopamine release is activated by stopping acetylcholine release. The coordinated timing or synchrony of the direct and indirect pathways is critical for refined movements. Changes in neurotransmitter ratios are considered a prominent factor in Parkinson's disease. In general, drugs increase striatal dopamine release, and others can potentiate both dopamine and acetylcholine release. Both neurotransmitters and their receptors show diurnal variations. Recently, it was observed that reward function is modulated by the circadian system, and behavioral changes (hyperactivity and hypoactivity during the light and dark phases, respectively) are present in an animal model of Parkinson's disease. The striatum is one of the key structures responsible for increased locomotion in the active (dark) period in mice lacking M4 muscarinic receptors. Thus, we propose here a hierarchical model of the interaction between dopamine and acetylcholine signaling systems in the striatum. The basis of this model is their functional morphology. The next highest mode of interaction between these two neurotransmitter systems is their interaction at the neurotransmitter/receptor/signaling level. Furthermore, these interactions contribute to locomotor activity regulation and reward behavior, and the topmost level of interaction represents their biological rhythmicity.

Social Isolation: How Can the Effects on the Cholinergic System Be Isolated?

Social species form organizations that support individuals because the consequent social behaviors help these organisms survive. The isolation of these individuals may be a stressor. We reviewed the potential mechanisms of the effects of social isolation on cholinergic signaling and vice versa how changes in cholinergic signaling affect changes due to social isolation.There are two important problems regarding this topic. First, isolation schemes differ in their duration (1-165 days) and initiation (immediately after birth to adulthood). Second, there is an important problem that is generally not considered when studying the role of the cholinergic system in neurobehavioral correlates: muscarinic and nicotinic receptor subtypes do not differ sufficiently in their affinity for orthosteric site agonists and antagonists. Some potential cholinesterase inhibitors also affect other targets, such as receptors or other neurotransmitter systems. Therefore, the role of the cholinergic system in social isolation should be carefully considered, and multiple receptor systems may be involved in the central nervous system response, although some subtypes are involved in specific functions. To determine the role of a specific receptor subtype, the presence of a specific subtype in the central nervous system should be determined using search in knockout studies with the careful application of specific agonists/antagonists.

Sexual dimorphism in stress-induced changes in adrenergic and muscarinic receptor densities in the lung of wild type and corticotropin-releasing hormone-knockout mice.

We tested the hypothesis that single and repeated immobilization stress affect densities of alpha(1)-adrenoceptor (alpha(1)-AR) and beta-AR subtypes, muscarinic receptors (MR), adenylyl cyclase activity (AC) and phospholipase C activity (PLC) in lungs of male and female wild type (WT) and corticotropin-releasing hormone gene (CRH-knockout (KO)) disrupted mice. We found sex differences in the basal levels of alpha(1)-AR subtypes (females had 2-3 times higher density of receptors than males) and MR (males had twice the density found in females). In marked contrast, beta-AR subtype densities did not differ between sexes. CRH gene disruption decreased all three studied receptors in intact mice (to 20-50% of WT) in both sexes (except beta(1)-AR in females). Stress induced sexually dimorphic responses, while all alpha(1)-AR subtypes decreased in females (to 30% of control approximately), only alpha(1A)-AR level diminished (about 50%) in males. beta(1)-AR decreased in males (to about 40%) but remained stable in females. beta(2)-AR diminished in females (to about 20-60%) and also in males (to about 30-60%). MR decreased in both sexes (approximately to 50%). AC activity diminished in males (to < 50%) while PLC activity was not changed. In CRH-KO mice, the stress response was severely diminished. Paradoxically, the receptor response to stress was less affected by CRH-KO in males than in females. AC activity did not change in CRH-KO mice. In conclusion, in mice the stress reaction is sexually dimorphic and an intact hypothalamo-pituitary-adrenocortical system is required for the normal reaction of pulmonary adrenergic and MR to stress.

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.

Acute restraint stress modifies the heart rate biorhythm in the poststress period.

We studied the changes in the heart and the activity biorhythms in mice exposed to acute (one 120-minute session) and repeated (7 two-hour sessions) restraint stress in 129J1/CF1 mice (WT) and in mice without M2 muscarinic receptors (M2KO) during the prestress period, during stress (STR) and for five days after the last stress session (POST). There were changes in the mesor (a midline based on the distribution of values across the circadian cycles; decreased in M2KO by 6% over all POST), day means (inactive period of diurnal rhythm in mice; higher in M2KO and further increased on STR and on the second to the fifth POST) and night means (active period; lower by 13% in M2KO and remained decreased in STR and in POST). The total area under the curve was decreased both in the WT and M2KO on STR and in all POST. Repeated stress caused changes over all days of STR, but the initial values were restored in POST. The average night values were decreased, and the day means were increased by 16% over all STR in M2KO. The day means decreased by 14% in the 4 POST in WT. The activity biorhythm parameters were almost unchanged. We show here that stress can specifically affect heart biorhythm in M2KO mice, especially when the stress is acute. This implies the role of M2 muscarinic receptor in stress response.

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.

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles.

Mammal heart tissue has long been assumed to be the exclusive domain of the M(2) subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M(2) subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M(1) muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M(1)-M(4) receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M(2) subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M(2), with the majority of these belonging to the M(1) subtype. We were also able to detect a marginal fraction (6 +/- 2%) of receptors that, based on other findings, belong mainly to the M(5) muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M(1)-M(4) receptors and can not therefore be used as a method for M(5) muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M(1) muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M(5) muscarinic receptors but not to that of the M(3) receptors.