Evaluation of Ouabain's Tissue Distribution in C57/Black Mice Following Intraperitoneal Injection, Using Chromatography and Mass Spectrometry.

Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC-MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: Cmax = 882.88 ± 21.82 ng/g; Tmax = 0.08 ± 0.01 h; T1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: Cmax = 145.24 ± 44.03 ng/g (undetectable at 60 min); Tmax = 0.08 ± 0.02 h; T1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: Cmax = 1072.3 ± 260.8 ng/g; Tmax = 0.35 ± 0.19 h; T1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: Cmax = 2558.0 ± 382.4 ng/g; Tmax = 0.35 ± 0.13 h; T1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS.

Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations.

Neurophotonic technology is a rapidly growing group of techniques that are based on the interactions of light with natural or genetically modified cells of the neural system. New optical technologies make it possible to considerably extend the tools of neurophysiological research, from the visualization of functional activity changes to control of brain tissue excitability. This opens new perspectives for studying the mechanisms underlying the development of human neurological diseases. Epilepsy is one of the most common brain disorders; it is characterized by recurrent seizures and affects >1% of the world's population. However, how seizures occur, spread, and terminate in a healthy brain is still unclear. Therefore, it is extremely important to develop appropriate models to accurately explore the causal relationship of epileptic activity. The use of neurophotonic technologies in epilepsy research falls into two broad categories: the visualization of neural epileptic activity, and the direct optical influence on neurons to induce or suppress epileptic activity. An optogenetic variant of the classical kindling model of epileptic seizures, in which activatable cells are genetically defined, is called optokindling. Research is also underway concerning the application of neurophotonic techniques for suppressing epileptic activity, aiming to bring these methods into clinical practice. This review aims to systematize and describe new approaches that use combinations of different neurophotonic methods to work with in vivo models of epilepsy. These approaches overcome many of the shortcomings associated with classical animal models of epilepsy and thus increase the effectiveness of developing new diagnostic methods and antiepileptic therapy.

Na+,K+-ATPase and Cardiotonic Steroids in Models of Dopaminergic System Pathologies.

In recent years, enough evidence has accumulated to assert that cardiotonic steroids, Na+,K+-ATPase ligands, play an integral role in the physiological and pathophysiological processes in the body. However, little is known about the function of these compounds in the central nervous system. Endogenous cardiotonic steroids are involved in the pathogenesis of affective disorders, including depression and bipolar disorder, which are linked to dopaminergic system dysfunction. Animal models have shown that the cardiotonic steroid ouabain induces mania-like behavior through dopamine-dependent intracellular signaling pathways. In addition, mutations in the alpha subunit of Na+,K+-ATPase lead to the development of neurological pathologies. Evidence from animal models confirms the neurological consequences of mutations in the Na+,K+-ATPase alpha subunit. This review is dedicated to discussing the role of cardiotonic steroids and Na+,K+-ATPase in dopaminergic system pathologies-both the evidence supporting their involvement and potential pathways along which they may exert their effects are evaluated. Since there is an association between affective disorders accompanied by functional alterations in the dopaminergic system and neurological disorders such as Parkinson's disease, we extend our discussion to the role of Na+,K+-ATPase and cardiotonic steroids in neurodegenerative diseases as well.

Evaluation of Approach to a Conspecific and Blood Biochemical Parameters in TAAR1 Knockout Mice.

It is known that the trace amine-associated receptor 1 (TAAR1) receptor is involved in limbic brain functions by regulating dopamine transmission and putative reward circuitry. Moreover, other TAARs are expressed in the olfactory system of all studied vertebrate species, sensing innate socially-relevant odors, including pheromones. Therefore, one can assume that TAARs may play a role in rodent social and sexual behavior. A comparative behavioral and biochemical analysis of TAAR1 knockout (TAAR1-KO) and wild-type mice is also important for the preliminary evaluation of the potential side effects of future TAAR1-based therapies. In our studies, we adapted a sexual incentive motivation test for mice to evaluate the sexual behavior of TAAR1-KO and wild-type mice. Previously, similar methods were primarily applied to rats. Furthermore, we measured testosterone and other biochemical parameters in the blood. As a result, we found only minimal alterations in all of the studied parameters. Thus, the lack of TAAR1 does not significantly affect sexual motivation and routine lipid and metabolic blood biochemical parameters, suggesting that future TAAR1-based therapies should have a favorable safety profile.

Trace Amine-Associated Receptor 2 Is Expressed in the Limbic Brain Areas and Is Involved in Dopamine Regulation and Adult Neurogenesis.

Trace amines are a group of biogenic amines that are structurally and functionally close to classical monoamine neurotransmitters. Trace amine-associated receptors (TAARs) are emerging as promising targets for treating neuropsychiatric disorders. It has been documented that all TAARs, apart from TAAR1, function as olfactory receptors involved in sensing innate odors encoded by volatile amines. However, recently, brain expression and function of TAAR5 were also demonstrated. In this study, we assessed the behavior, brain neurochemistry, and electrophysiology changes in knock-out mice lacking Trace amine-associated receptor 2 (TAAR2) but expressing beta-Galactosidase mapping expression of TAAR2 receptors. As expected, we detected beta-Galactosidase staining in the glomerular layer of the olfactory bulb. However, we also found staining in the deeper layers of the olfactory bulb and several brain regions, including the hippocampus, cerebellum, cortex, raphe nuclei, hypothalamus, and habenula, indicating that TAAR2 receptors are not only expressed in the olfactory system but are also present in the limbic brain areas that receive olfactory input. In behavioral experiments, TAAR2 knock-out (TAAR2-KO) mice showed increased locomotor activity and less immobility in the forced swim test, with no changes in anxiety level. Furthermore, TAAR2-KO mice showed alterations in brain electrophysiological activity-particularly, decreased spectral power of the cortex and striatum in the 0, 9-20 Hz range. TAAR2-KO mice also had elevated tissue dopamine levels in the striatum and an increased dopaminergic neuron number in the Substantia Nigra. In addition, an increased brain-derived neurotrophic factor (BDNF) mRNA level in the striatum and Monoamine Oxidase B (MAO-B) mRNA level in the striatum and midbrain was found in TAAR2-KO mice. Importantly, TAAR2-KO mice demonstrated an increased neuroblast-like and proliferating cell number in the subventricular and subgranular zone, indicating increased adult neurogenesis. These data indicate that in addition to its role in the innate olfaction of volatile amines, TAAR2 is expressed in limbic brain areas and regulates the brain dopamine system, neuronal electrophysiological activity, and adult neurogenesis. These findings further corroborated observations in TAAR1-KO and TAAR5-KO mice, indicating common for TAAR family pattern of expression in limbic brain areas and role in regulating monoamine levels and adult neurogenesis, but with variable involvement of each subtype of TAAR receptors in these functions.

A New Paradigm for Training Hyperactive Dopamine Transporter Knockout Rats: Influence of Novel Stimuli on Object Recognition.

Attention deficit hyperactivity disorder (ADHD) is believed to be connected with a high level of hyperactivity caused by alterations of the control of dopaminergic transmission in the brain. The strain of hyperdopaminergic dopamine transporter knockout (DAT-KO) rats represents an optimal model for investigating ADHD-related pathological mechanisms. The goal of this work was to study the influence of the overactivated dopamine system in the brain on a motor cognitive task fulfillment. The DAT-KO rats were trained to learn an object recognition task and store it in long-term memory. We found that DAT-KO rats can learn to move an object and retrieve food from the rewarded familiar objects and not to move the non-rewarded novel objects. However, we observed that the time of task performance and the distances traveled were significantly increased in DAT-KO rats in comparison with wild-type controls. Both groups of rats explored the novel objects longer than the familiar cubes. However, unlike controls, DAT-KO rats explored novel objects significantly longer and with fewer errors, since they preferred not to move the non-rewarded novel objects. After a 3 months' interval that followed the training period, they were able to retain the learned skills in memory and to efficiently retrieve them. The data obtained indicate that DAT-KO rats have a deficiency in learning the cognitive task, but their hyperactivity does not prevent the ability to learn a non-spatial cognitive task under the presentation of novel stimuli. The longer exploration of novel objects during training may ensure persistent learning of the task paradigm. These findings may serve as a base for developing new ADHD learning paradigms.

Putative TAAR5 agonist alpha-NETA affects event-related potentials in oddball paradigm in awake mice.

Trace amines have been reported to be neuromodulators of monoaminergic systems. Trace amines receptor 5 (TAAR5) is expressed in several regions of mice central nervous system, such as amygdala, arcuate nucleus and ventromedial hypothalamus, but very limited information is available on its functional role. The purpose of this study is to examine the effect of TAAR5 agonist alpha-NETA on the generation of mismatch negativity (MMN) analogue in C57BL/6 mice. Event-related potentials have been recorded from awake mice in oddball paradigms before and after the alpha-NETA administration. Alpha-NETA has been found to decrease N40 MMN-like difference, which resulted from the increased response to standard stimuli. An opposite effect has been found for the P80 component: the amplitude increased in response both to standard and deviant stimuli. A significant increase in N40 peak latency after the alpha-NETA administration has been found. This may suggest a reduced speed of information processing similar to the increase in P50 and N100 components latencies in schizophrenia patients. These results provide new evidence for a role of TAAR5 in cognitive processes.

A low-cost and customizable alternative for commercial implantable cannula for intracerebral administration in mice.

Stereotaxic intracerebral cannula implantation for neuroactive agent administration is a wide-spread method for chronic experiments requiring bypassing the blood-brain barrier in rodents. However, commercially available cannula are bulky and may interfere with animal movement or lead to their dislodging during grooming. As the number of cannula needed in one experiment, and the accompanying costs can be high, it is in the interest of researchers to produce them on their own. Custom cannula manufacturing also offers the flexibility of different cannula lengths, which is required for agent delivery to various brain structures. In this article we present a protocol for making guide cannula along with the accompanying systems required for injection, which are small, cost-effective, light, easy to make, reusable, and can be made from easily procured materials.

Effect of alpha-NETA on auditory event related potentials in sensory gating study paradigm in mice.

The potential contribution of trace amines (TA) to the pathophysiology of neuropsychiatric disorders makes it interesting to examine the effect of TA receptor ligands on schizophrenia biomarkers. We studied the effect of systemic administration of a putative Trace Amine-Associated Receptor 5 (TAAR5) agonist, alpha-NETA (2-(alpha-naphthoyl) ethyltrimethylammonium iodide), on the amplitude of the N40 event related potentials component and on the sensory gating (SG) index in C57BL/6 mice. It was found that low doses of alpha-NETA (2.5 mg/kg and 5 mg/kg) do not elicit a significant effect on the parameters of the N40 component and the SG index. However, the higher dose of alpha-NETA (10 mg/kg) induces a significant effect on the N40 component, but since a decrease in amplitude is observed on both the first and second stimuli in the pair, the SG index does not change. Thus, alpha-NETA administration causes a steady decrease in the N40 amplitude in response to both the first and second stimuli in the paired-click paradigm, and an increase in the N40 peak latency.

Effect of trace amine-associated receptor 1 agonist RO5263397 on sensory gating in mice.

The trace amine-associated receptor 1 (TAAR1) agonist RO5263397 effect on sensory gating in C57BL/6 mice was studied. Sensory gating is a mechanism for dosing and filtering the incoming information, by which the brain regulates the responses to sensory stimuli coming from the environment. Sensory gating deficit is considered to be one of the schizophrenia endophenotypes. TAAR1 agonist at a 1 mg/kg dosage contributed to the sensory gating index (S1-S2) increase. Sensory gating index rose due to the N40 amplitude increase in response to the first stimulus in a pair, whereas the amplitude of the second stimulus remained unchanged. These results suggest that the sensory gating in mice may be modulated through TAAR1-dependent processes, indicating potential contribution of TAAR1 and trace amines in general to the neuropharmacology of cognitive processes.

Trace Amine-Associated Receptor 1 Agonist Modulates Mismatch Negativity-Like Responses in Mice.

The trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor widely expressed in the mammalian brain, particularly in limbic system and monoaminergic areas. It has proven to be an important modulator of dopaminergic, serotoninergic, and glutamatergic neurotransmission and is considered to be a potential useful target for the pharmacotherapy of neuropsychiatric disorders, including schizophrenia. One of the promising schizophrenia endophenotypes is a deficit in neurocognitive abilities manifested as mismatch negativity (MMN) deficit. This study examines the effect of TAAR1 partial agonist RO5263397 on the MMN-like response in freely moving C57BL/6 mice. Event-related potentials (ERPs) were recorded from awake mice in the oddball paradigm before and after RO5263397 administration. The RO5263397 (but not saline) administration increased the N40 amplitude in response to deviant stimuli. That provided the MMN-like difference at the 36-44 ms interval after the injection. The pitch deviance-elicited changes before the injection and in the control paradigm were established for the P68 component. After TAAR1 agonist administration the P68 amplitude in response both to standard and deviant stimuli was increased. These results suggest that the MMN-like response in mice may be modulated through TAAR1-dependent processes (possibly acting through the direct or indirect glutamate NMDA receptor modulation), indicating the TAAR1 agonists potential antipsychotic and pro-cognitive activity.

Age-Dependent Electrocorticogram Dynamics and Epileptogenic Responsiveness in Rats Subjected to Prenatal Hypoxia.

Using electrocorticogram (ECoG) analysis, we compared age-related dynamics of general neuronal activity and convulsive epileptiform responsiveness induced by intracortical microinjections of 4-aminopyridine (4-AP) in control Wistar rats and those subjected to prenatal hypoxia (Hx; E14; 7% O2, 3 h). The studies were carried out in three age periods roughly corresponding to childhood (P20-27), adolescence (P30-45), and adulthood (P90-120). It was found that in the process of postnatal development of the control rats, the peak of the ECoG power spectrum density (PSD) of the theta rhythm during wakefulness shifted from the low to the higher frequency, while in the Hx rats this shift had the opposite direction. Moreover, the Hx rats had different frequency characteristics of the ECoG PSD and longer episodes of spike-and-wave discharges caused by 4-AP injections compared to the controls. The total ECoG PSD of slow-wave sleep (1-5 Hz) was also dramatically decreased in the process of development of the Hx rats. Such alterations in PSD could be explained by the changes in balance of the excitation and inhibition processes in the cortical networks. Analyzing protein levels of neurotransmitter transporters in the brain structures of the Hx rats, we found that the content of the glutamate transporter EAAT1 was higher in the parietal cortex in all age groups of Hx rats while in the hippocampus it decreased during postnatal development compared to controls. Furthermore, the content of the vesicular acetylcholine transporter in the parietal cortex, and of the inhibitory GABA transporter 1 in the hippocampus, was also affected by prenatal Hx. These data suggest that prenatal Hx results in a shift in the excitatory and inhibitory balance in the rat cortex towards excitation, making the rat's brain more vulnerable to the effects of proconvulsant drugs and predisposing animals to epileptogenesis during postnatal life.

Identification of TAAR5 Agonist Activity of Alpha-NETA and Its Effect on Mismatch Negativity Amplitude in Awake Rats.

Mismatch negativity (MMN) is a well-defined component of human event-related potentials that reflects the pre-attentive, stimulus-discrimination process and is associated with involuntary switching of attention. MMN-like responses detected in animal models provide an opportunity to investigate the neural mechanisms of this process that involves several neurotransmitter and neuromodulator systems. Trace amines are believed to play a significant role in neuromodulation of synaptic transmission. The present study aimed to determine the role of trace amine-associated receptor 5 (TAAR5) in the MMN-like response in rats. First, using a bioluminescence resonance energy transfer (BRET) cAMP biosensor, we performed unbiased screening of TAAR5 ligands from a commercially available compound library (661 compounds) and identified 2-(alpha-naphthoyl)ethyltrimethylammonium iodide (alpha-NETA) as a potent (EC50 150 nM) TAAR5 agonist. Then, we recorded auditory event-related potentials during an oddball paradigm in awake freely moving rats that were intraperitoneally injected with a vehicle or two doses of the putative TAAR5 agonist alpha-NETA. The MMN-like response was increased by alpha-NETA 3 mg/kg dose, but not by 1 mg/kg dose or 0.9% saline solution. These results suggest that the MMN-like response in rats may be modulated, at least in part, through TAAR5-dependent processes.

TAAR5 receptor agonist affects sensory gating in rats.

Trace amines are structurally close to classical monoamines and dysregulation in trace amines and/or their receptors might contribute to pathology of mental disorders. The study was aimed to investigate the effect of recently identified Trace Amine-Associated Receptor 5 (TAAR5) agonist 2-(alpha-naphthoyl)ethyltrimethylammonium iodide (alpha-NETA) on sensory gating (SG) in awake freely moving rats. SG was studied in paired-click paradigm and SG index was calculated as difference in event related potentials component N40 amplitudes to the first and second stimulus in the pair. The 1 mg/kg dose of alpha-NETA as well as the control injection of saline had no significant effects on the SG index. However, higher doses of alpha-NETA (3 and 5 mg/kg) significantly decreased the SG index. The change in the SG index was mainly due to a decrease in the N40 amplitude, and the 5 mg/kg dose caused the N40 decrease both in response to the first and second stimulus in the pair. Thus, TAAR5 activation can influence SG, indicating the potential role of trace amines and TAAR5 in sensory information dosing.

Advantages and limitations of brain imaging methods in the research of absence epilepsy in humans and animal models.

The purpose of this review is to analyze research possibilities and limitations of several methods, technical tools and their combinations for elucidation of absence epilepsy mechanisms, particularly the childhood absences. Despite the notable collection of simultaneous recording of clinical electroencephalography (EEG) and behavioral changes in relation to absence seizures, shortcomings of scalp EEG in both spatial resolution and precise detection of subcortical centers have limited the understanding of the fundamental mechanisms of altered brain function during and after recurrent epileptic paroxysms. Therefore, in the past decade, EEG recordings have often been combined with simultaneous imaging methods in epilepsy studies. Among imaging methods, the following ones are used regularly: functional magnetic resonance imaging (fMRI), positron-emission tomography (PET), low-resolution electromagnetic tomography (LORETA), single photon emission spectroscopy (SPECT), near-infrared spectroscopy (NIRS), and optical imaging of intrinsic signals (IOS). In addition, voltage-sensitive dye optical imaging method and even photoacoustic microscopy can be applied to animal models of epilepsy. Samplings of some of the most relevant data obtained by the above methods are presented. It appears that the elaboration of more adequate animal models of the patterns of absence seizures during the early postnatal period is necessary for better correspondence of human and animal absence phenomena.