Stanislav Rozov

Long-term cognitive and neurochemical effects of “bath salt” designer drugs methylone and mephedrone

INTRODUCTION/AIMS The use of cathinone-derivative designer drugs methylone and mephedrone has increased rapidly in recent years. Our aim was to investigate the possible long-term effects of these drugs on a range of behavioral tests in mice. Further, we investigated the long-term effects of these drugs on brain neurochemistry in both rats and mice. METHODS We treated animals with a binge-like regimen of methylone or mephedrone (30 mg/kg, twice daily for 4 days) and, starting 2 weeks later, we performed behavioral tests of memory, anxiety and depression and measured brain levels of dopamine (DA), serotonin (5-HT), their metabolites and norepinephrine (NE). 5-HT and DA transporter (5-HTT and DAT) levels were also measured in rats by [(3)H]paroxetine and [(3)H]mazindol binding. RESULTS Mephedrone reduced working memory performance in the T-maze spontaneous alternation task but did not affect neurotransmitter levels aside from a 22% decrease in striatal homovanillic acid (HVA) levels in mice. Methylone had little effect on behavior or neurotransmitter levels in mice but produced a widespread depletion of 5-HT and 5-HTT levels in rats. CONCLUSIONS Both methylone and mephedrone appeared to have a long-term effect on either behavioral or biochemical gauges of neurotoxicity in rodents.

Dopamine from the Brain Promotes Spinal Motor Neuron Generation during Development and Adult Regeneration

Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish

The histaminergic and hypocretin/orexin (hcrt) neurotransmitter systems play crucial roles in alertness/wakefulness in rodents. We elucidated the role of histamine in wakefulness and the interaction of the histamine and hcrt systems in larval zebrafish. Translation inhibition of histidine decarboxylase (hdc) with morpholino oligonucleotides (MOs) led to a behaviorally measurable decline in light‐associated activity, which was partially rescued by hdc mRNA injections and mimicked by histamine receptor H1 (Hrh1) antagonist pyrilamine treatment. Histamine‐immunoreactive fibers targeted the dorsal telencephalon, an area that expresses histamine receptors hrh1 and hrh3 and contains predominantly glutamatergic neurons. Tract tracing with DiI revealed that projections from dorsal telencephalon innervate the hcrt and histaminergic neurons. Translation inhibition of hdc decreased the number of hcrt neurons in a Hrh1‐dependent manner. The reduction was rescued by overexpression of hdc mRNA. hdc mRNA injection alone led to an up‐regulation of hcrt neuron numbers. These results suggest that histamine is essential for the development of a functional and intact hcrt system and that histamine has a bidirectional effect on the development of the hcrt neurons. In summary, our findings provide evidence that these two systems are linked both functionally and developmentally, which may have important implications in sleep disorders and narcolepsy.—Sundvik, M., Kudo, H., Toivonen, P., Rozov, S., Chen, Y.‐C., Panula, P. The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish. FASEB J. 25, 4338–4347 (2011). www.fasebj.org

Evidence for the role of histamine H3 receptor in alcohol consumption and alcohol reward in mice

Recent research suggests that histamine H3 receptor (H3R) antagonism may diminish motivational aspects of alcohol dependence. We studied the role of H3Rs in alcohol-related behaviors using H3R knockout (KO) mice and ligands. H3R KO mice consumed less alcohol than wild-type (WT) mice in a two-bottle free-choice test and in a ‘drinking in the dark’ model. H3R antagonist ciproxifan suppressed and H3R agonist immepip increased alcohol drinking in C57BL/6J mice. Impairment in reward mechanisms in H3R KO mice was confirmed by the lack of alcohol-evoked conditioned place preference. Plasma alcohol concentrations of H3R KO and WT mice were similar. There were no marked differences in brain biogenic amine levels in H3R KO mice compared with the control animals after alcohol drinking. In conclusion, the findings of this study provide evidence for the role of H3R receptor in alcohol-related behaviors, especially in alcohol drinking and alcohol reward. Thus, targeting H3Rs with a specific antagonist might be a potential means to treat alcoholism in the future.

Histamine Release in the Basal Forebrain Mediates Cortical Activation through Cholinergic Neurons

The basal forebrain (BF) is a key structure in regulating both cortical activity and sleep homeostasis. It receives input from all ascending arousal systems and is particularly highly innervated by histaminergic neurons. Previous studies clearly point to a role for histamine as a wake-promoting substance in the BF. We used in vivo microdialysis and pharmacological treatments in rats to study which electroencephalogram (EEG) spectral properties are associated with histamine-induced wakefulness and whether this wakefulness is followed by increased sleep and increased EEG delta power during sleep. We also investigated which BF neurons mediate histamine-induced cortical activation. Extracellular BF histamine levels rose immediately and remained constant throughout a 6 h period of sleep deprivation, returning to baseline levels immediately afterward. During the spontaneous sleep–wake cycle, we observed a strong correlation between wakefulness and extracellular histamine concentrations in the BF, which was unaffected by the time of day. The perfusion of histamine into the BF increased wakefulness and cortical activity without inducing recovery sleep. The perfusion of a histamine receptor 1 antagonist into the BF decreased both wakefulness and cortical activity. Lesioning the BF cholinergic neurons abolished these effects. Together, these results show that activation of the cholinergic BF by histamine is important in sustaining a high level of cortical activation, and that a lack of activation of the cholinergic BF by histamine may be important in initiating and maintaining nonrapid eye movement sleep. The level of histamine release is tightly connected to behavioral state, but conveys no information about sleep pressure.

Acute ethanol treatment upregulates th1, th2, and hdc in larval zebrafish in stable networks

Earlier studies in zebrafish have revealed that acutely given ethanol has a stimulatory effect on locomotion in fish larvae but the mechanism of this effect has not been revealed. We studied the effects of ethanol concentrations between 0.75 and 3.00% on 7-day-old larval zebrafish (Danio rerio) of the Turku strain. At 0.75-3% concentrations ethanol increased swimming speed during the first minute. At 3% the swimming speed decreased rapidly after the first minute, whereas at 0.75 and 1.5% a prolonged increase in swimming speed was seen. At the highest ethanol concentration dopamine levels decreased significantly after a 10-min treatment. We found that ethanol upregulates key genes involved in the biosynthesis of histamine (hdc) and dopamine (th1 and th2) following a short 10-min ethanol treatment, measured by qPCR. Using in situ hybridization and immunohistochemistry, we further discovered that the morphology of the histaminergic and dopaminergic neurons and networks in the larval zebrafish brain was unaffected by both the 10-min and a longer 30-min treatment. The results suggest that acute ethanol rapidly decreases dopamine levels, and activates both forms or th to replenish the dopamine stores within 30 min. The dynamic changes in histaminergic and dopaminergic system enzymes occurred in the same cells which normally express the transcripts. As both dopamine and histamine are known to be involved in the behavioral effects of ethanol and locomotor stimulation, these results suggest that rapid adaptations of these networks are associated with altered locomotor activity.

Periodic properties of the histaminergic system of the mouse brain

Brain histamine is involved in the regulation of the sleep–wake cycle and alertness. Despite the widespread use of the mouse as an experimental model, the periodic properties of major markers of the mouse histaminergic system have not been comprehensively characterized. We analysed the daily levels of histamine and its first metabolite, 1‐methylhistamine, in different brain structures of C57BL/6J and CBA/J mouse strains, and the mRNA level and activity of histidine decarboxylase and histamine‐N‐methyltransferase in C57BL/6J mice. In the C57BL/6J strain, histamine release, assessed by in vivo microdialysis, underwent prominent periodic changes. The main period was 24 h peaking during the activity period. Additional 8 h periods were also observed. The release was highly positively correlated with active wakefulness, as shown by electroencephalography. In both mouse strains, tissue histamine levels remained steady for 24 h in all structures except for the hypothalamus of CBA/J mice, where 24‐h periodicity was observed. Brain tissue 1‐methylhistamine levels in both strains reached their maxima in the periods of activity. The mRNA level of histidine decarboxylase in the tuberomamillary nucleus and the activities of histidine decarboxylase and histamine‐N‐methyltransferase in the striatum and cortex did not show a 24‐h rhythm, whereas in the hypothalamus the activities of both enzymes had a 12‐h periodicity. These results show that the activities of histamine‐metabolizing enzymes are not under simple direct circadian regulation. The complex and non‐uniform temporal patterns of the histaminergic system of the mouse brain suggest that histamine is strongly involved in the maintenance of active wakefulness.

A novel developmental role for dopaminergic signaling to specify hypothalamic neurotransmitter identity

Hypothalamic neurons expressing histamine and orexin/hypocretin (hcrt) are necessary for normal regulation of wakefulness. In Parkinsons disease, the loss of dopaminergic neurons is associated with elevated histamine levels and disrupted sleep/wake cycles, but the mechanism is not understood. To characterize the role of dopamine in the development of histamine neurons, we inhibited the translation of the two non-allelic forms of tyrosine hydroxylase (th1 and th2) in zebrafish larvae. We found that dopamine levels were reduced in both th1 and th2 knockdown, but the serotonin level and number of serotonin neurons remained unchanged. Further, we demonstrated that th2 knockdown increased histamine neuron number and histamine levels, whereas increased dopaminergic signaling using the dopamine precursor l-DOPA (l-3,4-dihydroxyphenylalanine) or dopamine receptor agonists reduced the number of histaminergic neurons. Increases in the number of histaminergic neurons were paralleled by matching increases in the numbers of hcrt neurons, supporting observations that histamine regulates hcrt neuron development. Finally, we show that histaminergic neurons surround th2-expressing neurons in the hypothalamus, and we suggest that dopamine regulates the terminal differentiation of histamine neurons via paracrine actions or direct synaptic neurotransmission. These results reveal a role for dopaminergic signaling in the regulation of neurotransmitter identity and a potential mechanism contributing to sleep disturbances in Parkinsons disease.

Altered Electroencephalographic Activity Associated with Changes in the Sleep-Wakefulness Cycle of C57BL/6J Mice in Response to a Photoperiod Shortening

Aim: Under natural conditions diurnal rhythms of biological processes of the organism are synchronized with each other and to the environmental changes by means of the circadian system. Disturbances of the latter affect hormonal levels, sleep-wakefulness cycle and cognitive performance. To study mechanisms of such perturbations animal models subjected to artificial photoperiods are often used. The goal of current study was to understand the effects of circadian rhythm disruption, caused by a short light-dark cycle regime, on activity of the cerebral cortex in rodents. Methods: We used electroencephalogram to assess the distribution of vigilance states, perform spectral analysis, and estimate the homeostatic sleep drive. In addition, we analyzed spontaneous locomotion of C57BL/6J mice under symmetric, 22-, 21-, and 20-h-long light–dark cycles using video recording and tracking methods. Results and Conclusions: We found that shortening of photoperiod caused a significant increase of slow wave activity during non-rapid eye movement sleep suggesting an elevation of sleep pressure under such conditions. While the rhythm of spontaneous locomotion was completely entrained by all light–dark cycles tested, periodic changes in the power of the θ- and γ-frequency ranges during wakefulness gradually disappeared under 22- and 21-h-long light–dark cycles. This was associated with a significant increase in the θ–γ phase-amplitude coupling during wakefulness. Our results thus provide deeper understanding of the mechanisms underlying the impairment of learning and memory retention, which is associated with disturbed circadian regulation.

On the Role of Histamine Receptors in the Regulation of Circadian Rhythms

Several lines of evidence suggest a regulatory role of histamine in circadian rhythms, but little is known about signaling pathways that would be involved in such a putative role. The aim of this study was to examine whether histamine mediates its effects on the circadian system through Hrh1 or Hrh3 receptors. We assessed both diurnal and free-running locomotor activity rhythms of Hrh1 -/- and Hrh3 -/- mice. We also determined the expression of Per1, Per2 and Bmal1 genes in the suprachiasmatic nuclei, several areas of the cerebral cortex and striatum under symmetric 24 h light-dark cycle at zeitgeber times 14 and 6 by using radioactive in situ hybridization. We found no differences between Hrh1 -/- and wild type mice in the length, amplitude and mesor of diurnal and free-running activity rhythms as well as in expression of Per1, Per2 and Bmal1 genes in any of the examined brain structures. The amplitude of free-running activity rhythm of the Hrh3 -/- mice was significantly flattened, whereas the expression of the clock genes in Hrh3 -/- mice was similar to the wild type animals in all of the assessed brain structures. Therefore, the knockout of Hrh1 receptor had no effects on the circadian rhythm of spontaneous locomotion, and a knockout of Hrh3 receptor caused a substantial reduction of free-running activity rhythm amplitude, but none of these knockout models affected the expression patterns of the core clock genes in any of the studied brain structures.