Adenosine A1 Receptors Participate in Excitability Changes after Cortical Epileptic Afterdischarges in Immature Rats.

Background: Postictal refractoriness, i.e., the inability to elicit a new epileptic seizure immediately after the first one, is present in mature animals. Immature rats did not exhibit this refractoriness, and it is replaced by postictal potentiation. In addition to the immediate postictal potentiation, there is a delayed potentiation present at both ages. These phenomena were studied using cortical epileptic afterdischarges as a model. Objective: We aimed to analyze participation of adenosine A1 receptors in postictal potentiation and depression. Methods: Adenosine A1 receptors were studied by means of Western blotting in the cerebral cortex with a focus on the age groups studied electrophysiologically. Stimulation and recording electrodes were implanted epidurally in 12- and 25-day-old rats. The first stimulation always induced conditioning epileptic afterdischarge (AD), and 1 min after its end, the stimulation was repeated to elicit the second, testing AD. Then, the drugs were administered and paired stimulations were repeated 10 min later. A selective agonist CCPA (0.5 and 1 mg/kg i.p.) and a selective antagonist DPCPX (0.1, 0.5 and 1 mg/kg i.p.) were used to examine the possible participation of adenosine A1 receptors. Results: Control younger animals exhibited potentiation of the testing AD and a moderate increase in both conditioning and testing ADs after an injection of saline. The A1 receptor agonist CCPA shortened both post-drug ADs, and neither potentiation was present. The administration of an antagonist DPCPX resulted in marked prolongation of the conditioning AD (delayed potentiation), and the second testing AD was shorter than the post-drug conditioning AD, i.e., there was no longer immediate potentiation of ADs. To eliminate effects of the solvent dimethylsulfoxide, we added experiments with DPCPX suspended with the help of Tween 80. The results were similar, only the prolongation of ADs was not as large, and the testing ADs were significantly depressed. The older control group exhibited a nearly complete suppression of the first testing AD. There was no significant change in the conditioning and testing ADs after CCPA (delayed potentiation was blocked). Both groups of DPCPX-treated rats (with DMSO or Tween) exhibited significant augmentation of delayed potentiation but no significant difference in the immediate depression. Adenosine A1 receptors were present in the cerebral cortex of both age groups, and their quantity was higher in 12- than in 25-day-old animals. Conclusions: An agonist of the A1 receptor CCPA suppressed both types of postictal potentiation in 12-day-old rats, whereas the A1 antagonist DPCPX suppressed immediate potentiation but markedly augmented the delayed one. Immediate postictal refractoriness in 25-day-old rats was only moderately (non-significantly) affected; meanwhile, the delayed potentiation was strongly augmented.

Adenosine Kinase Isoforms in the Developing Rat Hippocampus after LiCl/Pilocarpine Status Epilepticus.

LiCl/pilocarpine status epilepticus (SE) induced in immature rats leads, after a latent period, to hippocampal hyperexcitability. The excitability may be influenced by adenosine, which exhibits anticonvulsant activity. The concentration of adenosine is regulated by adenosine kinase (ADK) present in two isoforms-ADK-L and ADK-S. The main goal of the study is to elucidate the changes in ADK isoform expression after LiCl/pilocarpine SE and whether potential changes, as well as inhibition of ADK by 5-iodotubercidin (5-ITU), may contribute to changes in hippocampal excitability during brain development. LiCl/pilocarpine SE was elicited in 12-day-old rats. Hippocampal excitability in immature rats was studied by the model of hippocampal afterdischarges (ADs), in which we demonstrated the potential inhibitory effect of 5-ITU. ADs demonstrated significantly decreased hippocampal excitability 3 days after SE induction, whereas significant hyperexcitability after 20 days compared to controls was shown. 5-ITU administration showed its inhibitory effect on the ADs in 32-day-old SE rats compared to SE rats without 5-ITU. Moreover, both ADK isoforms were examined in the immature rat hippocampus. The ADK-L isoform demonstrated significantly decreased expression in 12-day-old SE rats compared to the appropriate naïve rats, whereas increased ADK-S isoform expression was revealed. A decreasing ADK-L/-S ratio showed the declining dominance of ADK-L isoform during early brain development. LiCl/pilocarpine SE increased the excitability of the hippocampus 20 days after SE induction. The ADK inhibitor 5-ITU exhibited anticonvulsant activity at the same age. Age-related differences in hippocampal excitability after SE might correspond to the development of ADK isoform levels in the hippocampus.

Anticonvulsive Effects and Pharmacokinetic Profile of Cannabidiol (CBD) in the Pentylenetetrazol (PTZ) or N-Methyl-D-Aspartate (NMDA) Models of Seizures in Infantile Rats.

In spite of use of cannabidiol (CBD), a non-psychoactive cannabinoid, in pediatric patients with epilepsy, preclinical studies on its effects in immature animals are very limited. In the present study we investigated anti-seizure activity of CBD (10 and 60 mg/kg administered intraperitoneally) in two models of chemically induced seizures in infantile (12-days old) rats. Seizures were induced either with pentylenetetrazol (PTZ) or N-methyl-D-aspartate (NMDA). In parallel, brain and plasma levels of CBD and possible motor adverse effects were assessed in the righting reflex and the bar holding tests. CBD was ineffective against NMDA-induced seizures, but in a dose 60 mg/kg abolished the tonic phase of PTZ-induced generalized seizures. Plasma and brain levels of CBD were determined up to 24 h after administration. Peak CBD levels in the brain (996 ± 128 and 5689 ± 150 ng/g after the 10- and 60-mg/kg doses, respectively) were reached 1-2 h after administration and were still detectable 24 h later (120 ± 12 and 904 ± 63 ng/g, respectively). None of the doses negatively affected motor performance within 1 h after administration, but CBD in both doses blocked improvement in the bar holding test with repeated exposure to this task. Taken together, anti-seizure activity of CBD in infantile animals is dose and model dependent, and at therapeutic doses CBD does not cause motor impairment. The potential risk of CBD for motor learning seen in repeated motor tests has to be further examined.

Tuberous Sclerosis (tsc2+/-) Model Eker Rats Reveals Extensive Neuronal Loss with Microglial Invasion and Vascular Remodeling Related to Brain Neoplasia.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by frequent noncancerous neoplasia in the brain, which can induce a range of severe neuropsychiatric symptoms in humans, resulting from out of control tissue growth. The causative spontaneous loss-of-function mutations have been also identified in rats. Herein, we studied histopathological and molecular changes in brain lesions of the Eker rat model carrying germline mutation of the tsc2 gene, predisposed to multiple neoplasias. Predominant subcortical tumors were analyzed, along with a rare form occurring within the pyriform lobe. The uniform composition of lesions supports the histochemical parity of malformations, with immunofluorescence data supporting their neuro-glial origin. Massive depletion of mature neurons and axonal loss were evident within lesions, with occasional necrotic foci implying advanced stage of pathology. Enrichment of mesenchymal-derived cell markers with hallmarks of neurogenesis and active microglia imply enhanced cell proliferation, with local immune response. The depletion of capillaries within the core was complemented by the formation of dense mesh of nascent vessels at the interface of neoplasia with healthy tissue, implying large-scale vascular remodeling. Taken as a whole, these findings present several novel features of brain tumors in Eker rat model, rendering it suitable for studies of the pathobiology and progression of primary brain tumors, with therapeutic interventions.

New Design of the Electrophoretic Part of CLARITY Technology for Confocal Light Microscopy of Rat and Human Brains.

BACKGROUND: CLARITY is a method of rendering postmortem brain tissue transparent using acrylamide-based hydrogels so that this tissue could be further used for immunohistochemistry, molecular biology, or gross anatomical studies. Published papers using the CLARITY method have included studies on human brains suffering from Alzheimer's disease using mouse spinal cords as animal models for multiple sclerosis. METHODS: We modified the original design of the Chung CLARITY system by altering the electrophoretic flow-through cell, the shape of the platinum electrophoresis electrodes and their positions, as well as the cooling and recirculation system, so that it provided a greater effect and can be used in any laboratory. RESULTS: The adapted CLARITY system is assembled from basic laboratory components, in contrast to the original design. The modified CLARITY system was tested both on rat brain stained with a rabbit polyclonal anti-Iba-1 for microglial cells and on human nucleus accumbens stained with parvalbumin and tyrosine hydroxylase for visualization of specific neurons by confocal laser scanning microscopy. CONCLUSIONS: Our design has the advantage of simplicity, functional robustness, and minimal requirement for specialized additional items for the construction of the CLARITY apparatus.

Adenosine A1 Receptor Agonist 2-chloro-N6-cyclopentyladenosine and Hippocampal Excitability During Brain Development in Rats.

Objective: The adenosinergic system may influence excitability in the brain. Endogenous and exogenous adenosine has anticonvulsant activity presumably by activating A1 receptors. Adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) may thus bolster anticonvulsant effects, but its action and the number of A1 receptors at different developmental stages are not known. Methods: Hippocampal epileptic afterdischarges (ADs) were elicited in 12-, 15-, 18-, 25-, 45-, and 60-day-old rats. Stimulation and recording electrodes were implanted into the dorsal hippocampus. The A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 0.5 or 1 mg/kg) was administered intraperitoneally 10 min before the first stimulation. Control animals were injected with saline. All rats were stimulated with a 2-s series of 1-ms biphasic pulses delivered at 60 Hz with increasing stepwise intensity (0.05-0.6 mA). Each age and dose group contained 9-14 animals. The AD thresholds and durations were evaluated, and the A1 receptors were detected in the hippocampus in 7-, 10-, 12-, 15-, 18-, 21-, 25-, 32-, and 52-day-old rats. Results: Both CCPA doses significantly increased hippocampal AD thresholds in 12-, 15-, 18-, and 60-day-old rats compared to controls. In contrast, the higher dose significantly decreased AD threshold in the 25-day-old rats. The AD durations were significantly shortened in all age groups except for 25-day-old rats where they were significantly prolonged. A1 receptor expression in the hippocampus was highest in 10-day-old rats and subsequently decreased. Significance: The adenosine A1 receptor agonist CCPA exhibited anticonvulsant activity at all developmental stages studied here except for 25-day-old rats. Age-related differences might be due to the development of presynaptic A1 receptors in the hippocampus.

Behavioral and Pharmacokinetic Profile of Indole-Derived Synthetic Cannabinoids JWH-073 and JWH-210 as Compared to the Phytocannabinoid Δ9-THC in Rats.

Synthetic cannabinoid compounds are marketed as "legal" marijuana substitutes, even though little is known about their behavioral effects in relation to their pharmacokinetic profiles. Therefore, in the present study we assessed the behavioral effects of systemic treatment with the two synthetic cannabinoids JWH-073 and JWH-210 and the phytocannabinoid Δ9-THC on locomotor activity, anxiety-like phenotype (in the open field) and sensorimotor gating (measured as prepulse inhibition of the acoustic startle response, PPI), in relation to cannabinoid serum levels. Wistar rats were injected subcutaneously (sc.) with JWH-073 (0.1, 0.5, or 5 mg/kg), JWH-210 (0.1, 0.5, or 5 mg/kg), Δ9-THC (1 or 3 mg/kg) or vehicle (oleum helanti) in a volume of 0.5 ml/kg and tested in the open field and PPI. Although JWH-073, JWH-210, Δ9-THC (and its metabolites) were confirmed in serum, effects on sensorimotor gating were absent, and locomotor activity was only partially affected. Δ9-THC (3 mg/kg) elicited an anxiolytic-like effect as suggested by the increased time spent in the center of the open field (p < 0.05). Our results further support the potential anxiolytic-like effect of pharmacological modulation of the endocannabinoid system.

Corrigendum: Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits.

[This corrects the article on p. 42 in vol. 9, PMID: 29487522.].

Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits.

The role of NMDA receptors in learning, memory and hippocampal function has long been recognized. Post-mortem studies have indicated that the expression or subunit composition of the NMDA glutamate receptor subtype might be related to the impaired cognitive functions found in schizophrenia patients. NMDA receptor antagonists have been used to develop animal models of this disorder. There is accumulating evidence showing that not only the acute but also the chronic application of NMDA receptor antagonists may induce schizophrenia-like alterations in behavior and brain functions. However, limited evidence is available regarding the consequences of NMDA receptor blockage during periods of adolescence and early adulthood. This study tested the hypothesis that a 2-week treatment of male Long-Evans and Wistar rats with dizocilpine (MK-801; 0.5 mg/kg daily) starting at postnatal days (PD) 30 and 60 would cause a long-term cognitive deficit and changes in the levels of NMDA receptor subunits. The working memory version of the Morris water maze (MWM) and active place avoidance with reversal on a rotating arena (Carousel) requiring cognitive coordination and flexibility probed cognitive functions and an elevated-plus maze (EPM) was used to measure anxiety-like behavior. The western blot method was used to determine changes in NMDA receptor subunit levels in the hippocampus. Our results showed no significant changes in behaviors in Wistar rats. Slightly elevated anxiety-like behavior was observed in the EPM in Long-Evans rats with the onset of treatment on PD 30. Furthermore, Long-Evans rats treated from PD 60 displayed impaired working memory in the MWM. There were; however, no significant changes in the levels of NMDA receptor subunits because of MK-801 administration. These findings suggest that a 2-week treatment starting on PD 60 in Long-Evans rats leads to long-term changes in working memory, but this deficit is not paralleled by changes in NMDA receptor subunits. These results support the face validity, but not construct validity of this model. We suggest that chronic treatment of adolescent and adult rats does not constitute a plausible animal model of schizophrenia.

Pharmacokinetic, Ambulatory, and Hyperthermic Effects of 3,4-Methylenedioxy-N-Methylcathinone (Methylone) in Rats.

Methylone (3,4-methylenedioxy-N-methylcathinone) is a synthetic cathinone analog of the recreational drug ecstasy. Although it is marketed to recreational users as relatively safe, fatalities due to hyperthermia, serotonin syndrome, and multi-organ system failure have been reported. Since psychopharmacological data remain scarce, we have focused our research on pharmacokinetics, and on a detailed evaluation of temporal effects of methylone and its metabolite nor-methylone on behavior and body temperature in rats. Methylone [5, 10, 20, and 40 mg/kg subcutaneously (s.c.)] and nor-methylone (10 mg/kg s.c.) were used in adolescent male Wistar rats across three behavioral/physiological procedures and in two temporal windows from administration (15 and 60 min) in order to test: locomotor effects in the open field, sensorimotor gating in the test of prepulse inhibition (PPI), and effects on rectal temperature in individually and group-housed rats. Serum and brain pharmacokinetics after 10 mg/kg s.c. over 8 h were analyzed using liquid chromatography mass spectrometry. Serum and brain levels of methylone and nor-methylone peaked at 30 min after administration, both drugs readily penetrated the brain with serum: brain ratio 1:7.97. Methylone dose-dependently increased overall locomotion. It also decrease the amount of time spent in the center of open field arena in dose 20 mg/kg and additionally this dose induced stereotyped circling around the arena walls. The maximum of effects corresponded to the peak of its brain concentrations. Nor-methylone had approximately the same behavioral potency. Methylone also has weak potency to disturb PPI. Behavioral testing was not performed with 40 mg/kg, because it was surprisingly lethal to some animals. Methylone 10 and 20 mg/kg s.c. induced hyperthermic reaction which was more pronounced in group-housed condition relative to individually housed rats. To conclude, methylone increased exploration and/or decreased anxiety in the open field arena and with nor-methylone had short duration of action with effects typical for mixed indirect dopamine-serotonin agonists such as 3,4-metyhlenedioxymethamphetamine (MDMA) or amphetamine. Given the fact that the toxicity was even higher than the known for MDMA and that it can cause hyperthermia it possess a threat to users with the risk for serotonin syndrome especially when used in crowded conditions.

Pharmacokinetic and behavioural profile of THC, CBD, and THC+CBD combination after pulmonary, oral, and subcutaneous administration in rats and confirmation of conversion in vivo of CBD to THC.

Metabolic and behavioural effects of, and interactions between Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are influenced by dose and administration route. Therefore we investigated, in Wistar rats, effects of pulmonary, oral and subcutaneous (sc.) THC, CBD and THC+CBD. Concentrations of THC, its metabolites 11-OH-THC and THC-COOH, and CBD in serum and brain were determined over 24h, locomotor activity (open field) and sensorimotor gating (prepulse inhibition, PPI) were also evaluated. In line with recent knowledge we expected metabolic and behavioural interactions between THC and CBD. While cannabinoid serum and brain levels rapidly peaked and diminished after pulmonary administration, sc. and oral administration produced long-lasting levels of cannabinoids with oral reaching the highest brain levels. Except pulmonary administration, CBD inhibited THC metabolism resulting in higher serum/brain levels of THC. Importantly, following sc. and oral CBD alone treatments, THC was also detected in serum and brain. S.c. cannabinoids caused hypolocomotion, oral treatments containing THC almost complete immobility. In contrast, oral CBD produced mild hyperlocomotion. CBD disrupted, and THC tended to disrupt PPI, however their combination did not. In conclusion, oral administration yielded the most pronounced behavioural effects which corresponded to the highest brain levels of cannabinoids. Even though CBD potently inhibited THC metabolism after oral and sc. administration, unexpectedly it had minimal impact on THC-induced behaviour. Of central importance was the novel finding that THC can be detected in serum and brain after administration of CBD alone which, if confirmed in humans and given the increasing medical use of CBD-only products, might have important legal and forensic ramifications.

Mephedrone (4-Methylmethcathinone): Acute Behavioral Effects, Hyperthermic, and Pharmacokinetic Profile in Rats.

Mephedrone (MEPH) is a synthetic cathinone derivative with effects that mimic MDMA and/or cocaine. Our study in male Wistar rats provides detailed investigations of MEPH's and its primary metabolite nor-mephedrone's (nor-MEPH) pharmacokinetics and bio-distribution to four different substrates (serum, brain, lungs, and liver), as well as comparative analysis of their effects on locomotion [open field test (OFT)] and sensorimotor gating [prepulse inhibition of acoustic startle reaction (PPI ASR)]. Furthermore, in order to mimic the crowded condition where MEPH is typically taken (e.g., clubs), the acute effect of MEPH on thermoregulation in singly- and group-housed rats was evaluated. Pharmacokinetics of MEPH and nor-MEPH after MEPH (5 mg/kg, sc.) were analyzed over 8 h using liquid chromatography with mass spectrometry. MEPH (2.5, 5, or 20 mg/kg, sc.) and nor-MEPH (5 mg/kg, sc.) were administered 5 or 40 min before the behavioral testing in the OFT and PPI ASR; locomotion and its spatial distribution, ASR, habituation and PPI itself were quantified. The effect of MEPH on rectal temperature was measured after 5 and 20 mg/kg, sc. Both MEPH and nor-MEPH were detected in all substrates, with the highest levels detected in lungs. Mean brain: serum ratios were 1:1.19 (MEPH) and 1:1.91 (nor-MEPH), maximum concentrations were observed at 30 min; at 2 and 4 h after administration, nor-MEPH concentrations were higher compared to the parent drug. While neither of the drugs disrupted PPI, both increased locomotion and affected its spatial distribution. The effects of MEPH were dose dependent, rapid, and short-lasting, and the intensity of locomotor stimulant effects was comparable between MEPH and nor-MEPH. Despite the disappearance of behavioral effects within 40 min after administration, MEPH induced rectal temperature elevations that persisted for 3 h even in singly housed rats. To conclude, we observed a robust, short-lasting, and most likely synergistic stimulatory effect of both drugs which corresponded to brain pharmacokinetics. The dissociation between the duration of behavioral and hyperthermic effects is indicative of the possible contribution of nor-MEPH or other biologically active metabolites. This temporal dissociation may be related to the risk of prolonged somatic toxicity when stimulatory effects are no longer present.