What is the role of subcutaneous single injections on the behavior of adult male rats exposed to drugs?

Psychostimulants, as well as cannabinoids, have been shown to significantly affect a great variety of behaviors in both humans and laboratory animals. Our previous studies have repeatedly demonstrated that the application of the vehicle for psychostimulants, i.e. saline, to control groups, generated different behavioral test results compared to absolute naive controls (i.e. without any injection). Therefore, our present study has set three goals: (1) to evaluate the effect of three different psychostimulant drugs, (2) to evaluate the effect of three doses of delta 9-tetrahydrocannabinol (THC), and (3) to evaluate the effect of saline and ethanol injections vs sham injections and no injection on spontaneous behavior of adult male rats. The LABORAS test (Metris B.V., Netherlands) was used to examine spontaneous locomotor activity and exploratory behavior in an unknown environment over 1 h. In Experiment 1, psychostimulant drugs were tested: single subcutaneous (s.c.) injections of amphetamine (5 mg/kg), cocaine (5 mg/kg), and 3,4-methylenedioxymethamphetamine (MDMA) (5 mg/kg) were applied prior to testing. Control animals received the same volume (1 ml/kg) of s.c. saline. In Experiment 2, the effect of three doses of THC (1, 2, and 5 mg/kg, s.c.) were examined. An s.c. injection of vehicle (ethanol) was used as a control. In Experiment 3, injections of saline and ethanol were compared to the group receiving a sham s.c. injection and to a group of absolute "naive" controls. Our results demonstrated that (1) all psychostimulants increased locomotion time, distance traveled, and speed while decreasing immobility time of adult male rats relative to saline controls. The most prominent effect was associated with MDMA; (2) The effect of THC was dose-dependent and was most apparent within the first 10 min of the LABORAS test. (3) With regard to the effect of injection: absolute controls (without injection) compared to animals injected with ethanol, saline, or sham-injected displayed reduced immobility time, traveled longer distances, and had increased speed. In conclusion, our data showed drug dependent behavioral changes in adult male rats after application of psychostimulants and cannabinoids. Our findings also suggest that not only drugs but the actual single injection per se also affects the behavior of laboratory animals in an unknown environment. This effect seems to be associated with the acute stress associated with the injection.

Changes of cortical perfusion in the early phase of subarachnoid bleeding in a rat model and the role of intracranial hypertension.

Brain perfusion is reduced early after subarachnoid hemorrhage (SAH) due to intracranial hypertension and early vasospasm. The contribution of these two mechanisms is unknown. By performing a prophylactic decompressive craniectomy (DC) in a rat model of SAH we aimed to study brain perfusion after the component of intracranial hypertension has been eliminated. We used 2x2 factorial design, where rats received either decompressive craniectomy or sham operation followed by injection of 250 microl of blood or normal saline into prechiasmatic cistern. The cortical perfusion has been continually measured by laser speckle-contrast analysis for 30 min. Injection of blood caused a sudden increase of intracranial pressure (ICP) and drop of cerebral perfusion, which returned to baseline within 6 min. DC effectively prevented the rise of ICP, but brain perfusion after SAH was significantly lower and took longer to normalize compared to non-DC animals due to increased cerebral vascular resistance, which lasted throughout 30 min experimental period. Our findings suggest that intracranial hypertension plays dominant role in the very early hypoperfusion after SAH whilst the role of early vasospasm is only minor. Prophylactic DC effectively maintained cerebral perfusion pressure, but worsened cerebral perfusion by increased vascular resistance.

Morphine decreases social interaction of adult male rats, while THC does not affect it.

The aim of the present study was to compare effect of three low doses of morphine (MOR) and delta9-tetrahydrocannabinol (THC) on social behavior tested in Social interaction test (SIT). 45 min prior to testing adult male rats received one of the drugs or solvents: MOR (1; 2.5; 5 mg/kg); saline as a solvent for MOR; THC (0.5; 1; 2 mg/kg); ethanol as a solvent for THC. Occurrence and time spent in specific patterns of social interactions (SI) and non-social activities (locomotion and rearing) was video-recorded for 5 min and then analyzed. MOR in doses of 1 and 2.5 mg/kg displayed decreased SI in total. Detailed analysis of specific patterns of SI revealed decrease in mutual sniffing and allo-grooming after all doses of MOR. The highest dose (5 mg/kg) of MOR decreased following and increased genital investigation. Rearing activity was increased by lower doses of MOR (1 and 2.5 mg/kg). THC, in each of the tested doses, did not induce any specific changes when compared to matching control group (ethanol). However, an additional statistical analysis showed differences between all THC groups and their ethanol control group when compared to saline controls. There was lower SI in total, lower mutual sniffing and allo-grooming, but higher rearing in THC and ethanol groups than in saline control group. Thus, changes seen in THC and ethanol groups are seemed to be attributed mainly to the effect of the ethanol. Based on the present results we can assume that opioids affect SI more than cannabinoid.

Does effect from developmental methamphetamine exposure on spatial learning and memory depend on stage of neuroontogeny?

Psychostimulants, including methamphetamine (MA), have neurotoxic effect, especially, if they are targeting CNS during its critical periods of development. The present study was aimed to examine cognitive changes after prenatal and neonatal MA treatment in combination with chronic MA exposure in adulthood of male rats. Eight groups of male rats were tested in adulthood: males whose mothers were exposed to MA (5 mg/kg) or saline (SA, 1 ml/kg) during the first half of gestation period (GD 1-11), the second half of gestation period (GD 12-22) and neonatal period (PD 1-11). In addition, we compared indirect neonatal application via the breast milk with the group of rat pups that received MA or SA directly by injection (PD 1-11). Males were tested in adulthood for cognitive changes in the Morris Water Maze (MWM). MWM experiment lasted for 12 days: Learning (Day 1-6), Probe test (Day 8) and Retrieval Memory test (Day 12). Each day of the MWM animals were injected with MA (1 mg/kg) or SA (1 ml/kg). Prenatal MA exposure did not induce changes in learning abilities of male rats, but neonatal exposure to MA leads to an increase search errors and latencies to find the hidden platform. Prenatal and also neonatal MA exposure impaired cognitive ability to remember the position of the platform in Retrieval Memory test in adulthood. Animals exposed to the prenatal treatment within the second half of gestation (ED 12-22) swam longer, slower and spent more time to find the hidden platform in Retrieval Memory test than animals exposed throughout other periods. The present study demonstrated that stage of development is crucial for determination the cognitive deficits induced by prenatal or neonatal MA exposure.

Early changes of brain perfusion after subarachnoid hemorrhage - the effect of sodium nitroprusside.

Causes of early hypoperfusion after subarachnoid hemorrhage (SAH) include intracranial hypertension as well as vasoconstriction. The aim of the study was to assess the effect of intracerebroventricular (ICV) administration of sodium nitroprusside (SNP) on early hypoperfusion after SAH. Male Wistar rats (220-240 g) were used, SAH group received 250 microl of fresh autologous arterial blood into the prechiasmatic cistern; sham-operated animals received 250 microl of isotonic solution. Therapeutic intervention: ICV administration of 10 microg SNP; 5 microl 5 % glucose (SNP vehicle) and untreated control. Brain perfusion and invasive blood pressure were monitored for 30 min during and after induction of SAH. Despite SNP caused increase of perfusion in sham-operated animals, no response was observed in half of SAH animals. The other half developed hypotension accompanied by brain hypoperfusion. There was no difference between brain perfusion in SNP-treated, glucose-treated and untreated SAH animals during the monitored period. We did not observe expected beneficial effect of ICV administration of SNP after SAH. Moreover, half of the SNP-treated animals developed serious hypotension which led to brain hypoperfusion. This is the important finding showing that this is not the option for early management in patient after SAH.

Direct measurement of free radical levels in the brain after cortical ischemia induced by photothrombosis.

Tissue ischemia is connected with the production of free radicals (FR). This study was designed to directly measure of the amount of FR in rat brains related to a photothrombotic ischemic event shortly after establishing the lesion. A model of left hemisphere photothrombosis ischemia was used in the experiment. Brains of animals from the experimental group were removed and placed in liquid N(2) for 60 min after the green laser exposure, the control group brains, exposed to the photosensitive dye Rose Bengal (RB), were placed in liquid N(2) for 80 min after RB application, naive control brains were also briefly stored in liquid N(2). Spectroscopy of electron paramagnetic (spin) resonance was used to directly measure FR (hydroxyl (OH(.)) and nitroxyl (NO(.)). Compared to naive controls, both the ischemia and RB groups had significantly higher levels of OH(.), however, there were no differences between them. Comparison of hemispheres, i.e., with and without ischemia, in the experimental group did not show any significant difference in OH(.). NO(.) were elevated in the ischemia and RB groups compare to naive controls. Higher levels of NO(.) were found in hemispheres with ischemia compared to unexposed hemispheres. Increases in OH(.) were probably associated with the action of RB itself in this model of ischemia. Increases in NO(.) were closely related to the pathogenesis of photothrombotic ischemia and could be related to the activity of nitric oxide synthases.

How various drugs affect anxiety-related behavior in male and female rats prenatally exposed to methamphetamine.

Different forms of anxiety-related behavior have been reported after a single drug use of many abused substances, however, less is known about how males and females are affected differently from exposure to various drugs. Furthermore, chronic prenatal methamphetamine (MA) exposure was shown to predispose the animal to an increased sensitivity to drugs administrated in adulthood. Using the Elevated plus-maze test (EPM), the first aim of the present study was to examine how male and female rats are affected by acute drug treatment with subcutaneously (s.c.) administrated (a) MA (1mg/kg); (b) drugs with a similar mechanism of action to MA: amphetamine (AMP, 1mg/kg), cocaine (COC, 5mg/kg), 3,4-methylenedioxymethamphetamine (MDMA, 5mg/kg); and (c) drugs with different mechanisms of action: morphine (MOR, 5mg/kg), and Δ 9-tetrahydrocannabinol (THC, 2mg/kg). The second aim was to determine if prenatally MA-exposed (5mg/kg) animals show an increased sensitivity to adult drug treatment. The parameters analyzed were divided into two categories: anxiety-related behavior and anxiety-unrelated/exploratory behavior. Our results showed in female rats a decreased percentage of the time spent in the closed arms (CA) after MA, and an increased percentage of the time spent in the open arms (OA) after MA, AMP, and COC treatment, indicating an anxiolytic-like effect. In females, MDMA and THC treatment increased the percentage of the time spent in the CA. An increased percentage of the time spent in the CA was also seen after MOR treatment in females as well as in males, indicating an anxiogenic-like effect. As far as the interaction between prenatal MA exposure and adult drug treatment is concerned, there was no effect found. In conclusion, it seems that: (a) in some cases female rats are more vulnerable to acute drug treatment, in terms of either anxiogenic- or anxiolytic-like effects; (b) prenatal MA exposure does not sensitize animals to the anxiety-related effects of any of the drugs.

[The role of nitric oxide and NO-synthase in the pathogenesis of cerebral damage after subarachnoid hemorrhage; laboratory models of subarachnoid hemorrhage]. / Role oxidu dusného a NO-syntázy v patofyziologii poskozeni mozku po subarachnoidálním krváceni; laboratorni modely subarachnoidálního krváceni.

Subarachnoid hemorrhage (SAH) of CNS is acute life-threating condition. In addition to its well understood sequential increase in intracranial pressure and decreased cerebral perfusion pressure, there is also early and late vasoconstriction. Mechanism of vasoconstriction is complex and one of important roles play changes in the amount of nitric oxide (NO). Present work overviews known pathogenesis of non-traumatic SAH, with stress on NO regulation of cerebral blood flow and its changes during SAH. It also describes mechanisms of early and late brain damage following subarachnoid hemorrhage. We discuss possible pharmacological prevention of the damage and laboratory models of nontraumatic SAH.

Gender differences in the effect of adult amphetamine on cognitive functions of rats prenatally exposed to methamphetamine.

Psychostimulants have been shown to affect brain regions involved in the process of learning and memory consolidation. It has been shown that females are more sensitive to the effects of drugs than males. The aim of our study was to investigate how prenatal methamphetamine (MA) exposure and application of amphetamine (AMP) in adulthood would affect spatial learning of adult female and male rats. Mothers of the tested offspring were exposed to injections of MA (5mg/kg) or saline (SA) throughout the entire gestation period. Cognitive functions of adult rats were evaluated in the Morris Water Maze (MWM) tests. Adult offspring were injected daily with AMP (5mg/kg) or SA through the period of MWM testing. Our data from the MWM tests demonstrates the following. Prenatal MA exposure did not change the learning ability of adult male and female rats. However, AMP administration to adult animals affected cognitive function in terms of exacerbation of spatial learning (increasing the latency to reach the hidden platform, the distance traveled and the search error) only in female subjects. There were sex differences in the speed of swimming. Prenatal MA exposure and adult AMP treatment increased the speed of swimming in female groups greater than in males. Overall, the male subjects showed a better learning ability than females. Thus, our results indicate that the adult AMP treatment affects the cognitive function and behavior of rats in a sex-specific manner, regardless of prenatal exposure.

Exposure to methamphetamine during first and second half of prenatal period and its consequences on cognition after long-term application in adulthood.

It is known that psychostimulants including methamphetamine (MA) have neurotoxic effect, especially, if they are targeting CNS during its critical periods of development. The present study was aimed on evaluation of cognitive changes following scheduled prenatal MA exposure in combination with long-term exposure in adulthood of male rats. Two periods of gestation were targeted: 1(st) half - the embryonic day (ED) 1-11 and 2(nd) half - ED 12-22. Rat mothers received subcutaneously a daily injection of MA (5 mg/kg) or saline (SAL, 1 ml/kg) throughout scheduled periods. Male offspring were tested for cognitive changes in the Morris Water Maze (MWM) in adulthood. Each day of the experiment animals received an injection of MA (1 mg/kg) or SAL (1 ml/kg) during 12 days. Our results demonstrated that in the group of animals exposed to the drug during ED 1-11, neither prenatal MA exposure, nor adult MA treatment changed the performance in the MWM test. Only the velocity was increased in group with long-term MA treatment (SAL/MA and MA/MA). In the group of animals exposed to the drug during ED 12-22, rats exposed to MA prenatally and also in adulthood (MA/MA) swam faster but learned the position of the platform slower in the Place Navigation Test than animals exposed to SAL in adulthood (MA/SAL). In the Probe Test, MA/SAL had decreased velocity and swam shorter distance than MA/MA or SAL/SAL rats suggesting increased floating of these animals. In the Memory Retention Test, SAL/MA rats swam shorter distance than SAL/SAL or MA/MA animals suggesting changes in used strategies in memory recall. As conclusion, our results suggest differences in the effect of combination of prenatal and adult exposure to MA. These effects further depend on the stage of CNS development and schedule of MA exposure affecting intrauterine development in male rats.

Prenatal methamphetamine exposure induces long-lasting alterations in memory and development of NMDA receptors in the hippocampus.

Since close relationship was shown between drug addiction and memory formation, the aim of the present study was to investigate the effects of interaction between prenatal methamphetamine (MA) exposure and MA treatment in adulthood on spatial and non-spatial memory and on the structure of the N-methyl-D-aspartate (NMDA) receptors in the hippocampus. Adult male rats prenatally exposed to MA (5 mg/kg) or saline were tested in adulthood. Non-spatial memory was examined in the Object Recognition Test (ORT) and spatial memory in the Object Location Test (OLT) and in the Memory Retention Test (MRT) conducted in the Morris Water Maze (MWM), respectively. Based on the type of the memory test animals were injected either acutely (ORT, OLT) or long-term (MWM) with MA (1 mg/kg). After each testing, animals were sacrificed and brains were removed. The hippocampus was then examined in Western Blot analysis for occurrence of different NMDA receptors' subtypes. Our results demonstrated that prenatal MA exposure affects the development of the NMDA receptors in the hippocampus that might correspond with improvement of spatial memory tested in adulthood in the MWM. On the other hand, the effect of prenatal MA exposure on non-spatial memory examined in the ORT was the opposite. In addition, we showed that the effect of MA administration in adulthood on NMDA receptors is influenced by prenatal MA exposure, which seems to correlate with the spatial memory examined in the OLT.

Oxidative stress induced by epileptic seizure and its attenuation by melatonin.

An epileptic seizure and postictal period in addition to well-known features are also characterized by massive consumption of energy. This is thought to lead to oxidative stress and increased generation of free radicals, which is reflected by increased levels of oxidative products. Our previous work described the neuroprotective effects of melatonin in preventing cognitive worsening after a single epileptic seizure. This work was aimed on direct measurement of free radicals in brain tissue using the EPR method 1, 15 and 60 minutes after seizure. The measurement was performed in adult male Wistar rats at the mentioned intervals after a single tonic-clonic seizure induced by flurothyl. In comparison to control animals there was a significant increase in hydroxyl and nitroxyl radicals 60 minutes after the seizure. The levels of hydroxyl radicals were significantly lower in animals that received melatonin 60 minutes before seizure induction compared to animals without preventive treatment. Therefore, melatonin affected the generation of the measured free radicals differently. An important finding was the delayed increase in free radicals after a single seizure in the later phases of recovery.

[Selected problems of behavioral tests]. / Nekteré problémy behaviorálního testování.

Changes in experimental animals' behavior as well as humans' behavior reflect even mild alterations in function of central nervous system. In order to detect those changes variety of tests' batteries were developed. With help of such unified tests it is possible to determine and differentiate individual behavioral parameters and establish their relation to specific CNS alterations. Behavioral tests results from animal studies thereafter can be linked to certain type of behavioral changes in humans, because biochemical composites and reactions of the brain are significantly alike in all of mammals. The advantages of behavioral tests include its non-invasive methods of detection and possibility to determine changes of function even before there are any morphological or biochemical representations. The following work overviews specific aspects of behavioral testing, e.g., animals' preparation to the test, behavioral categories, possibility of tests' measurements and their limitations.