Lack of tolerance for the anti-dyskinetic effects of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in rats

7-Nitroindazole (7-NI) inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS). The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson’s disease.

Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Behavioral and physiological methods for early quantitative assessment of spinal cord injury and prognosis in rats

Methods for reliable evaluation of spinal cord (SC) injury in rats at short periods (2 and 24 h) after lesion were tested to characterize the mechanisms implicated in primary SC damage. We measured the physiological changes occurring after several procedures for producing SC injury, with particular emphasis on sensorimotor functions. Segmental and suprasegmental reflexes were tested in 39 male Wistar rats weighing 250-300 g divided into three control groups that were subjected to a) anesthesia, b) dissection of soft prevertebral tissue, and c) laminectomy of the vertebral segments between T10 and L1. In the lesion group the SC was completely transected, hemisected or subjected to vertebral compression. All animals were evaluated 2 and 24 h after the experimental procedure by the hind limb motility index, Bohlman motor score, open-field, hot-plate, tail flick, and paw compression tests. The locomotion scale proved to be less sensitive than the sensorimotor tests. A reduction in exploratory movements was detected in the animals 24 h after the procedures. The hot-plate was the most sensitive test for detecting sensorimotor deficiencies following light, moderate or severe SC injury. The most sensitive and simplest test of reflex function was the hot-plate. The hemisection model promoted reproducible moderate SC injury which allowed us to quantify the resulting behavior and analyze the evolution of the lesion and its consequences during the first 24 h after injury. We conclude that hemisection permitted the quantitation of behavioral responses for evaluation of the development of deficits after lesions. Hind limb evaluation scores and spontaneous exploration events provided a sensitive index of immediate injury effects after SC lesion at 2 and 24 h. Taken together, locomotion scales, open-field, and hot-plate tests represent reproducible, quantitatively sensitive methods for detecting functional deficiencies within short periods of time, indicating their potential...

Ginkgo biloba leaf extract (EGb 761) enhances catalepsy induced by haloperidol and L-nitroarginine in mice

Ginkgo biloba extract EGb 761 has been reported to have therapeutic effects which have been attributed to anti-oxidant and free radical-scavenging activities, including a direct action on nitric oxide production. L G-nitro-arginine (L-NOARG), a nitric oxide synthase inhibitor, and haloperidol, a drug that blocks dopamine receptors, are both known to induce catalepsy in rodents. Nitric oxide has been shown to influence dopaminergic transmission in the striatum. The purpose of the present study was to evaluate the effect of the extract obtained from leaves of Ginkgo biloba tree EGb 761 on catalepsy induced by haloperidol or by L-NOARG. Albino Swiss mice (35-45 g, N = 8-12) received by gavage a single or repeated oral dose (twice a day for 4 days) of EGb 761 followed by ip injection of haloperidol or L-NOARG. After the treatments, the animals were submitted to behavioral evaluation using the catalepsy test. Acute treatment with 80 mg/kg EGb did not modify the catalepsy induced by L-NOARG but, the dose of 40 mg/kg significantly enhanced haloperidol-induced catalepsy measured at the 10th min of the test. After repeated treatment with 80 mg/kg EGb 761, a significant increase in the cataleptic effect produced by both haloperidol and L-NOARG was observed. These data show that repeated EGb 761 administration increases the effects of drugs that modify motor behavior in mice. Since the catalepsy test has predictive value regarding extrapyramidal effects, the possibility of pharmacological interactions between haloperidol and Ginkgo biloba extracts should be further investigated in clinical studies.

Routine post-weaning handling of rats prevents isolation rearing-induced deficit in prepulse inhibition

Rats reared under isolation conditions from weaning present a number of behavioral changes compared to animals reared under social conditions (group housing). These changes include deficits in prepulse inhibition (PPI) of the startle reflex to a loud sound. PPI refers to the reduction of the magnitude of the startle reflex when a relatively weak stimulus (the prepulse) precedes by an appropriate time interval the intense startle-elicing stimulus (the pulse). PPI is useful for studying sensorimotor integration. The present study evaluated the effect of handling on the impairment of PPI induced by isolation-rearing. Male Wistar rats (N = 11-15/group) were housed in groups (5 per cage and handled three times a week) or isolated (housed individually) since weaning (21 days) for 10 weeks when they reach approximately 150 g. The isolated rats were divided into "minimally handled" animals (handled once a week for cleaning purposes only) or "handled" animals (handled three times a week). This handling consisted of grasping the rat by the tail and moving it to a clean cage (approximately 5 s). A statistically significant reduction (52 percent) in the PPI test was found only in the isolated group with minimal handling while no difference was seen between grouped animals and isolated handled animals. These results indicate that isolation rearing causes disruption in the PPI at adult age, which serves as an index of attention deficit. This change in the sensory processing of information induced by post-weaning isolation can be prevented by handling during the development of the animal.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models

The effect of acute (120 mg/kg) and chronic (25 mg/kg, twice a day, for 4 days) intraperitonial injection of the nitric oxide (NO) synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NOARG) was evaluated on seizure induction by sound drugs such as pilocarpine and pentylenetetrazole (PTZ) and by stimulation of audiogenic seizure-resistant (R) and audiogenic seizure-susceptible (S) rats. Seizures were elicited by a subconvulsant dose of pilocarpine (100 mg/kg) only after NOS inhibition. NOS inhibition also simultaneously potentiated the severity of PTZ-induced limbic seizures (60 mg/kg) and protected against PTZ-induced tonic seizures (80 mg/kg). The audiogenic seizure susceptibility of S or R rats did not change after similar treatments. In conclusion, proconvulsant effects of NOS inhibition are suggested to occur in the pilocarpine model and in the limbic componentes of PTZ-induced seizures, while an anticonvulsant role is suggested for the tonic seizures induced by higher doses of PTZ, revealing inhibitor-specific interations with convulsant dose and also confirming the hypothesis that the effects of NOS inhibitors vary with the model of seizure.

Pinealectomy attenuates the effect of restraint on plus maze exploration in rats

To investigate a possible stress modulation role of the pineal gland, male Wistar albino rats (200-250 g) were submitted to pinealectomy and divided into four groups one week after surgery: i) sham-operated unrestrained animals (N = 14); ii) pinealectomized unrestrained animals (N = 22); iii) sham-operated animals submitted to 2 h of restraint (N = 52); iv) pinealectomized animals submitted to 2 h of restraint (N = 56). Twenty-four hours later the animals were tested in the elevated plus maze for 5 min. Pinealectomized rats submitted to restraint explored the open arms of the maze to a greater extent than sham-operated restrained rats (mean percent of open arm entries = 26.4 ñ 2.3 vs 18.0 ñ 2.1, mean percent of time spent in the open arms = 11.8 ñ 2.1 vs 6.8 ñ 1.2). Pinealectomized animals not submitted to restraint showed no difference in maze exploration when compared to sham-operated rats (mean percent of open arm entries = 29.3 ñ 3.8 vs 31.1 ñ 5.8, mean percent of time spent in the open arms = 8.8 ñ 1.8 vs 12.5 ñ 2.2). The results, therefore, suggest that the pineal gland may play a modulatory role in the behavioral consequences of stress.

Intraventricular cycloheximide attenuates the restraint-induced long-lasting effect on plus maze exploration

Rats submitted to 2 h of restraint stress show reduced open arm exploration in the elevated plus maze 24 h later. To determine if this effect is dependent on protein synthesis during or after the restraint period, cycloheximide, a protein synthesis inhibitor, was injected into the right cerebral ventricle of male Wistar rats (200-250 g), immediately before (N = 19 animals per group), immediately after (N = 7 animals per group) or 2 h (N = 10 animals per group) following a 2h period of forced restraint. Twenty-four hours later the animals were tested in the elevated plus maze. Non-stressed control groups received saline (SAL, N = 8-9 per group) or cycloheximide (CHX, N = 8-9 per group) and were tested 1 h or 24 h later in the maze. Pre- but not post-stress microinjections of cycloheximide (20 mug in 2 mul) increased exploration in the elevated plus maze (per cent of time spent in open arms, pre-stress injection: SAL = 4.6 ñ 1.2, CHX = 10.7 2.3; number of enclosed arms entries: SAL = 3.6 ñ 0.5, CHX = 5.6 0.4). No drug effect was observed in non-stressed animals. These results suggest that blockade of protein synthesis during the restraint period may attenuate the behavioral consequences of stress.

Stress-induced expression of the c-fos proto-oncogene in the hippocampal formation

To investigate the effects of stress on c-fos mRNA expression, rats were submitted to forced immobilization for 15, 30, 60 or 120 min before sacrifice. In situ hybridization was performed on sections containing the dorsal hippocampus with a 32P-labelled 50-base oligonucleotide probe (10(7)-10(9) cpm/micrograms) complementary to nucleotides 370-319 of rat c-fos. Forced restraint induced a time-dependent increase in c-fos mRNA expression which was most pronounced in the dentate gyrus and CA1-CA3 regions of the hippocampal formation, and which peaked after 30 min of immobilization (72.7 +/- 1.0 vs 24.1 +/- 0.8 cpm/mm2 in unrestrained animals). A positive but weaker signal was also detected in the amygdala, pyriform cortex and other parts of the cerebral cortex and habenulae. These results suggest that the hippocampal formation is activated during stress.

Induction of the c-fos proto-oncogene in the rat pineal gland during stress

To investigate the effects of stressful stimuli on pineal gland activity, male Wistar albino rats (200-250 g, 2-4 per group) were submitted to 30 min of forced immobilization or to unilateral vibrissotomy 30 min before sacrifice. In situ hybridization was performed with a 35S-labelled 50-base oligonucleotide probe complementary to nucleotides 270-319 of rat c-fos on sections containing the pineal gland. Autoradiograms were quantified using a JAVA microdensitometer. Stressful stimuli induced a significant increase in the expression of c-fos mRNA in the pineal gland (restraint = 144.3 +/- 14.4 cpm/mm2; hemivibrissotomy = 206.7 +/- 29.5 cpm/mm2) as compared to no restraint animals (30.6 +/- 5.1 cpm/mm2), animals displaying tonic-clonic seizures after an ip (64 mg/kg) injection of pentylenetetrazole (34.0 +/- 4.7 cpm/mm2), or competition (70.6 +/- 11.4 cpm/mm2) and RNAase-treated (52.7 +/- 9.1 cpm/mm2) controls. These results raise the possibility that stressful stimuli may interfere with pineal gland function