Transgenic mice with neuronal overexpression of bcl-2 gene present navigation disabilities in a water task.

In the CNS, Bcl-2 is an antiapoptotic gene involved in the regulation of neuronal death. Transgenic mice overexpressing the human gene Bcl-2 (Hu-bcl-2 mice) showed delayed acquisition in two tasks requiring them to find a hidden platform starting from either a random or a constant starting location. The same mice were not deficient in another task requiring them to find a visible platform suggesting that the delay observed was not due to motor, visual or motivational deficits in the water. The delay observed in Hu-bcl-2 mice was more important in the random starting test in which the allocentric demand for navigation was stronger. The results suggested that allocentric navigation is particularly sensitive to abnormal CNS maturation following the overexpression of the bcl-2 gene. The specific deficits (motor learning, fear-related behavior and allocentric navigation) observed in Hu-bcl-2 mice suggest that the regulation of developmental neuronal death is crucial for multisensorial learning and emotional behavior.

Carbon monoxide regulates cerebral blood flow in epileptic seizures but not in hypercapnia.

Carbon monoxide (CO) is an endogenously produced gas sharing many properties with nitric oxide (NO), notably activating soluble guanylate cyclase and relaxing blood vessels. The brain can generate high quantities of CO from a constitutive enzyme, haem oxygenase (HO-2). To determine whether CO is involved in the regulatory mechanisms of cerebral blood flow (CBF), two conditions associated with a reproducible CBF increase were studied in rats: epileptic seizures induced by kainate, and hypercapnia. The HO inhibitor tin protoporphyrin (Sn-PP) did not modify the basal level of CBF, significantly reduced the increase in CBF during status epilepticus, and did not affect the cerebrovascular response to hypercapnia. It is concluded that CO participates in the regulation of CBF in specific conditions, notably those associated with glutamate release.

The selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole, reduces the delayed neuronal damage due to forebrain ischemia in rats.

BACKGROUND AND PURPOSE: The present study was designed to investigate whether neuronally derived nitric oxide (NO) plays a toxic role in the cascade of cellular events triggered by global cerebral ischemia in rats. METHODS: 7-Nitroindazole (7-NI) was used as a selective inhibitor of neuronal NO synthase. Global ischemia was induced for 20 minutes in anesthetized rats following the four-vessel occlusion model. Electroencephalogram and brain and body temperatures were continuously monitored. All rats were thermoregulated for the entire duration of anesthesia. 7-NI (25 mg/kg) or its vehicle was given intraperitoneally just after the carotid clamping and again 1 hour later. Rats were randomly divided into four groups: (1) vehicle (n = 7); (2) 7-NI (n = 7); (3) L-arginine (300 mg/kg IP) +7-NI (n = 7); and (4) 7-NI associated with warming to 37 degrees C for 7 hours after disruption of anesthesia to compensate for the decrease in temperature induced by 7-NI (n = 9). Seven days after ischemia, hippocampal CA1 damage was evaluated by classic histology. The lesion was scored with the use of a point scale, and the surviving neurons were counted. RESULTS: Lesion scores were significantly lower and neuron counts higher in the two (warmed and unwarmed) groups of rats in which 7-NI was given alone than in vehicle- and L-arginine +7-NI-treated rats. CONCLUSIONS: The results indicate that 7-NI was neuroprotective in 20-minute global ischemia in rats and that the neuroprotective effect of 7-NI was mostly due to the blockade of NO synthesis, suggesting that NO released from neurons in ischemic conditions has a deleterious influence on hippocampal pyramidal neurons.

Inhibition of neuronal (type 1) nitric oxide synthase prevents hyperaemia and hippocampal lesions resulting from kainate-induced seizures.

The possible roles for nitric oxide produced by neurons in epileptic conditions have been investigated from two different aspects: microcirculation and delayed damage. Our aim was to determine whether the selective inhibition of neuronal (type 1) nitric oxide synthase by 7-nitroindazole, during seizures induced by systemic kainate, modifies hippocampal blood flow and oxygen supply and influences the subsequent hippocampal damage. Experiments were performed in conscious Wistar rats whose electroencephalogram was recorded. 7-Nitroindazole (25 mg/kg, i.p.) or its vehicle was injected 30 min before kainate administration (10 mg/kg, i.p.) and then twice at 1-h intervals. Kainate triggered typical limbic seizures evolving into status epilepticus, identified by uninterrupted electroencephalographic spike activity. The seizures were stopped by diazepam (5 mg/kg, i.p.) after 1 h of status epilepticus. Three types of experiments were performed in vehicle- and 7-nitroindazole-treated rats. (1) Hippocampal nitric oxide synthase activity was measured under basal conditions, at 1 h after the onset of the status epilepticus and at 24 h after its termination (n = 4-6 per group). (2) Hippocampal blood flow and tissue partial pressure of oxygen were measured simultaneously by mass spectrometry for the whole duration of the experiment, while systemic variables and body temperature were monitored (n = 6 per group). (3) Hippocampal damage was revealed by Cresyl Violet staining and evaluated with a lesion score seven days after status epilepticus (n = 12 per group). Hippocampal nitric oxide synthase activity was not significantly modified during status epilepticus or the following day in vehicle-treated rats. In contrast, it was inhibited by 57% in 7-nitroindazole-treated rats, both in basal conditions and after 1 h of status epilepticus, but was not different from its basal level 24 h later. 7-Nitroindazole significantly decreased basal hippocampal blood flow and tissue partial pressure in oxygen by 30% and 35%, respectively without affecting any systemic or thermal variable. During status epilepticus, 7-nitroindazole significantly reduced the increase in hippocampal blood flow by 70% and prevented any increase in the tissue partial pressure of oxygen. Seven days later, the hippocampal damage in the CA1 and CA3 layers was significantly less in 7-nitroindazole-treated rats than in vehicle-treated rats. These results indicate that the inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects neurons from seizure-induced toxicity despite reducing blood flow and oxygen supply to the hippocampus.

Nitric oxide (NO): in vivo electrochemical monitoring in the dorsal horn of the spinal cord of the rat.

NO synthase (NOS) is largely distributed in the superficial and deep laminae of the dorsal horn as well as in dorsal root ganglion cells. It has been proposed that nitric oxide (NO) participates in the transmission of sustained, and possibly brief, nociceptive, inputs at the spinal level. The aim of this study was to check the ability of in vivo electrochemical monitoring of NO within the dorsal horn of the lumbar spinal cord (L3-L4 level) of chloral hydrate anesthetized or decerebrated spinalized rats. 30 microm diameter and 450 microm length treated carbon fiber electrodes coated with nickel(II) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrine and NafionR, and associated with differential normal pulse voltammetry, gave a peak of oxidation current around 650 mV (vs. Ag-AgCl) in vitro in NO solutions between 0.125 and 1.25 microM. In vivo, a 650 mV peak appeared which was stable (recording interval 2 min) for up to 3 h (+/-6%). Comparison between in vitro calibration and in vivo voltammograms gave an estimated in vivo extracellular concentration of 0.50 microM. In vivo, peaks decreased by 95% at 90 min and for up to 3 h after an i.p. injection of 100 mg/kg of the NOS inhibitor (NOSI) L-arginine-p-nitroanilide (L-ANA). At the same dose i.p., N(G)-nitro-L-arginine methyl ester (L-NAME) was almost ineffective after 90 min in animals paralyzed with pancuronium bromate or gallamine trethiodide. However, in non-curarized decerebrated spinalized animals, L-NAME depressed the voltammograms by 36% at 90 min. S-Ethylthiourea (80 mg/kg i.p.), also decreased the voltammograms by 45% at 140 min, and finally, 7-nitroindazole (7-NI, 90 mg/kg i.p), induced a important decrease of the 650 mV peak (23% of control) at 120 min. These results are in agreement with biochemical data showing the decrease of NOS activity within the lumbar spinal cord by L-NAME (45% of control at 90 min) and 7-NI (20% of control at 90 min). The NO donor hydroxylamine (30 mg/kg i.p.) significantly increased the peaks (140% at 90 min), and sodium nitroprusside (SNP, 20 mM) when directly superfused upon the spinal cord (200-300 microl min(-1)) induced a large increase in the peak (300% at 90 min). Moreover, SNP 60 min after L-ANA, or 90 min after L-NAME, rapidly restored the 650 mV peak up to control values. These results demonstrate the validity of electrochemical monitoring of NO within the dorsal horn of the spinal cord. The in vivo electrochemical detection of NO is in progress to study the implication of this messenger in the transmission of nociceptive messages at the spinal level.

Nitric oxide of neuronal origin is involved in cerebral blood flow increase during seizures induced by kainate.

In a previous study, we reported that the sustained increase in CBF concomitant with seizures induced by kainate is mainly due to the potent vasodilator nitric oxide (NO). However, the production site of NO acting at cerebral vessels was undetermined. In the present study, we investigated whether NO responsible for the cerebral vasodilation is of either neuronal or endothelial origin. We used a putative selective inhibitor of neuronal NO synthase, 7-nitro indazole (7-NI). CBF was measured continuously in parietal cortex by means of laser Doppler flowmetry in awake rats. Systemic variables and electroencephalograms were monitored. Kainate (10 mg/kg i.p.) was given to rats previously treated with saline (n = 8) or 7-NI (25 mg/kg i.p., n = 8) or L-arginine (300 mg/kg i.p., n = 8) followed 30 min later by 7-NI (25 mg/kg i.p.). Under basal conditions, 7-NI decreased CBF by 27% without modifying the mean arterial blood pressure. Under kainate, 7-NI prevented significant increases in CBF throughout the seizures despite sustained paroxysmal electrical activity. L-arginine, the substrate in the production of NO, prevented any decrease in CBF under 7-NI in basal conditions and partially, but nonsignificantly, reversed the cerebrovascular influence of 7-NI during seizures. In a separate group of rats (n = 6), inhibition of cortical NO synthase activity by 7-NI was assayed at 73%. The present results show that neurons are the source of NO responsible for the cerebrovascular response to seizure activity after kainate systemic injection.