Activation of the 10-Hz sympathetic generator during the second phase of severe hypoxia-hypercapnia and Cushing reaction

Under urethane anesthesia, as in freely moving cats, the sympathetic nerve discharge (SND) contains a 10-Hz component, either as a single peak in the autospectra or in addition to the cardiac-related or 2-6-Hz rhythm. In this study, we examined the changes in these rhythmic SND components during the reaction to asphyxia and to sudden elevation in the intracranial pressure (Cushing reaction). In all cats included in this study, resting SND was dominated by 2-6-Hz rhythm, but a peak at 10 Hz was also present in the coherence functions. During asphyxia or Cushing reaction, the 2-6-Hz component exhibited the usual two-phase pattern of activation followed by suppression. In phase 2, however, the SND did not desynchronize, as in chloralose-urethane anesthetized cats [Am. J. Physiol. 256 (1989) R120; J. Physiol. (London) 469 (1993) 37], and the massive SND activation and the resulting pressor reaction was due to strengthening of the 10-Hz rhythm. After the ischemic reaction, the 10-Hz component diminished and 2-6-Hz rhythm recovered. These findings suggest that two-phase response to hypoxia-hypercapnia is not due to hypoxic neuronal damage but represents a physiological sympathetic reaction involving different patterns of SND.     

Role of medullary excitatory amino acid receptors in mediating the 10-Hz rhythm in sympathetic nerve discharge of cats

We tested the hypothesis that excitatory amino acid (EAA)-mediated transmission plays a role in generating the 10-Hz rhythm in sympathetic nerve discharge (SND) of baroreceptor-denervated, urethane-anesthetized cats. Microinjection of either an N-methyl-d-aspartate (NMDA) or non-NMDA receptor antagonist into any one of three medullary regions (lateral tegmental field, rostral, or caudal ventrolateral medulla) essentially eliminated the 10-Hz rhythm in inferior cardiac SND. We conclude that EAA receptors in the medulla are critical for generation of the 10-Hz rhythm.     

Coupling of arterial pulse and 10-Hz rhythm in sympathetic nerve discharge

We used time series analysis to characterize the relationships among the arterial pulse (AP) and the cardiac-related and 10-Hz rhythms in sympathetic nerve discharge (SND) of urethane-anesthetized cats. We found that 10-Hz activity was more tightly coupled to the AP than to the cardiac-related rhythm. These data support the view that the dynamic coupling of AP and the 10-Hz rhythm in SND involves a direct influence of baroreceptor activity on the 10-Hz oscillator.     

Selective immunotoxin-induced cholinergic deafferentation alters blood flow distribution in the cerebral cortex

Adult rats received intracerebroventricular (i.c.v.) administration of either phosphate buffer (PBS) or 192 IgG-saporin (Toxin), 3.6 μg rat⁻¹, a cholinergic immunotoxin. Six to eight weeks later, the animals received a continuous intravenous (i.v.) infusion of either physostigmine (4.2 μg kg⁻¹ min⁻¹) or saline, followed by measurement of cerebral cortical blood flow (CBF) with the autoradiographic Iodo-¹⁴C-antipyrine methodology in four groups of animals: Toxin i.c.v.+saline i.v. (n=9), Toxin i.c.v.+physostigmine i.v. (n=6), PBS i.c.v.+saline i.v. (n=6) and PBS i.c.v.+physostigmine i.v. (n=6). Choline acetyltransferase activity (ChAT) was assessed with Fonnum's method in samples of cortical tissue adjacent to the sites of CBF measurement. ChAT decreased in all regions of the Toxin groups when compared to PBS (% decrease: hippocampus=93%, neocortex=80–84%, entorhinal-piriform cortex=42%, amygdala=28%). CBF decreased globally in Toxin+SAL, most severely in posterior parietal and temporal regions (24–40% decrease from PBS+saline). Physostigmine enhanced CBF predominantly in these same areas both in PBS and Toxin animals although to a lesser extent in the latter. Our results demonstrate the importance of cholinergic mechanisms in the control of CBF. The similarity between the topography of CBF decrease following administration of the immunotoxin to that observed in Alzheimer's disease suggests that the CBF pattern observed in this disease may be the result of cholinergic deafferentation.     

Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats

The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5–25 μg: 5 μl) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 μg) increased MAP and HR by 32 ± 3 mmHg and 45 ± 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 μg; i.c.v.) blocked the effects of physostigmine (25 μg; i.c.v.). Also, the pre-administration of the M₂ muscarinic antagonist, methoctramine (25 μg; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M₁ muscarinic antagonist, pirenzepine (100 μg; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M₃ muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 μg; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25–30 mg/kg), physostigmine (25 μg; i.c.v.) evoked a decrease in MAP and HR of 13 ± 6 mmHg and 15 ± 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats arepossibly mediated through stimulation of central M₂ muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.     

Cardiovascular effects of central administration of clonidine in conscious cats

The effects on arterial blood pressure and heart rate after an intracerebroventricular (i.c.v.) administration of clonidine were investigated using conscious normotensive cats. Injection of clonidine (5–10 μg; 5 μl; i.c.v.) elicited a decrease in mean arterial pressure (MAP) and heart rate (HR) in a dose-dependent manner. The highest dose of 10 μg of clonidine decreased MAP and HR by 39 ± 3 mmHg and 74 ± 5 b.p.m., respectively (n = 7). Pretreatment with yohimbine, the α₂-adrenoceptor antagonist (8 μg; 5 μl; i.c.v.) blocked the cardiovascular responses to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). Furthermore, preadministration of cimetidine (100 μg; 5 μl; i.c.v.), the H₂ histamine receptor antagonist with imidazoline receptor activating properties, prevented the decreases in MAP and HR to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). By contrast, pretreatment with the specific I₁ imidazoline receptor blocker, efaroxan (100–500 μg; 5 μl; i.c.v.), failed to inhibit the cardiovascular effects of an i.c.v. administration of 10 μg clonidine (n = 7). These results suggest that the effects of centrally administered clonidine on MAP and HR are probably not mediated through activation of the I₁ subtype of imidazoline receptors in conscious cats. However, the cardiovascular effects elicited by i.c.v. administration of clonidine appear to result from stimulation of central α₂-adrenergic or the H₂ histaminergic-like receptors.     

Neurons of the rostral para-ambiguual field have activity correlated to the 10-Hz rhythm in sympathetic nerve discharge of urethane-anesthetized cat

Using the techniques of time domain correlation (mid-signal spike-triggered averaging) and frequency domain correlation (neuron-to-nerve coherence), 24% (54) of a sample of 229 neurons of the rostral para-ambiguual field have shown to have activity correlated to the 8- to 13-Hz rhythm in the inferior cardiac sympathetic nerve discharge (SND) of urethane-anesthetized cat. This correlation existed in both baroreceptor-innervated and -denervated animals. Of the correlated neurons, 37% (20) were non-rhythmically firing and displayed flat autospectra, while 63% (34) fired rhythmically and contained well-defined peaks in their autospectra. The group firing rate of these neurons was 4.3±0.4 spikes/s, indicating that they are not pacemaker neurons for the 10-Hz SND rhythm. The group time of firing of these neurons to the next peak of the SND slow wave was 52±4 ms. Correlation of the activity of medullary neurons with the 8–13-Hz rhythm of the SND was previously claimed only for rostral ventrolateral medulla, caudal raphe, and rostral caudal ventrolateral medulla. This present finding suggests that this behavior may be more widely spread throughout the medulla.     

Evidence for a central depressor action of postsynaptic alpha 1-adrenergic receptor antagonists

The effects of alpha-adrenergic receptor antagonists on sympathetic nervous discharge (SND) recorded from the external carotid and splanchnic nerves were studied in baroreceptor intact and denervated cats. Prazosin (50 microgram/kg, i.v.) produced a rapid fall in mean arterial pressure (MAP) and no significant change in heart rate (HR) in baroreceptor denervated cats. Prazosin administration was also associated with a prolonged inhibition of SND. Nerve activity was significantly reduced within 5 min of prazosin administration remained depressed throughout the 2 h observation period. Like prazosin, WB-4101 (0.5 mg/kg, i.v.) also produced significant reductions in MAP and SND. In addition, WB-4104 produced a transient bradycardia. The decreases in MAP and SND were reversed by piperoxane (0.5 mg/kg, i.v.). Both prazosin and WB-4101 inhibited the pressor response to i.v. norepinephrine. In baroreceptor intact cats, prazosin decreased MAP and SND, but did not affect HR. In contrast, phentolamine (1 mg/kg, i.v.) decreased MAP but increased SND and HR. These data indicate that the sympatholytic action of WB-4101 and prazosin results from a centrally mediated reduction in SND as well as a peripheral blockade of alpha-adrenergic receptors. These data further suggest that noradrenergic neurons normally facilitate the outflow of sympathetic nerve activity from the central nervous system.     

Is CRF a ganglionic transmitter or modulator in the cat sudomotor pathway?

The presence of corticotrophin releasing factor (CRF)-like immunoreactivity distinguishes a subset of cat sympathetic preganglionic neurons which supplies sweat glands. It is abundant in their terminals in the stellate ganglion. We sought first to determine whether this immunoreactivity is due to true CRF, then to test whether CRF plays any role in ganglionic transmission in the cat sudomotor pathway. CRF-immunoreactive material extracted from cat stellate ganglia and hypothalamus were eluted on HPLC at equivalent retention times, slightly less than that of standard sheep CRF. In chloralose-anaesthetised cats, sheep CRF (0.13, 1.3 and 13 μg/kg, i.v.) raised plasma immunoreactive ACTH levels by between 3- and 300-fold. Submaximal stimulus trains delivered to pre- or postganglionic nerves of the right stellate ganglion evoked electrodermal response (EDR, a measure of sweat gland activity) in the right forepaw pad as well as increases in heart rate and blood pressure. Exogenous sheep CRF (dose range 130 ng/kg to 13 mg/kg) given close-arterially to the stellate ganglion in 5 chloralose-anaesthetised cats had no consistent effect on either baseline or preganglionically-evoked EDR. Given i.v. at 13 μg/kg to four further cats, sheep CRF caused no significant change in mean baseline or mean preganglionically-evoked EDP (P > 0.05; CUSUM test). Hexamethonium (10 or 30 mg/kg i.v.) abolished the EDR to preganglionic nerve stimulation (7/7 cats). Increasing preganglionic stimulus voltage, frequency and train duration failed to show any hexamethonium-resistant component of the EDR, although such effects were evident in the cardioaccelerator pathway. We conclude that cat preganglionic sudomotor neurons contain true CRF, although the cat peptide is not identical to that of the sheep. Exogenous CRF had no measurable effect on postganglionic neurons or on ganglionic transmission in the cat sudomotor pathway. Ganglionic transmission in the sudomotor pathway, unlike that to the heart, is entirely nicotinic. The function of CRF in cat sudomotor pathway remains unknown.     

Cardiovascular effects of intravenous administration of clonidine in conscious cats

We previously reported that cardiovascular effects elicited by intracerebroventricular (i.c.v.) administration of clonidine result from stimulation of central α₂-adrenergic and/or H₂-histaminergic receptors, but not via activation of I₁-imidazoline receptors in conscious cats. In this study, we investigated the effects on arterial blood pressure (MAP) and heart rate (HR) following an intravenous (i.v.) administration of clonidine using conscious cats. Injection of clonidine (2–10 μg/kg i.v.) elicited a decrease in mean arterial pressure (MAP) and heart rate (HR) dose-dependently. The dose of 10 μg/kg of clonidine decreased MAP and HR by 30±4 mmHg and 62±15 bpm, respectively. Intravenous or i.c.v. pretreatment with yohimbine, the α₂-adrenoceptor and 5-HT_1A receptor antagonist, blocked the cardiovascular responses to a subsequent i.v. injection of 10 μg/kg clonidine. However, i.v. or i.c.v. preadministration of cimetidine, the H₂-histamine receptor antagonist, failed to antagonize the decreases in MAP and HR to a subsequent i.v. injection of 10 μg/kg clonidine. In addition, i.c.v. or i.v. pretreatment with the I₁-imidazoline receptor blocker, efaroxan, failed to inhibit the cardiovascular effects of an i.v. administration of clonidine. These results demonstrate that i.v. clonidine evokes decreases in MAP and HR possibly via central α₂-adrenoceptor and/or 5-HT_1A receptors and not through H₂-histamine or I₁-imidazoline receptors.     

The suprachiasmatic nuclei: Two circadian clocks?

Unilateral lesions of the bilaterally paired suprachiasmatic nuclei of the hypothalamus were made in golden hamsters maintained in constant light (LL). Prior to surgery, the circadian rhythm of locomotor activity had dissociated or ‘split’ into two distinct components in 19 out of 30 hamsters, while in the remaining 11 animals (i.e. non-splitters) a normal rhythm with a single bout of activity was maintained. Following destruction of a major portion (i.e. 50–100%) of a single suprachiasmatic nucleus (SCN) in 6 ‘split’ and 5 ‘nonsplit’ hamsters, a single circadian bout of activity was observed in all animals. In 9 of these 11 animals, the circadian period of the activity rhythm was altered following unilateral SCN lesions. In two split animals sustaining lesions which missed the SCN, but disrupted efferents caudal to the SCN, the split condition was also abolished. Lesions which destroyed less than 50% of a single SCN did not abolish the split condition (n= 6) and had little effecy on the circadian pattern of the non-splitters (n= 5). In contrast, bilateral destruction of the suprachiasmatic nuclei eliminated the circadian pattern of activity in both ‘split’ (n= 5) and ‘non-split’ (n= 1) animals. These results demonstrate that each SCN is capable by itself of maintaining circadian rhythmicity and that at least two circadian oscillators, or oscillating systems, reside within the suprachiasmatic nuclei.     

The peripheral nociceptive actions of intravenously administered 5-HT in the rat requires dual activation of both 5-HT2 and 5-HT3 receptor subtypes

In 16-week-old Sprague-Dawley rats lightly anesthetized with pentobarbital, 5-HT (3–96 μg/kg, i.v.;n = 6) produced distinct pseudaffective responses and a dose-dependent (slope= 17.2 ± 6.8s/log₁₀dose) inhibition of the tail-flick (TF) reflex (ED₅₀ = 32.6 ± 9.2 μg/kg). In the same rats, a 1:1 combination of α-methyl 5-HT (a 5-HT₂ receptor selective agonist) and 2-methyl 5-HT (a 5-HT₃ receptor selective agonist) (3–192 μg/kg, i.v.), produced the same profile of pseudaffective responses and also resulted in a dose-dependent (slope= 34.0± 7.0s/log₂dose) inhibition of the TF reflex (ED₅₀ = 88.4 ± 20.5 μg/kg). In contrast, administration of α-methyl 5-HT (3–192 μg/kg, i.v.) or 2-methyl 5-HT (3–192 μg/kg, i.v.) alone did not produce any pseudaffective responses or any change in TF latency from baseline. In conscious 16-week-old male Sprague-Dawley rats, administration of 5-HT (48 μg/kg, i.v.;n = 5), or a 1:1 combination of α-methyl 5-HT and 2-methyl 5-HT (total dose= 120 μg/kg, i.v.;mn = 5), resulted in a passive avoidance behavior assessed in a step-down paradigm (slopes= 139.7 ± 58.2and154.9 ± 63.9s/trial, respectively), and the same profile of distinct pseudaffective responses exhibited by the lightly pentobarbital-anesthetized rats. However, administration of either α-methyl 5-HT (96 μg/kg, i.v.;n = 4) or 2-methyl 5-HT (96 μg/kg, i.v.;n = 4), while producing significant 5-HT receptor-mediated cardiovascular responses, produced a learned behavior not different from saline (0.25 ml, i.v.;n = 6) (slopes= 7.6 ± 2.5, 6.3 ± 1.8and7.4 ± 3.6s/trial, respectively). These results are consistent with the hypothesis that the peripheral nociceptive responses to i.v. 5-HT requires dual activation of 5-HT₂ and 5-HT₃ receptor subtypes.     

Inhibition of sympathetic activity by stimulating in the raphe nuclei and the role of 5-hydroxytryptamine in this effect

The possibility that the putative transmitter 5-hydroxytryptamine (5-HT) is involved in the mediation of long latency to onset raphe-spinal inhibition of sympathetic preganglionic neurones was investigated in anaesthetized cats by stimulating sites located in nucleus raphe pallidus and obscurus and recording sympathetic discharge in T₃ or T₁₀ white rami evoked either reflexively or by intraspinal stimulation at cervical level. Several putative 5-HT anttagonists were administered intravenously (i.V.) or topically to the spinal cord. In 7 cats lysergic acid diethylamide (LSD) in a dose range 25–50 μg/kg i.v. or 0.6 μg topically, reversibly reduced the raphe spinal inhibition by 40–100%. Topical application was more effective than i.v. administration. In 5 cats stimulating within the ventromedial reticular formation at sites unlikely to involved 5-HT neurons produced a short latency to onset inhibition which was unaffected by LSD. Methysergide, cinanserin and cyproheptadine depressed sympathetic discharge in the absence of brain stimulation in cats with CNS intact and in unanaesthetized decerebrate spinal cats. The results are discussed in the light of the known actions of the putative 5-HT antagonists.     

Cortical spreading depression-associated hyperemia in rats: involvement of serotonin

We investigated whether the vasoactive neurotransmitter serotonin (5-HT) is involved in cortical spreading depression (CSD)-associated hyperemia in the rat. We focused on the 5-HT₂ receptor, which is engaged in 5-HT induced small arteriolar relaxation in cats, as well as on the 5-HT_1D/1B receptor, the binding site of the potent antimigraine drug sumatriptan. In male barbiturate anaesthetized Wistar rats (n=25) CSDs were elicited by brain topical application of 1 M KCl, and the DC-potential and regional cerebral blood flow (rCBF, by Laser Doppler flowmetry) were measured over the same hemisphere through dura and thinned bone, respectively. Intravenous application of 8 mg/kg of the 5-HT_2A/2C receptor antagonist ritanserin (group I; n=8) significantly reduced the hyperperfusion amplitude during CSD by 44% (p<0.05, from 342±124 to 194±97%, baseline before CSD=100%), and prolonged its duration by approx. 30%. Vehicle alone (group II; n=4) did not affect CSD hyperperfusion. The highly selective 5-HT_1D/1B receptor agonist 311C90 was given in two doses: 100 μg/kg i.v. (n=5) had no effect on CSD hyperperfusion, while 800 μg/kg (n=5) increased hyperperfusion significantly (p<0.05, from 224±86 to 310±148%). We conclude that serotonin is, probably via 5-HT₂ receptors, involved in the modulation of the regional cerebral blood flow increase during CSD. Novel highly selective receptor antagonists may help to discriminate the differential contribution of various 5-HT receptor subspecies.     

Vagal afferent-mediated inhibition of a nociceptive reflex by intravenous serotonin i the rat. I. Characterization

The effect of intravenous (i.v.) serotonin (5-HT) on nociception and blood pressure in male Sprague-Dawley rats. Intravenous 5-HT produced a dose-dependent (6–192 μg/kg, i.v.) inhibition of the nociceptive tail-flick (TF) reflex in lightly pentobarbital-anesthetized (ED50 = 40 μg/kg) and conscious rats (ED50 = 44 μg/kg). In the lightly pentobarbital-anesthetized rat, the blood pressure response to i.v. 5-HT was typically a triphasic response with a marked Bezold-Jarisch reflex-induced decrease in pressure (associated with a brief period of apnea) followed by a pressor phase and a subsequent delayed hypotension. In the conscious rat, the response was typically biphasic with the late hypotensive phase absent. A variety of anatomical and pharmacological manipulations were to characterize the 5-HT-induced inhibition of the TF reflex and associated changes in blood pressure. Prevention of 5-HT-induced reflex apnea by artificial ventilation did not affect inhibition of the TF reflex produced by 5-HT. Pharmacological manipulations were performed to mimic, as closely as possible, the acute increases and decreases in blood pressure associated with i.v. 5-HT. Nitroprusside (8 μg/kg, i.v.) produced a decrease in blood pressure of similar magnitude and rate as that associated with the Bezold-Jarish reflec-induced decrease in pressure produced by 72 μg/kg 5-HT, but did not change TF latency from baseline. Similarly, acute increases in pressure produced by phenylphrine (8 μg/kg, i.v.), intended to mimic the secondary pressor effect of 5-HT, did not change TF latency. The short-acting ganglion blocker trimethaphan (5 mg/kg, i.v.) closely mimicked the late hypotensive phase produced by 5-HT but again resulted in no change in TF latency. Pretreatment with the ganglion blocker chlorisondamine (2.5 mg/kg) abolished all depressor responses to 72 μg/kg 5-HT, but did not significantly affect the TF reflex. These results indicate that acute changes in blood pressure and respiration associated with i.v. 5-HT do not contribute to inhibition of the TF reflex. This conclusion was confirmed in experiments in which bilateral vagotomy abolished approximately 70% of the 5-HT-induced inhibition of the TF reflex (and all depressor responses), and resulted in a significantly greater pressor response. Finally, low thoracic spinal cord transection (T_9–10) abolished the inhibition of the TF reflex produced by i.v. 5-HT. Therefore, 5-HT stimulates vagal afferents and inhibits the TF reflex by activating descending inhibitory system from the brainstem. Taken together with the behavioral responses observed, these results support the notion that 5-HT, when administered i.v., is a noxious stimulus.     

Pharmacological analysis of a cholinergic receptor mediated regulation of brain norepinephrine neurons

Summary The significance of the cholinergic receptors within the noradrenergic nucleus locus coeruleus (LC) was analyzed by using single unit recording techniques and microiontophoretic drug application of various cholinergic and anticholinergic drugs. Physostigmine (25–50g/kg), intravenously administered, caused increased firing of LC neurons and this effect was blocked by scopolamine but not by methylscopolamine. Also nicotine (10–50g/kg i.v.) caused activation of LC neurons, an effect which was antagonized by the presumed nicotinic antagonist mecamylamine (8 mg/kg) as well as scopolamine (0.5 mg/kg i.v.). The cholinergic receptor within the LC showed specific muscarinic characteristics since microiontophoretic application of various muscarinic agonists caused excitation of LC units, whereas microiontophoretically applied nicotine had no effect. In addition, the acetylcholine (ACh)-induced excitation of the LC neurons was not blocked by nicotinic antagonists but was totally antagonized by the muscarinic agonist scopolamine. Scopolamine, when microiontophoretically applied onto the LC neurons, antagonized the stimulatory effect of systemically injected physostigmine but not that of nicotine. These results suggest that the stimulation of noradrenaline (NA) neurons in the LC by cholinergic drugs such as physostigmine is mediated via cholinergic, muscarinic receptors within this nucleus. The LC activation by nicotine is, however, an indirect effect, probably involving central ACh release and mediated via a non-cholinergic LC input.Parts of these results were reported at a symposium on The Effects of Nicotine on Central Nervous System in Stockholm, Sweden, November 28–29, 1978, and at the Third European Neuroscience Meeting, Rome, Italy, September 11–14, 1979.     

Dose-response analysis of the effect of 21-aminosteroid tirilazad mesylate (U-74006F) upon neurological outcome and ischemic brain damage in permanent focal cerebral ischemia

The protective effects of the 21-aminosteroid tirilazad mesylate (U-74006F), one of the most efficacious inhibitors of free radical-initiated lipid peroxidation, against ischmic brain damage particularly in permanent focal cerebral ischemia still remain controversial. The present study was designed to determine the degree of neuroprotection produced by various doses of U-74006F in permanent middle cerebral artery occlusion. Focal cerebral ischemia was achieved mby permanent occlusion of the left middle cerebral artery in male Sprague-Dawley rats. Four groups of rats were studied: viz. vehicle-administered controls (n = 7), and U-74006F-treated animals at doses of 0.3 mg/kg (n = 7), 1.0 mg/kg (n = 7) and 3.0 mg/kg (n = 7) (i.v. 15 min, 2 h and 6 h post occlusion, and 3.3 times higher than the first 3 doses, i.p. 12 h post occlusion). Twenty-four hours after surgery, the animals were subjected to neurological examination using a grading scale of 0 to 3, and sacrificed to assess ischemic damage by means of tetrazolium chloride staining. A dose-related attenuation of neurological deficits and ischemic damage was observed. At the two highest doses, the volume of ischemic damage in the cerebral hemisphere was reduced by 25.3% (P < 0.05) and 32.9% (P < 0.005), compared to the controls. The neurological deficit score for animals treated with U-74006F (3.0 mg/kg) was also significantly lower than for the controls (1.7 ± 1.1 vs. 2.7 ± 0.8; P < 0.05). This study shows that U-74006F ameliorates postischemic neurologic deficits and provides dose-dependent neuroprotection against ischemic brain damage in focal cerebral infarction.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

Behavioral state-related changes of extracellular serotonin concentration in the dorsal raphe nucleus: a microdialysis study in the freely moving cat

For direct measurement of the extracellular concentration of serotonin (5-HT) in the dorsal raphe nucleus (DRN) over the sleep-wake cycle we used the technique of in vivo microdialysis in six freely moving, naturally sleeping cats whose behavioral state was polygraphically determined. Perfusate samples from microdialysis probes histologically localized to the DRN showed the following significantly different levels of extracellular 5-HT, with the numbers in parentheses indicating successively the mean value in fmol/5 μl perfusate sample, the % level relative to waking, and the sample n:waking (4.02, 100%, n = 38) > slow wave sleep (2.02, 50%, n = 30) > REM sleep (1.61, 38%, n = 17). These data, to our knowledge the first direct DRN 5-HT measurements during behavioral state changes, directly parallel the levels of serotonergic neuronal action potential activity and suggest that DRN extracellular 5-HT is determined by this action potential activity through synaptic release by recurrent axonal collaterals in the DRN.     

Neuroprotective effects of the glutamate release inhibitor 619C89 in temporary middle cerebral artery occlusion

619C89 is a use-dependent Na⁺ channel antagonist that decreases the release of glutamate during ischemia. The efficacy of this drug in reducing infarction volume 72 h after occlusion of the middle cerebral artery (MCA) for 2 h in rats (n = 93) was determined by analysis of TTC-stained coronal section of the brain. Doses of 10, 20, 30 and 50 mg/kg of study drug given i.v. prior to MCA occlusion significantly (P < 0.05−0.01) reduced infarction volume in cortex compared to vehicle controls. Only the 50 mg/kg dose reduced infarction volume in the striatum (P < 0.05). Administration of 50 mg/kg of 619C89 30 and 60 min after the onset of ischemia reduced cortical infarction volume (P < 0.05), but there was no effect when the drug was given 5 min after reperfusion. No post-treatment regimen reduced striatal infarction volume. These results confirm the neuroprotective effects of 619C89 in temporary focal ischemia.