Neuronal activity-related coupling in cortical arterioles: involvement of astrocyte-derived factors.

Neuronal activity-evoked dilatation was investigated in cortical arterioles in brain slices from mature rats maintained in vitro at 31-33 degrees C. In the presence of the thromboxane A2 agonist U46619 (75 nM) to preconstrict vessels, internal diameter decreased by 14.2% and rhythmic contractile activity (vasomotion) developed. Addition of the epoxygenase inhibitor miconazole (20 microm) produced a further decrease in diameter and increase in the frequency of vasomotion, suggesting that tonic release of epoxygenase products maintains a level of cerebrovascular dilator tone. Addition of 1 mum AMPA for 5 min evoked a 15.4 +/- 3.7% increase in diameter and the frequency of vasomotion decreased by -6.7 +/- 1.4 contractions min(-1). The response persisted in the presence of 1 mum TTX, indicating that it was independent of neuronal activity and thus likely to have been evoked by activation of AMPA receptors on astrocytes rather than neurones. The response to the brief (5 min) application of AMPA remained unchanged in the presence of miconazole (20 microm). Prolonged (30 min) application of AMPA produced a +12.1 +/- 1.5% increase in internal diameter and reduction in vasomotion (-8.4 +/- 1.7 contractions min(-1)) that were sustained throughout the stimulation period. However, when AMPA was applied in the presence of miconazole (20 microm) it evoked only a transient increase in diameter (+9.8 +/- 3.1%) and decrease in vasomotion (-6.6 +/- 1.5 contractions min(-1)) that lasted for less than 10 min despite continued application of AMPA. The results suggest that products of epoxygenase activity, probably epoxyeicosatrienoic acids (EETs) are involved in activity-related dilatation in cortical arterioles. Whilst epoxygenase activity is not required to initiate dilatation, it appears to be involved in sustaining the response. Thus EETs released from membrane stores could contribute to the initial stages, but once these have been depleted de novo synthesis of EETs is required to maintain the effect.

Inhibition of vasomotion in hippocampal cerebral arterioles during increases in neuronal activity.

The activity of small arterioles, internal diameter 9.9 +/- 0.8 microm (SEM), was investigated in the CA1 region of hippocampal slices maintained in vitro at 34 degrees C. Under resting conditions, the vessels were quiescent. However, in the presence of the thromboxane A2 agonist U46619 (75-100 nM), rhythmic contractile activity (vasomotion, 1.1-9.9 min(-1), mean 4.1 +/- 0.7 min(-1) SEM) developed in the smooth muscle cells of the vessel walls. Electrical stimulation of the Schaffer collateral fibre pathway was used to evoke increases in neuronal activity in CA1 in the vicinity of the vessels under investigation. A 3-min period of electrical stimulation of the Schaffer collateral fibre pathway produced a significant reduction in vasomotion in 8/8 vessels. During stimulation, vasomotion either ceased completely (n = 5) or the frequency decreased from 7.1, 3.3 and 3.2 min(-1) to 1.2, 0.4 and 0.6 min(-1), respectively (n = 3). In addition, the amplitude of the residual contractions was reduced by 66%, 12% and 52%. In the presence of 1 microM tetrodotoxin (TTX) (n = 4) to block the generation of action potentials, vasomotion was still present. However, the inhibition of vasomotion evoked by increased neuronal activity was blocked concomitant with the abolition of the field potentials recorded in CA1 in response to the stimulation of the Schaffer collaterals. These findings suggest that a reduction in vasomotion may contribute to the local hyperaemia, which accompanies increases in synaptic activity in the brain.

Fluorescent imaging of nitric oxide production in neuronal varicosities associated with intraparenchymal arterioles in rat hippocampal slices.

The fluorescent indicator 4,5-diaminofluorescein (DAF-2) has been used to investigate the production of nitric oxide in the vicinity of intraparenchymal cerebral blood vessels. Slices of rat hippocampus 300-350 microm thick, were loaded with 5 microM DAF-2 diacetate. On exposure to light of 450-490 nm wavelength, point sources of fluorescence, 1.8+/-0.2 microm in diameter (mean+/-SEM), were observed in close apposition to the outer surface of the vascular smooth muscle wall of 10/15 arterioles. In fixed slices, resectioned and processed for nicotinamide adenine dinucleotide phosphate-dependent diaphorase, stained varicose fibres were also seen in close association with the smooth muscle wall of small arterioles. These findings suggest that tonic activity in perivascular nitrergic nerve fibres lying in close proximity to intraparenchymal microvessels may be a source of dilator tone within the parenchyma.

Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein.

4,5-Diaminofluorescein (DAF-2) was used to identify individual nitric oxide (NO)-producing neurones in brain slices in vitro. Coronal slices of midbrain or hippocampus, 300 microm thick from young adult rats, were incubated for 30 min in 1 microM DAF-2 diacetate (DAF-2 DA) and maintained in ACSF at 33 degrees C. Illumination at 450-490 nm revealed punctate fluorescence in neurones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral periaqueductal grey matter, deep collicular layers and cortical areas. Neurones in the hippocampal pyramidal cell layer, molecular layer of the dentate gyrus and the hilus fluoresced also. The fluorescence was abolished by pre-incubation of slices with L-NAME (100 microM-1 mM), the inhibitor of constitutive nitric oxide synthase (NOS), but not by D-NAME (100 microM) or L-NIL (5-50 microM), an inhibitor of inducible NOS. In some superficially located arterioles, there were small regions of bright fluorescence close to the outer smooth muscle wall and diffuse fluorescence within the adjacent smooth muscle cells. A diffuse fluorescence was also seen in some superficially located capillaries. Basal production of NO was not seen within deeper blood vessels. DAF-2 DA offers a sensitive indicator for visualising basal production of NO with high spatial resolution and could provide a means of identifying NOS-containing neurones in brain slices in vitro prior to neurophysiological study.

Neurovascular relationships in hippocampal slices: physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels.

Experiments were carried out to investigate the functional and anatomical relationships between neuronal elements and cerebral microvessels in 300-350-microm thick coronal hippocampal slices maintained at 33-35 degrees C, obtained from 150-200 g male Wistar rats. Cerebral arterioles (9-22 microm in diameter) were visualized in situ and pre-constricted by 22.0+/-6.6% by the addition of the thromboxane A2 agonist U46619 (75 nM), to the bathing medium. The glutamate agonist N-methyl-D-aspartate (0.01-1 mM) produced a dose-related increase in luminal diameter of pre-constricted vessels. In the presence of 4 microM haemoglobin to scavenge nitric oxide from the extravascular environment of the slice, the increase in diameter evoked by 0.1 mM N-methyl-D-aspartate was significantly reduced from 17.5+/-4.6% to 4.8+/-1.7% indicating that N-methyl-D-aspartate-induced vasodilatation of cerebral microvessels is mediated via a mechanism which involves neuronally-derived nitric oxide. In a parallel anatomical study, beta-nicotinamide adenine dinucleotide phosphate-dependent diaphorase staining was used to reveal the enzyme nitric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (< 11 cells per slice) of darkly-stained multipolar neurons, 21-32 microm in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Morphometric analysis of the relationship between cerebral microvessels, beta-nicotinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase-reactive neuronal elements and individual pyramidal layer neurons, identified by filling with biocytin, revealed that for a given point on a pyramidal layer neuron, the proximity of the nearest diaphorase-reactive nerve fibre was less than 10 microm, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 microm. Such a distance would probably preclude diffusion of vasoactive metabolites in effective concentrations from the area of increased neuronal activity. We therefore propose that the diaphorase-reactive nerve network constitutes the functional link. It is possible that during periods of increased neuronal activity, spillover of glutamate from synapses may activate the diaphorase-reactive network. Release of nitric oxide from the network in the vicinity of local cerebral arterioles may then produce relaxation of the vascular smooth muscle, enabling increased blood flow into the capillary network supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activity elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arterioles. Stereological analysis of the relationship between neuronal and vascular elements within hippocampal slices suggested that a small subpopulation of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in close apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism.

Inhibitory effect of nitric oxide on neuronal activity in the periaqueductal grey matter of the rat.

Experiments were carried out in urethane-an-anesthetized rats to examine the effect of nitric oxide (NO) on neuronal activity within the dorsolateral sector of the midbrain periaqueductal grey matter (PAG), an area which is rich in NO-synthesizing neurones. NADPH-dependent diaphorase histochemistry revealed small NO synthase-containing perikarya, 15.4 +/- 3.1 microns (mean +/- SEM) in diameter, in a longitudinal column in the dorsolateral sector of the PAG. The labelled cell bodies were surrounded by a dense meshwork of stained fibres and processes in which unlabelled neurones were embedded. In order to establish whether NO was generated when NO donors were ejected iontophoretically from micropipettes, a chemiluminescence method was used to estimate the output of NO in vitro after iontophoresis of two chemically different classes of NO donor: the sydnonimine 3-morpholino-sydnonimin-hydrochloride (SIN 1) and the nitrosothiol S-nitroso glutathione (SNOG). Iontophoresis of both NO donors into 200 microliters aliquots of 165 mM NaCl using ejection currents between 6000 and 18,000 nA.min produced a current-related increase in the concentration of NO. Iontophoresis of SIN 1 in vivo produced a reproducible, current-related inhibition of firing in 40 of 59 neurones in the dorsolateral PAG. In 8 of 10 neurones the effect of SIN 1 was significantly reduced after iontophoresis of methylene blue (10-30 nA for 2.7-5 min). The inhibition took up to 7 min to develop and lasted for up to 13 min. Inhibitory responses to GABA were not affected by methylene blue. Iontophoresis of SNOG also inhibited ongoing activity of 18 of 24 neurones tested in the PAG. The experiments demonstrate firstly that NO donors can be used in vivo to deliver NO in the vicinity of neurones by iontophoresis from micropipettes. Secondly, NO appears to inhibit neuronal activity within the PAG.

Inhibition of prostacyclin release from cultured endothelial cells by nitrovasodilator drugs.

Pretreatment (18 h) of the bovine aortic endothelial cell line AG4762 to 500 microM sodium nitroprusside (SNP), glyceryl trinitrate (GTN) or 3-morpholino-sydnonimine (SIN-1) significantly inhibited 100 nM bradykinin-stimulated prostacyclin (PGI2) release. SIN-1 produced the greatest reduction (67 +/- 6%), followed by SNP (47 +/- 12%) and GTN (45 +/- 9%). Only SIN-1 and GTN inhibited basal PGI2 release where again the effect of SIN-1 (66 +/- 6%) was greater than that of GTN (31 +/- 15%). There was no effect of SNP on basal PGI2 release. We have demonstrated this inhibition of bradykinin-stimulated PGI2 release is not the result of cell death. In addition, 8-bromo-cyclic GMP, whilst having no effect on basal PGI2 release, demonstrated a small but significant inhibition (15 +/- 6%) of the enhanced response to 100 nM bradykinin. These studies may reflect a mechanism by which the release of vasodilators from endothelial cells is altered during therapy with nitrovasodilators and thus may contribute to the development of tolerance to these drugs.

Distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase staining in intraparenchymal blood vessels of the rat brain.

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase (nitric oxide synthase, NOS) in the endothelial lining of intraparenchymal blood vessels was examined in sections of rat brain prepared from 500 microns thick slices of brain fixed by immersion or from blocks of tissue taken from whole brains fixed by vascular perfusion. In immersion-fixed tissue, a network of stained vessels, many as small as 3 microns in diameter was seen throughout the grey and white matter. In tissue fixed by perfusion small calibre vessels less than 5 microns were less prevalent. The results indicate that NOS is normally present in the endothelial lining throughout the cerebrovascular tree, including the capillaries. Endothelium-derived nitric oxide could have a more widespread role in the regulation of cerebral blood flow than considered previously.

The influence of the ventrolateral medulla on thermoregulatory circulations in the rat.

The rostral ventrolateral medulla (RVLM) was stimulated electrically and chemically (0.1-0.2 microliter, 0.4 M DL-homocysteic acid) in urethane-anaesthetised rats. Changes in the vasomotor tone of the cutaneous circulation of the tail, front and hind feet, the nose and the fur covered areas of the back, proximal hind leg and neck were assessed indirectly by measurement of skin temperatures. Electrical stimulation of the RVLM at 25 Hz (2-5 min) produced sustained decreases in skin temperatures and differed from the effect induced by stimulation at 100 Hz or chemical stimulation, in that the concomitant increases in blood pressure and respiration, as well as the accompanying bradycardia or tachycardia, were minimal and within 10-15% of basal control levels. In the hyperthermic animal changes in the skin temperatures of the tail and feet were observed on stimulation of sites lateral to the rostral third of the inferior olive (IO) and which extended through the nuc. paragigantocellularis lateralis to an area medial to the caudal half of the facial nucleus (FN). There was some degree of topographical organisation with the front and hind feet areas overlying the more extensive tail area in a rostro-caudal orientation. The representation of the other areas of the body was more limited. Neck and nose temperatures changed on stimulation of a small area of the RVLM adjacent to the caudal pole of FN, while the skin of the back and upper hind limb responded to stimulation of a limited area lateral to the rostral pole of IO. The unequal representation of the cutaneous surface probably reflects the degree of vascularisation and the importance of the tail and plantar foot surfaces in thermoregulatory responses.

Changes in the tail surface temperature of the rat following injection of 5-hydroxytryptamine into the ventrolateral medulla.

5-Hydroxytryptamine (5-HT) was injected into the rostral ventrolateral medulla (RVLM) in urethane-anaesthetized rats and its effect assessed on thermoregulatory and non-thermoregulatory cutaneous circulations by the measurement of skin surface temperatures. 5-Hydroxytryptamine (5-50 nmol) produced a dose-related fall in blood pressure (5-20 mmHg) and an increase in tail and plantar foot surface temperatures, indicative of dilatation in the underlying cutaneous circulations. If heat was not applied to the animal, the body temperature fell by 1-2 degrees C within 15-25 min. The decrease in tail and foot temperatures, produced by low frequency (25 Hz, 5 min) electrical stimulation, was antagonized by the injection of 5-HT at the site of stimulation. 5-Carboxyamidotryptamine (2.5-20 nmol) and flesinoxan (5-25 nmol) produced responses similar to 5-HT. The 5-HT2 receptor agonist, alpha-methyl 5-hydroxytryptamine (alpha-methyl 5-HT, 5.5-100 nmol) was only effective in increasing tail and plantar foot temperatures, at dose levels above 25 nmol. However, in a few sites restricted to the anterior region of the RVLM, alpha-methyl 5-HT (11 nmol) evoked a small decrease in tail and foot temperatures, indicative of a constrictor effect, without influencing resting cardiovascular parameters. The results are discussed in relation to the central mechanisms which underly the hypothermia and hyperthermia produced by 5-HT1A and 5-HT2 receptor agonists.

Involvement of 5-hydroxytryptamine in the blood pressure changes evoked by electrical stimulation of the median raphe nucleus.

Electrical stimulation of the median raphe nucleus (MRN) in urethane-anaesthetised rats decreased systemic blood pressure at low intensities of stimulation (5-25 microA) and increased it with higher intensities (20-150 microA). Prazosin (0-5-5.0 micrograms/Kg i.v.) dose-dependently attenuated pressor responses concurrently with a reduction in the responsiveness of peripheral alpha-adrenoceptors to phenylephrine (500ng i.v.). Methiothepin (5-10 micrograms/Kg i.v.) abolished depressor responses and reduced the pressor effects without altering the response to phenylephrine. Ketanserin (5-10 micrograms/Kg i.v.) abolished depressor changes and potentiated pressor responses. High doses (20-200 micrograms/Kg) produced a decrease in pressor responses but correspondingly lowered BP and reduced the response to phenylephrine. The results suggest the presence of 5-HT-containing links between the MRN and the peripheral cardiovascular effector systems.

Antagonism of the effect of delta sleep-inducing peptide by naloxone in the rat.

The somnogenic properties of delta sleep-inducing peptide (DSIP) were investigated using electroencephalographic criteria. The peptide caused a significant increase in both REM and non-REM sleep at the expense of waking when injected into the lateral ventricle of the rat brain in four doses of 5 micrograms during the dark (waking) phase of the light/dark cycle. Furthermore this sleep promoting effect was blocked by pre-treatment with the opiate antagonist naloxone at a dose level (0.1 mg/Kg s.c.) considered selective for mu-receptors.

Vasomotor responses recorded using the in situ perfusion of a portion of the rat cerebral circulation.

A simple method whereby an area of the rat cerebral circulation can be perfused in situ in the dead animal has been developed. A modified Krebs/Henseleit perfusate was passed at a constant flow rate through one internal carotid artery and recovered from the transverse sinus following ligature of the arterial system of the opposite side and all venous returns to the heart. Rubber latex injections indicated that the main perfusion pathway was unilateral via the middle, anterior, and posterior cerebral arteries. The basilar and cerebellar arteries were not involved. Vasoconstrictor substances, such as 5-hydroxytryptamine (5HT), noradrenaline, and related catecholamines, gave reproducible, dose-related responses, when tested over experimental periods of 4-5 hrs. Although the preparation had no intrinsic vascular tone, vasodilator substances, such as acetylcholine, isoprenaline, histamine, or the purines, adenosine, ATP, ADP, and AMP, could be tested against various levels of tone induced by the addition of 5HT to the perfusate. On the basis of these preliminary results, the preparation appears suitable for qualitative and quantitative investigations of the responses of the rat cerebral circulation to a wide range of vasoactive substances.

Comparison of the electrocortical changes induced by (+)-amphetamine and chlorpromazine when perfused directly into the dorsal raphé nucleus of the cat.

1 (+)-Amphetamine mimicked the intermittent and sustained electrocortical desynchronization produced by (-)-noradrenaline (NA) when perfused directly into the dorsal raphé nucleus of cat encéphale isolé preparations. 2 The effects of amphetamine or NA were abolished or significantly attenuated by prior application of (-)-propranolol. 3 The effect of amphetamine, but not that of NA, was blocked by prior applications of guanethidine or chlorpromazine (CPZ). 4 Desmethylimipramine (DMI) produced dose-related changes in electrocortical activity which were similar to those induced by NA when applied to the same sites within the dorsal raphé nucleus. 5 DMI potentiated the effects of both amphetamine and NA, but guanethidine only abolished the DMI-induced potentiation of the amphetamine response. 6 (-)-Propranolol, guanethidine and CPA produced a short period of electrocortical desynchronization at the beginning of the perfusion period before antagonism of the amphetamine response was apparent. 7 The results suggest that CPZ and amphetamine have an action within the dorsal raphé nucleus possibly related to noradrenergic terminals.

A presynaptic site of action within the mesencephalic reticular formation for (+)-amphetamine-induced electrocortical desynchronization.

1. Changes induced in the electrocorticogram by the bilateral perfusion of (+)-amphetamine into the mesencephalic reticular formation (MRF) have been studied in cat encéphale isolé preparations. 2. (+)-Amphetamine, applied for 5 min in the MRF, mimicked the electrocortical desynchronization induced by the perfusion of (-)-noradrenaline (NA) or (-)-alpha-methylnoradrenaline (AMNA) into the same sites. 3. Perfusion of 6-hydroxydopamine (6-OHDA) also induced desynchronization but, over the 1 h perfusion period, slow wave activity gradually returned to the electrical record. 4. Following the application of 6-OHDA the effect of (+)-amphetamine was abolished or significantly attenuated, whereas the effect of NA or AMNA was not affected. 5. The electrocortical desynchronization induced by (+)-amphetamine could be restored if its application was preceded by perfusion with NA or AMNA. 6. Fluorescence studies using AMNA indicated that 6-OHDA depleted noradrenergic nerve terminals near the cannulae tips. However, the terminals were still capable of taking up exogenously applied AMNA. 7. These results suggest that (+)-amphetamine has a presynaptic action on noradrenergic nerve terminals within the MRF.

Electrocortical changes induced by the perfusion of noradrenaline, acetylcholine and their antagonists directly into the dorsal raphé nucleus of the cat.

1 The electrocortical changes induced by the perfusion of drugs directly into the dorsal raphé nucleus of the cat encéphale isolé preparation have been studied. 2 (-)-Noradrenaline (NA), (-)-adrenaline, or (-)-isoprenaline (Isop) produced intermittent or sustained electrocortical desynchronization during the perfusion period. 3 These changes were markedly attenuated or completely abolished by the prior perfusion of (+/-)-sotalol or (-)-propranolol, but not by equimolecular concentrations of (+)-propranolol. 4 The effects of NA or Isop were also blocked by phentolamine, whereas phenoxybenzamine either potentiated the responses to NA and Isop or mimicked the effects of these catecholamines. 5 The effect of dopamine was similar to that induced by NA, but could not be elicited at all of the perfusion sites where NA was effective. It could be blocked by (+/-)-sotalol or (-)-propranolol and also by the prior perfusion of fusaric acid. 6 Acetylcholine (ACh) increased, or initiated, electrocortical synchronization. These effects could be antagonized by sensory stimulation, the prior perfusion of atropine, or the simultaneous perfusion of NA at the same site. 7 Lignocaine, induced prolonged electrocortical desynchronization, behavioral alerting and an increased responsiveness to sensory stimulation. 8 The results have been discussed in relation to the possible involvement of inhibitory beta-adrenoceptors and facilitatory cholinoceptors (muscarinic) in the functioning of the dorsal raphé nucleus.