Jing-Yu Chang

Serotonin uptake into cerebrovascular nerve fibers of rat, visualization by immunohistochemistry, disappearance following sympathectomy, and release during electrical stimulation

Immunohistochemistry as well as in vitro uptake and release of [3H]5-HT were performed on pial arteries of rat to investigate the nature of 5-HT containing nerve fibers. Immunoreactive fibers were constantly found only in the basilar, vertebral and superior cerebellar arteries, while in the other parts of the circle of Willis, 5-HT immunofluorescent fibers were absent. After systemic treatment with tryptophan following inhibition of monoamine oxidase with nialamide the immunofluorescence intensity was markedly enhanced. The 5-HT immunoreactive fibers disappeared after superior cervical ganglionectomy or intraventricular administration of 6-hydroxydopamine, but persisted after administration of 5,6-dihydroxytryptamine. When isolated vessels were incubated in low concentration of 5-HT (1 nM) together with nialamide, a very dense plexus of 5-HT immunoreactive fibers appeared in all branches of the circle of Willis. Uptake and release experiments were carried out by incubation of arterial preparations with 3 nM [3H]5-HT (together with nialamide), followed by electrical field stimulation, or by exposure to tyramine or 124 mM potassium, all of which induced a 100%-350% increase in the tritium release over prestimulation values. Preincubation with cocaine and bilateral superior cervical ganglionectomy abolished or markedly attenuated the release upon all modes of stimulation. The results suggested that the 5-HT observed by immunohistochemistry in pial arteries is located in sympathetic nerve terminals where it may serve as a neuromodulator that is released during nerve activation.

Evidence for coexistence of serotonin and noredrenaline in sympathetic nerves supplying brain vessels of guinea pig

Nerve fibers containing 5-hydroxytryptamine (5-HT) were demonstrated immunohistochemically in the wall of pial vessels associated with the circle of Willis in the guinea pig. The fibers formed a network structure which was more dense in the rostral part of the arterial circle and its branches than in the caudal part. The 5-HT immunoreactive fibers disappeared in all arteries studied after bilateral superior cervical ganglionectomy, and unilateral ganglionectomy eliminated the 5-HT immunoreactivity in the ipsilateral part of the middle cerebral, posterior cerebral and superior cerebellar arteries. Decentralization of the superior cervical ganglion had no effect on the perivascular nerve plexus. Subsequent staining with dopamine-beta-hydroxylase (DBH) antiserum following elution of the first antibody revealed that 5-HT was present in the noradrenergic nerve fibers. Small intensive fluorescent cells with positive immunoreaction for 5-HT and DBH, respectively, were located in clusters within the ganglion, which showed no immunohistochemical evidence for the presence of serotonergic neurons. It is concluded that 5-HT is probably not synthesized in truly serotonergic fibers but rather taken up and stored together with noradrenaline in cerebrovascular sympathetic nerves originating in the superior cervical ganglia.

Changes in contractile response and effect of a calcium antagonist, nimodipine, in isolated intracranial arteries of baboon following experimental subarachnoid hemorrhage☆

Isolated pial arteries from a previously well-characterized model of experimental subarachnoid hemorrhage (SAH) in baboon were tested for their contractile response to 5-hydroxytryptamine (5-HT), norepinephrine (NE), and prostaglandin F2 alpha (PGF2 alpha) and the effect of the calcium antagonist, nimodipine. Autologous blood was injected cisternally at three times with one-day intervals to a total amount of 11.5-29.5 ml (mean: 18.5 ml), and the animals were killed 7 days after the first injection. Untreated animals served as controls. The degree of maximum contraction (EAm) with 5-HT and NE in the control situation was for the three arteries tested in the order middle cerebral greater than anterior cerebral greater than basilar artery. Experimental SAH markedly increased EAm, by 190-370 percent above control values (depending on type of vessel) for 5-HT and 170-185 percent for NE. In addition, the sensitivity to 5-HT was significantly increased, as evidenced by a left-shift of the concentration-response curve. Previous exposure of the artery to 10(-6) M nimodipine reduced the contractile response of both amines to approximately half, the inhibition being slightly less pronounced post-SAH. When vessels were contracted beforehand with the amines or with PGF2 alpha, followed by administration of increasing amount of nimodipine (10(-9) M to 10(-6) M), a concentration-dependent relaxation was obtained by up to 60 percent of the original level. This relaxing effect was significantly less following SAH in the experiments with NE and PGF2 alpha compared to 5-HT; the contraction in the presence of 5-HT did not differ before and after experimental SAH. The experiments show that SAH markedly enhances the intrinsic activity for both 5-HT and NE. Nimodipine inhibits the contractile response less efficiently following experimental SAH. The difference in the responsiveness to 5-HT on the one hand, and to NE and PGF2 alpha on the other, could be due to differences in the blood-induced alterations of those calcium channels that are influenced by the calcium antagonist, nimodipine.

Kinetic studies on uptake of serotonin and noradrenaline into pial arteries of rats

A population of cerebrovascular nerve fibers have recently been found to store serotonin (5-hydroxytryptamine; 5-HT). There is reason to assume that these 5-HT-containing fibers have a sympathetic rather than an intracerebral origin. This was further elucidated in the present study in which the uptake mechanisms of 5-HT and noradrenaline (NA) were characterized and compared in rat pial arteries by measuring the accumulation of [3H]5-HT and [14C]NA under various experimental conditions in vitro. Sympathectomized vessels served as blanks. The uptake into the perivascular sympathetic nerves was dependent on time as well as concentration and was saturable. The Km values were similar, 0.17 μM for 5-HT and 0.15 μM for NA, but the Vmax value was 10 times higher for NA (2.38 and 25 pmol/mg/15 min, respectively). The two amines competed with each other in the sympathetic uptake, as studied by inhibition of the accumulation of one labeled amine by the other nonlabeledamine. Corticosterone, acting on the extraneuronal process, significantly inhibited the 5-HT uptake but had no substantial effect on NA. Reserpine, blocking the intraaxonal vesicular stores, markedly attenuated the accumulation of NA, but not of 5-HT. The selective uptake blocker paroxetine reduced the 5-HT uptake with much higher potency than the NA uptake, whereas desipramine predominantly inhibited NA uptake. The pial 5-HT uptake was not significantly affected by lesion of the raphe complex, whereas it was reduced to half following superior cervical ganglionectomy. The results suggest that the 5-HT present in nerves associated with pial vessels at the base of the brain is taken up through an efficient axonal mechanism, functionally related but not identical to the uptake process for NA.

Serotonin potentiates noradrenaline-induced vasoconstriction through 5-HT1-type receptors in guinea pig basilar artery

Based on the previous finding that 5-hydroxytryptamine (5-HT) co-exists with norepinephrine (NE) in cerebrovascular sympathetic nerve fibers and can be released during electrical nerve stimulation, the postjunctional interaction between the two amines was studied in isolated basilar artery of guinea pig. A low concentration of 5-HT, which in itself has little or no constrictive effect, potentiated the weak contraction of NE by almost 300%. The amplification was antagonized by methiothepin, but not by ketanserin, and it could be mimicked by methysergide. The marked potentiation is thus probably associated with the 5-HT1-like receptors, which earlier have been found to mediate the direct vasoconstrictive action of 5-HT in this vessel preparation.

Ultrastructural cerebrovascular changes in a model of subarachnoid hemorrhage in baboon based on triple cisternal blood injection

In a subarachnoid hemorrhage model in the baboon, achieved through three cisternal blood injections with 1-day intervals, the cerebral arteries were dissected out 7 days after the first blood injection for electron microscopy All the animals showed ultrastructural changes in the cerebral arteries: two with severe, one with moderate, and three with mild alterations in the vessel walls. The most constant findings were seen in the muscle cells of the media layer. Fragmentation of the nuclei was frequently observed together with cytoplasmic vacuoles. Scattered groups or single degenerated muscle cells were also noted. In the intima the changes included rounding of the nuclei along with the appearance of cytoplasmic vacuoles. Desquamation or flattening of the endothelium and loss of tight junctions were encountered in some vessel areas. Degenerating mitochondria were a common finding.

Sympathetic Nerves Associated with Brain Vessels Store and Release Serotonin which Interacts with Noradrenaline in Cerebrovascular Contraction

Using the formaldehyde histofluorescence method, it was demonstrated some 20 years ago in our laboratory that the blood vessels of the brain are innervated by an extensive system of sympathetic nerves originating in the superior cervical ganglia (for references, see Owman, 1986). With the introduction of immunohistochemical techniques, whereby not only neuropeptides but also transmitter amines such as 5-hydroxytryptamine (5-HT) can be visualized microscopically, it has recently been shown that brain vessels of various species, including man, are supplied with fibres containing this amine (Griffith et al., 1982; Edvinsson et al., 1983; Griffith and Burnstock, 1983). Previously utilized histochemical methods (e.g. the formaldehyde technique) were obviously not sufficiently sensitive for microscopic visualization of the relatively low levels of 5-HT stored in perivascular nerve fibres. This finding has attracted considerable interest because 5-HT is a prominent vasoconstrictor substance in the brain circulation, and lias been implicated as one pathophysiological factor in serious clinical disorders such as vasospasm and migraine.

5-Hydroxytryptamine in Cerebrovascular Sympathetic Nerves and its Effect on Brain Vessels

Immunohistochemical studies have shown that the cerebral vascular bed is supplied with sympathetic fibres, in which 5-HT coexists with noradrenaline (and also neuropeptide Y), originating from the superior cervical ganglia. There is pharmacological and biochemical evidence that 5-HT is taken up locally rather than being synthetized in the nerves. It is released by electrical field stimulation of the perivascular nerve fibres, and it markedly amplifies the vasoconstrictor effect of noradrenaline. The serotonergic vasoconstriction is mediated by 5-HT1-like receptors in guinea-pigs and 5-HT2 receptors in monkey and rat, and the contraction is inhibited by the calcium antagonist, nimodipine. This is of particular interest in view of the fact that the cerebrovascular sympathetic fibres are able to modulate the autoregulation, of cerebral blood flow, whereas nimodipine impairs autoregulation resulting in pressure-dependent increment in the blood flow during hypertension. Moreover, 5-HT has been advocated as an important pathophysiological factor in cerebrovascular disorders, such as headache and vasospasm.