ABSTRACT
Octopaminergic sensitivity of the glia that form the blood-brain barrier of the cockroach was examined using microelectrode recordings of the potential developed across this barrier layer, the perineurium. Exposure to 10−4moll−1 DL-octopamine had no significant effect upon the changes in potential induced by substitution of Tris for external sodium. Treatment with concentrations ranging from 10−4moll−1 down to 5 × 10−7 mol 1”1 enhanced the resting level of the potential after 5 min, with an accompanying depression of the magnitude of potassium-induced steps. At 10−7moll−1, there was no significant effect upon the resting level, but there was a reduction in potassium sensitivity. In a study of the effect of other neurohormonal factors, it was found that DL-synephrine could depress the response to potassium but was less effective than octopamine. In contrast, carbamylcholine, histamine and some compounds related to octopamine enhanced the potassium sensitivity. The effect of octopamine was completely blocked by phentolamine, and partially blocked by propranolol. It is concluded that the perineurium possesses an octopamine receptor that could respond to octopamine circulating in the haemolymph, and which appears to mediate a reduction in the potassium sensitivity of the basolateral membrane of these glia.
INTRODUCTION
The blood-brain barrier of insects has been suggested by several investigations to be formed chiefly by the superficial layer of neuroglia of the central nervous system, the perineurium (see Treherne, 1974; Abbott & Treherne, 1977). This hypothesis has recently been confirmed for the perineurium of the cockroach by electrophysiological evidence (Schofield, Swales & Treherne, 1984a,6; Schofield & Treherne, 1984).
Hormonal control of the perineurium or other neuroglia in insects has never been studied. Neuroglia from other animals are known to be sensitive to a range of humoral factors, from experiments upon cultured cells (see Van Calker & Hamprecht, 1980) and a few in situ (see Villegas, 1981 ; Walz & Schlue, 1982; Reale, Evans & Villegas, 1986). There is some evidence to suggest that the function of perineurial cells is controlled by octopamine. The perineurial cells of the locust are known to contain an adenyl cyclase that is stimulated by 10−5moll−1 octopamine (Benedeczky & S-Rozsa, 1981) but the specificity of this effect is unknown. Also, at the abnormally high concentration of 10−4moll−1, octopamine has been observed to stimulate the Na-dependent respiration of cockroach nerve cords: since this effect was blocked by an agent that lowers Na permeability, it was suggested that octopamine raises the Na permeability of the blood-brain barrier (Steele & Chan, 1980) although the site of action of octopamine was not identified.
In this study we have examined the sensitivity of the perineurial glia to octopamine by characterizing effects upon the trans-perineurial potential generated by these glia (Schofield & Treherne, 1984), and then examining the specificity and antagonism of the responses. A preliminary account of this work has been published (Schofield & Treherne, 1985).
METHODS
Electrophysiological recordings were made from the abdominal nerve cord of adult male cockroaches, Periplaneta americana, reared in laboratory culture. The cord was mounted in a Perspex chamber, with the connectives between the fifth and sixth ganglia under flowing saline. Recordings were made of the trans-perineurial potential in one of these connectives, relative to the bathing medium, using a microelectrode inserted into the sub-perineurial interstitial system, essentially as described earlier (Schofield et al. 1984a). Different salines were passed for periods that were measured with the aid of an electronic timer.
Saline was as used in a previous investigation (Schofield & Treherne, 1984), having a pH of 7·2 and the following composition (mmoll−1): Na, 127; K, 3; Ca, 2; Mg, 2; mannitol, 50; trehalose, 5; Cl, 135; OH, 3; Hepes, 8·6. Low-Na saline (27 mmol l−1 Na) was made by substitution of 100 mmol l−1 Tris for 100 mmol l−1 Na (with a slight reduction in Cl content). Salines were filtered (0·45 μm Millipore) shortly before use. High-K saline (66·5 mmol l−1 K) was made by substitution of 63·5 mmol l−1 K for the equivalent amount of Na. In Tris-substituted saline, some Na was left to avoid any harmful effects of Na-free conditions (e.g. upon junctional permeability). In the high-K saline, K concentration was raised only as high as necessary to produce a response of useful size, again to avoid any harmful effects.
Glass microelectrodes were pulled from 1·0 mm o.d. thin-walled glass (Clark Electromedical), filled with filtered (0·2μm Millipore) 3 moll−1 KC1 at pH7·2 (3 mmol l−1 Hepes), and had a resistance of 4–8 MΩ.
Most pharmacological agents were supplied by Sigma, with the exception of DL-adrenaline HC1, which was obtained from Pfaltz & Bauer, Inc., and phentolamine mesylate which was kindly donated by Ciba Geigy Pharmaceuticals. All solutions were used within 8 h of preparation.
Values are given as median (mean ± S.E.M.). N=no. of observations = no. of preparations. Statistical analysis of differences was made using the Mann—Whitney U-test, and probability (P) was considered significant with a value less than 0·05.
RESULTS
Effect of octopamine upon trans-perineurial potential
The hypothesis that external application of 10−4moll−1 octopamine to the cockroach nervous system lowers the Na permeability of the blood-brain barrier (Steele & Chan, 1980) was tested by examining the effect of such exposure upon the interstitially recorded trans-perineurial potential (Vs). Changes in the p.d. (dVs) were evoked by exposure to low-Na (Tris) saline for 30 s at 5-min intervals, to give a measure of relative Na permeability (Pichón, Moretón & Treherne, 1971). In control experiments, in the absence of octopamine, there was a gradual fall in Vs and a gradual increase in the amplitude of the Tris-induced dVs. A standard procedure for experiments was therefore adopted in which cockroaches were obtained fresh from the culture, and impalements were made within 10–20 min after setting up the preparation. Decline in Vs and increase in dVs were steady by 45 min after setting up the preparation. Octopamine was applied at 60 min after set-up in six preparations, when the median value of Vs was 17·0mV (16·1 ± 1·86), not significantly different from the level in controls (N=7), 15·9mV (15·2 ± 1·81). At 5min before octopamine, dVs was 0·3 mV (0·5 ±0·23), not significantly different from the effect in controls at the comparable time (55 min), 0·2mV (0·04 ± 0·20).
Upon application of 10−4moll−1 DL-octopamine, there was a positive shift in the interstitial potential over a period of about 5 min, which was sometimes accompanied by an increase in the amplitude of Tris-induced changes (Fig. 1). Only the increase in Vs was significant: Vs increased by 2·5 mV (3·6 ± 1·62) during the first 5 min of octopamine, significantly greater (P< 0·01) than in controls between 60 and 65 min, –0·9 mV (–0·8 ± 0·096); while dVs increased by 0·6 mV (1·0 ± 0·42) during 30 min octopamine, no different to the change in controls over the comparable period, 0·4mV (0·5 ±0·12).
The effect upon Vs of 30 min exposure to octopamine was reversible (Fig. 1).
Effect of octopamine upon K-induced changes in potential
To provide further information about the effects of octopamine, preparations were exposed at 5-min intervals to a 15-s pulse of 67 mmol l−1 K, 45 s after a 15-s pulse of Tris. Pulses of high K have been used earlier to characterize the barrier (see Schofield et al. 1984a,b; Schofield & Treherne, 1984). In control experiments, Vs showed a similar time course to that in the preceding section, while K-induced dVs declined slowly. This decline was steady after about 45 min. Before octopamine (N = 7), values of Vs at 60min, 12·2mV (11·1 ± 1·41), Tris-induced dVs at 55 min, 0·4 mV (0·3 ±0·10) and K-induced dVs at 56 min, 32·7 mV (32·7 ± 1·84) were notsignificantly different from values at comparable times in controls, which showed a Vs of 10·7mV (10·8± 1·71) (N = 7), a Tris-induced dVs of 0·4mV (0·4±0·072) (N = 8) and a K-induced dVs of 32·5 mV (33·6 ±2·31) (N = 8).
Treatment with 10−4 mol I−1 octopamine was found to raise the level of Vs with no effect upon Tris-induced dVs, as in the preceding experiments, while a progressive reduction in the amplitude of the K-induced dVs was observed (Fig. 2). Vs increased over 5 min by 1·9 mV (2·0 ±0·59), more (P<0·01) than the change in controls, —0·6mV (–0·5 ±0·23). Tris-induced dV3 changed after 30min in octopamine by 0·2mV (0·2±0·13), no different to that in controls, 0·1 mV (0·2±0·074). After 31 min in octopamine, K-induced dVs was reduced by 6·9mV (–7·0 ± 1·3), significantly different (P< 0·05) from the reduction of –1·5 mV (–1·7 ± 0·41) observed in controls over a comparable period.
The rise in Vs was generally less than the fall in dVs, but sometimes the rise was more than adequate to account for the fall, such that the value of Vs in high K was carried to a higher absolute level than before octopamine (e.g. Fig. 2).
Concentration dependence of octopamine effects
The effects of DL-octopamine upon the interstitial potential and K-induced steps were then examined for a range of octopamine concentrations from 10−8moll−1 to 10−4moll − 1, employing 15-s pulses of 67mmoll−1 K at 5-min intervals, and octopamine application at 60 min after set-up. A pulse length of 15 s was adopted because the effects produced were nearly as great as those produced by 30-s pulses. Values of V3 and dVs before the test exposure were not significantly different from values in controls for these experiments (Table 1) and those in subsequent sections (Tables 2–4).
The effect upon the resting level of Vs was significant down to a concentration of 5× 10−7mol I−1 : during the first 5 min of 5× 10−7moll−1 octopamine application, Vs rose by 1·0 mV, compared to a value of –0·5 mV in controls (Table 1). An effect upon the K-induced dVs was found after only 5 min exposure to octopamine at concentrations down to 10−7moll−1 (Fig. 3). At this concentration, the effect appeared complete after 10 min, and a dose-response curve was prepared for the effect upon K-induced dVs after 10min octopamine (Fig. 4). At 10−smoll−1 and 10−4moll−1, there was a progressive decline in dVs during the exposure, and hysteresis afterwards, similar to that shown in the preceding section for 10−4mol l−1 (Fig. 2).
Amine specificity of the effect upon K-induced dVs
To examine the specificity of the response to octopamine, a range of biogenic amines was tested for similar effects. Since a marked reduction in the amplitude of K-induced steps in potential was found after 10 min exposure to octopamine at concentrations as low as 10−7moll−1 the amines were tested at this concentration using the same protocol as in the preceding section, comparing with the same controls (Table 2).
Of the amines tested, none had a significant effect upon the resting level of Vs, but several affected the K-induced dVs (Table 2).
Only DL-synephrine Was like octopamine in causing a reduction of dVs, but the effect was smaller, with a reduction of 4·1 mV compared to 5·7 mV during octopamine. The other phenolamine tested, tyramine, had no significant effect upon dVs.
Among the catecholamines, an increase in dVs was produced upon β -hydroxy-lation, and N -methylation appeared to enhance the effect: dopamine had no effect, DL-noradrenaline produced a slight increase of 0·5 mV and DL-adrenaline gave an increase of 1·2 mV.
With the three other compounds related to octopamine, phenylethylamine produced an increase in dVs of 1·1 mV, DL-phenylethanolamine had no significant effect, but DL-TV-methylphenylethanolamine caused a significant increase of 0·6 mV.
Glycine, glutamate, γ -aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) had no significant effect upon dVs, whereas histamine and carbamylcholine induced a slight but significant increase in dVs.
The effect of 10−7moll−1 DL-octopamine, to cause a fall in the size of the K-induced dVs in the absence of any effect upon resting Vs, thus appears to be very specific and is mimicked, only partially, by the A’-methylated derivative, synephrine.
Antagonists of octopamine action
To provide some characterization of the receptor upon which octopamine was acting, a comparison was made between the effects of phentolamine, an antagonist of α -adrenergic receptors, and propranolol, a β-blocker, upon the response to octopamine. Preparations were exposed to the agents at a concentration of 10 6 mol l−1 for 15 min prior to, and during, exposure to 10−7moll−1 DL-octopamine. Octopamine was applied at the standard time of 60 min after set-up. Effects were compared with those in controls at comparable times (Tables 3, 4).
Upon application of phentolamine (Fig. 5) or DL-propranolol (Fig. 6), the value of dVs ceased declining, but there was no effect upon resting Vs (Table 3).
In the presence of phentolamine, octopamine had no effect upon either Vs or dV9 (Fig. 5; Table 4). This contrasts with the reduction in dVs observed in octopamine in the absence of phentolamine (Table 1).
In contrast, in the presence of 10−6moll−1 DL-propranolol, IO−7moll−1 octopamine increased Vs by 1-4 mV, significantly different from the effect in controls, and depressed K-induced dVs by 2·7 mV (Fig. 6; Table 4). Thus there was a greater effect upon Vs and less effect upon dVs than in 10−7moll−1 octopamine alone (Table 1).
DISCUSSION
The glia which form the blood-brain barrier of the cockroach showed a response to octopamine that was unlike that given to a range of other biogenic amines. At a concentration of 10−7moll−1, the response was revealed as a reduction in the potassium sensitivity of the potential that is generated across the perineurium. Only synephrine produced a similar response, but was less effective. In invertebrates, several octopamine-sensitive systems also show a response to synephrine. The relative potency observed in the present system is consistent with that obtained for homogenates of whole cockroach brain (Harmar & Horn, 1979), and is also found for the induction of hypertrehalosaemia in cockroach haemolymph (Downer, 1979). Synephrine is more potent than octopamine in the threshold for effects in the locust upon myogenic rhythm (Evans & O’Shea, 1978) and muscle twitch tension (O’Shea & Evans, 1979). Some systems show equal sensitivities, such as locust neurosecretory cells (Orchard, Gole & Downer, 1983). A further similarity of the effect upon the perineurium with effects mediated by octopamine receptors in other invertebrates is the exhibition of a more effective blockage by phentolamine than by propranolol (see Evans, 1981). Such antagonism is shown by a-adrenergic receptors, but the substantial block by propranolol may indicate the presence of receptors with β-adrenergic characteristics. It should also be noted that an effect of octopamine upon the resting level of the interstitial potential was revealed in the presence of propranolol.
Most of the known octopamine receptors show a sensitivity to catecholamines and phenylamines as well as to phenolamines (see Harmar, 1980). In the present study, the only effect observed for such compounds was an increase in potassium sensitivity, whereas a decrease was observed with octopamine. Among these compounds, DL-adrenaline appeared most potent. This suggests that there is an adrenergic receptor upon the perineurium. Adrenergic receptors are found upon cultured vertebrate glia (Van Calker & Hamprecht, 1980). Insects have not been demonstrated to possess adrenergic receptors (see Brown & Nestler, 1985) or receptors for phenylamines (see Leake & Walker, 1980), but the nervous system may contain traces of adrenaline and noradrenaline (see Pitman, 1985).
The perineurium might also possess histaminergic and cholinergic receptors that mediate an increase in K sensitivity, since the K-induced potentials were enhanced by histamine and by carbachol. Cholinergic receptors have been found in squid glia (see Villegas, 1981) while receptors for both compounds are known in cultured vertebrate glia (see Van Calker & Hamprecht, 1980). The perineurium may therefore be the site of some of those adenylate cyclases in the insect nervous system that have been demonstrated to be sensitive to histamine (see Brown & Nestler, 1985) and acetylcholine (see Sattelle, 1985). Eserine-sensitive acetylcholinesterase activity has been detected in cockroach glial cells (Smith & Treherne, 1965). Also, binding of a-bungarotoxin has been observed in superficial regions of cockroach ganglia, but further investigation is required to see if this could include binding to the perineurium (Sattelle et al. 1983).
The observed effects of octopamine upon the perineurium indicate an action upon at least one of the electrical parameters that contribute to the trans-perineurial potential (Fig. 7; see also Schofield & Treherne, 1984): namely the e.m.f.s due to the paracellular shunt (Es), basolateral membrane (Eb) and apical membrane (Eb), and the resistances due to these structures (Rs, Rb and Ra, respectively). Es is likely to be small and have little influence (Schofield & Treherne, 1984). The potassium-induced positive steps in the potential (dVs) may be interpreted as due to the depolarization of the basolateral e.m.f., together with some reduction in basolateral resistance. A reduction in dVs, as observed at octopamine concentrations as low as 10−7molP’, can be seen in terms of the model to be simulated by certain effects upon the shunt or membranes (Fig. 8). Thus such an effect could be caused by a decrease in Rs (Fig. 8A) but would then be accompanied by reductions in Vs and Rt, which have not been seen: no significant effect upon Vs was observed at this concentration, and Rt has been found to rise during such treatment (Schofield & Treherne, 1985). A reduction in dVs could also be caused by a higher resistance of the basolateral membrane (Fig. 8B), but the effect upon the resting level of Vs would be the opposite to that observed. However, if the resistance of this membrane rose because the potassium conductance fell, there would be an accompanying reduction in e.m.f. Such a combined effect could produce a decrease in dVs with a small increase in both Vs and Rt (Fig. 8C), as seen. A rise in resistance of the adglial membrane would have much less effect (Fig. 8D) than an increase in Rb, while a rise in Ra and fall in Ea together would produce a small decrease in dVs that would be accompanied by a large drop in Vs (Fig. 8E). Thus the decrease in K-induced dVs produced by low concentrations of octopamine is best explained by a reduction in the potassium sensitivity of the basolateral membrane: at external concentrations of K below the perineurial intracellular concentration, the reduction in e.m.f. would give rise to a positive shift in trans-perineurial potential that would be countered by a negative shift due to the higher membrane resistance; at high K concentrations, both the decrease in potassium sensitivity and the higher resistance would depress the positive shift in trans-perineurial potential.
An effect of octopamine upon the basolateral membrane could easily be accompanied by an effect upon some other parameter of the perineurium. At high octopamine concentrations, the increase in the resting level of the trans-perineurial potential and the suggestion of more than one mechanism might reflect a reduction in basolateral e.m.f. becoming more important relative to the effect of a rise in Rb, but these data could also reflect an increase in the value of Rs during the octopamine exposure.
Octopamine has been shown in preliminary experiments to cause a reduction in the net K permeability of the perineurium (Schofield & Treherne, 1985). Such an effect could be caused by either of the above possibilities: by a fall in K permeability of the basolateral membrane or by a rise in intercellular resistance.
Octopamine, at the high concentration of 10−4 moll−1, did not appear to increase the sodium permeability of the perineurium as has been proposed by Steele & Chan (1980). If octopamine raised Na permeability selectively, the Tris-induced changes in potential could be expected to become more negative (Schofield & Treherne, 1984). No such effect was observed.
Sensitivity of glial cells to octopamine has been observed in only one other instance, for the Schwann cells of the squid giant axon (Reale et al. 1986), where the membrane is hyperpolarized at low external potassium concentrations. In the present experiments, the basolateral membrane of the perineurium was indicated to depolarize under such conditions. A further major difference between the two systems is that tyramine is an effective agonist in the Schwann cell but not in the cockroach perineurium.
It is not known whether the glial cells of the squid are exposed to octopamine in vivo. In the cockroach, the levels of octopamine that are attained in the haemolymph following activity (6× 10−8 mol I−1) and stress (9× 10−8 mol I−1) (Davenport & Evans, 1984) are indicated by the present results to be sufficient to stimulate the perineurium. Alternatively, the perineurium might respond to octopamine release from glia, or from that contained in haemocytes (Davenport & Evans, 1984) or neurones (see Evans, 1985). Some elements of the octopamine uptake mechanisms demonstrated by Evans (1978) may be involved in a physiological response to octopamine.
The action of octopamine upon the perineurium might involve the stimulation of adenyl cyclase, since such activation has been observed at an octopamine concentration of 10−5moll−1 in the locust perineurium (Benedeczky & S-Rozsa, 1981). Such stimulation is obtained in a variety of systems (see Evans, 1985) including the squid Schwann cell (Reale et al. 1986). Elevated levels of cyclic AMP are known to raise the resistance of intercellular occlusions (Duffey, Hainau, Ho & Bentzel, 1981), which may be involved in the present responses, and can be expected to have other important effects upon the glia which form the insect blood-brain barrier.
Further investigations are being made into the nature of the effects of octopamine upon the perineurium, whether the effect is sensitive to formamidines, and the role of other humoral factors.
ACKNOWLEDGEMENTS
We thank Dr P. D. Evans for much helpful advice.