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Noradrenergic effects on rat visual cortex: Single-cell microiontophoretic studies of alpha-2 adrenergic receptors
Life Sciences, Vol. 41, pp. 281-289 Printed in the U.S.A.
Pergamon Journal
NORADRENERGIC EFFECTS ON RAT VISUAL CORTEX: SINGLE-CELL MICROIONTOPHORETIC STUDIES OF ALPHA-2 ADRENERGIC RECEPTORS
Arlette Kolta, Laurent Diop and Tomas A. Reader
Centre de recherche en sciences neurologiques, D@partement de physiologie, Facult~ de M~decine, Universit~ de Montreal, C.P. 6128, Succursale A, Montr@al, Quebec H3C 3J7, Canada. (Received in final form May 7, 1987) Summary The catecholamine noradrenaline has been proposed to modulate the excitability of cortical neurons, and such a regulation may be mediated by specific adrenergic receptors. We characterized, using electrophysiological recordings, the types of responses of single cells in the rat visual cortex (areas 17 and 18) to the iontophoretic application of adrenergic agents. For the majority of spontaneous and visually-driven cells sampled, noradrenaline decreased the firing frequency, and in some cases of visuallydriven cells could increase the signal/noise ratio. These effects were also documented after the application of the alpha-2 adrenergic agonists clonidine and oxymetazoline, and could be reduced or blocked by a previous ejection of the specific alpha-2 antagonist idazoxan. The present study supports a role for alpha-2 adrenoceptors in the modulation of sensory inputs to the visual cortex.
The presence of chemically-defined monoamine-containing neurons in the mammalian central nervous system (CNS) has been well established; the existence of central catecholamine (CA) neurons has been thoroughly documented in histof|uorescent and biochemical surveys, including the determination of the endogenous levels and/or the enzymatic activities. At least two types of CA afferent systems have been shown to project to the cerebral cortex: dopamine (DA) and noradrenaline (NA) containing fibers, while only traces of adrenaline (AD) have been measured (1-7). In addition, radioautographic and histofluorescence observations have demonstrated the presence in the cerebral cortex of terminals containing NA (1,8) and DA (9), as well as the indoleamine serotonin (10,11). It has also been proposed that NA plays a role in the modulation of neuronal response properties of spontaneouslyactive cortical units (12). In adult cat, iontophoretic injections of NA in the visual cortex reduced the firing of a majority of cells stimulated by flashes of light (13). These findings were confirmed by Videen and coworkers (14), who found that NA iontophoresed induced an inhibition mainly of the background ("noise") firing rate of visually-driven (VD) neurons in
Copyright
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kittens and adult cats, thus enhancing the signal to noise (S/N) ratio. If NA modulates the excitability and background firing of cortical neurons, the types of adrenergic receptors involved have yet to be demonstrated. In a different type of electrophysiologlcal paradigm, the microiontophoresis of NA inhibited the firing of Locus coeruleus and raphe neurons (15), and these effects could be mimicked by the alpha-2 adrenergic agonist clonidine. Furthermore, these effects were receptor-specific, since they could be antagonized by alpha-2 antagonists. These observations suggest the involvement of alpha-2 adrenoceptors in the modulation of the excitability and/or firing of these brain-stem neurons. Since there is much less information regarding the adrenergic receptors of cortical neurons, the aims of the present investigation were: i) to characterize the responses to the microiontophoretic application of NA by means of electrophysiological unitary recordings in the occipital (visual) cortex of the rat, and 2) to determine the possible participation of the alpha-2 subtype of adrenoceptors in the mediation of the effects of noradrenaline on synaptically activated (VD; visually-driven) cortical neurons by microiontophoresis of clonidine (CLO), oxymetazoline (OXY) and idazoxan (IDA).
Material
and Methods
Adult male Sprague-Dawley rats (250-300 g) were used in this study. For the microiontophoretic recordings, the animals were anesthetized with urethane (1.25-1.5 g/kg, i.p.) and placed in a stereotaxic frame. The bone overlying the occipital cortex was removed, the dura mater retracted and the surface of the cerebral cortex covered with 2% agar in 0.9% NaCI. Standard microiontophoretic and extracelullar recording techniques were employed (16-18). Seven-barrel micropipettes, having an overall tip diameter 5-8 microns were filled with the following drugs dissolved in 0.1% ascorbic acid and at pH 4.0: noradrenaline HCI (0.5 M); clonidine HCI (0.1 M); idazoxan HCI (0.01 M); oxymetazoline HCI (0.I M); isoproterenol HCI (0.2 M) and propranolol HCI (0.2 M). The resistance of the recording barrel was 2-6 megohms. The central barrel, filled with 2-3 M NaC1 was used for recording, and one of the sides barrels (2 M NaCI) used as a balancing channel in conjunction with the operational amplifier (19) of the microiontophoresis programmer (BH-2 Neurophore system, Medical Systems, NY). Backing currents of -10 nA were used when not ejecting drugs. Extracellular unitary activity was amplified (P~511;Grass, MA), displayed on an oscilloscope, and the spikes discriminated by a voltage amplitude discriminator. The neuronal firing rates, integrated with a linear ratemeter over lO-sec intervals, were recorded on a six-channel recorder. Only neurons with a relatlvely stable rate of discharge were studied. On-line analysis of spontaneously-active (non-visually-driven; non-VD) and of visually-driven (VD) cells were also performed with an averager, digitized with an A/D converter (Adalab; Interactive Microware, PA) so that peri-stimulus histograms (PSH) could be generated using 128 bins of 0.5 to i0 msec/bin. VD cortical units were characterized by the PSH in response to a Grass P-20 photostimulator.
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Results
Throughout this study two populations of cells were sampled in the visual cortex of the rat; i.e.: neurons that could be synaptically activated by using a visual stimulation (VD; visually-driven cells) and spontaneously active (non-VD) units. In order to differentiate VD from non-VD neurons, we used the PSH in response to the photostimulator. However, it can not be ruled out that non-VD units were not visual neurons, since the stimulus used may have not been the most adequate to drive them specifically. Furthermore, non-VD units non-responsive to visual cues could also be participating in information processing by altering the excitability of synapticallydriven VD cells. For the majority of cortical neurons (non-VD and VD) sampled throughout this study NA and alpha-2 agonists exerted an inhibitory effect, and in only a few cases excitations could be documented. These results are summarized in TABLE I. As previously described for the rat frontoparietal cortex (somatosensory area), only in very few instances were biphasic responses obtained (12).
TABLE 1
Effects of adrenergic drugs on occipital cortex neurons. Drug
VD neurons n %
non-VD neurons n %
NORADRENALINE Inhibited Excited Biphasic* Unaffected
82 67 5 3 7
82 5 2 9
52 46 3 0 3
88 6 0 6
CLONIDINE Inhibited Excited Unaffected
42 26 3 13
62 7 31
31 27 2 2
87 6 6
OXYMETAZOLINE Inhibited Excited Unaffected
i0 7 I 2
70 I0 20
6 6 0 0
100 0 0
* These cells showed an inhibition followed by an excitation.
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The microiontophoresis of NA (ejection currents of 10-30 nA for 30-60 sec) on VD neurons induced a long-lasting inhibition of firing in 67 (82%) of the 82 VD cells recorded. This effect started at 15 ± 1.7 sec (Mean ±
NA 25 •
CLO 35 am
IDA 5O •
IDA 5O HI CLO 35 m
IDA 50 NA 25 IN
20t ~ ~o v
0
- 2 rain
FIG. 1 Ratemeter record (integrated over 10-sec intervals) of the firing rate of a cortical neuron in response to the photostimulator. The drugs noradrenaline (NA), clonidine (CLO) and idazoxan (IDA) were applied for the time and the dosage (in nA) indicated by the bars. The PSH are illustrated in Fig. 2.
S.E.M.), attained a maximum effect at 61 ± 5 sec and had an average duration of 199 ± 14 sec (See Fig. i for an example). Analysis of the PSH showed thnt NA inhibitory effects would firstly affect the late phase of excitation following the stimulus ( > 400 msec), and to a lesser degree the specific (earlier components) evoked response (Fig. 2). Since the effects of NA clearly outlasted not only the ejection and the period of maximum NA effect, there was an increase in the S/N ratio of the majority of VD cells, especially evident during the recovery or immediately after recovery to control levels of discharge. In the present study and using the same ejection parameters only 5 VD cells (5%) were excited while in 7 VD cells (9%) the firing frequency did not change. The excitations appeared during or after NA ejections, reached a maximum at 60 ± 30 sec and returned progressively to control levels (average duration 133 • 62 sec). In these cases there were no significant changes in the S/N ratio. Only three cells (2%) in this survey showed biphasic responses; i.e.: NA produced a first short period of inhibition, rapidly followed by an increase in firing. Responses to microiontophoretic applications of NA in visual cortex were also evaluated on the firing rate of non-VD neurons when encountered, and the effects were similar to the responses documented for the VD units. Indeed, NA ejections inhibited the spontaneous firing rate of 46 (88%) of 52 non-VD cells tested, and excited only 3 non-VD cells (6%). The durations of the inhibitions obtained for non-VD units were of the same order of
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magnitude as those documented for VD cells (non-VD: 158 ± 17 sec; VD: 14 sec). In only 3 non-VD cells (6%), the firing did not change application of NA.
N ~ R ~ [ N R L
C O N T R ~
x NE
E~
285
199 after
nR
~,,~,L~,,II,,,,,,l~i~,,..~ ,,,II,~,~,, CONTROL
RECOVeRy
I
~ON"t'mCR-
z DJeta:~ X~N CL.ID~IZD|NIE
es<>
~ ~1~
nR
I ]1 CONTMOL
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l[J i
FIG. 2 Peri-stimulus histograms (PSH) generated by 60 successive sweeps of a duration of 1,024 msec each, of the cell shown in Fig.l. The PSH represent the firing of the neuron before, during the effect of the drugs and the recovery period for: A = NA, B = CLO, C = IDA + CLO and D = IDA + NA.
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The ejection of the alpha-2 adrenergic agonists (CLO and OXY; ejection currents of 10-50 nA for 10-30 sec) produced long-lasting inhibitions of firing in the majority of the neurons recorded from the visual cortex. For VD cells CLO inhibited 62% of these neurons (TABLE i); the responses started at 52 ± 13 sec, attained a maximum at I13 ± 21 sec and lasted 194 ± 38 sec. Only 3 VD neurons were excited by CLO and the responses had a duration of 111 ± 50 sec. In i0 VD cells the agonist OXY was tested, and the inhibitory responses were gradual but of a much longer duration ( > 12 min) than those produced by NA or CLO. As was the case for the natural agonist NA, the effects of CLO and OXY seemed to be preferentially exerted on the late components of the PSH (Fig. 2). For non-VD neurons the main effects of the alpha-2 agonists was again a reduction in firing. After the microiontophoretic application of CLO, this inhibition of firing rate started at 15 ± 3 sec, attained a maximum at 82 ± 14 see, and had an average duration of 155 ± 27 sec. The pattern of the inhibitory effects of selective alpha-2 agonists was similar to that induced by NA; i.e.: a reduction of the firing frequency of discharge. Indeed, CLO and OXY mimicked the effects of the neurotransmitter on both non-VD and VD cortical neurons. These effects of adrenergic agents (NA, CLO and OXY) could be reduced or blocked by a previous ejection of the specific alpha-2 adrenergic antagonist IDA in 50% of cells recorded (Figs. I and 2). In three neurons during the long-lasting inhibition produced by OXY, the administration of IDA proved to be effective in re-establishing the initial discharge rate. In subsequent experiments, we investigated the effects of the betaadrenergic drugs isoproterenol (ISO) and propranolol (PRO). These drugs were applied with ejection currents of 25-50 nA for 30-60 sec on VD and non-VD neurons. For VD neurons (n=26) the agonist ISO inhibited 54% (average duration 131 ± 29 see), excited i1.5% (average duration 67 ± 17 sec) and had no effects in the remaining 34.5%. In the case of non-VD cells (n=17), ISO inhibited 53% (average duration 80 ± 15 see), had no effects on 41% and excited 6%. Although the inhibitory responses were of a similar duration as those observed with NA or CLO, their magnitude was of a lesser degree. The antagonist PRO inhibited 74% of VD neurons (average duration 130 ± 18 sec), excited 5% and had no effects on 21% (n=19). It was tested on only 6 non-VD units, and inhibited 5 (average duration 144 ± 4 sec). Because of the inhibitory effect of PRO by itself on neuronal firing, probably due to its lipophilic properties, we could not use it as a blocking agent in microiontophoretic experiments.
Discussion The NA nerve terminals (I) which constitute the main CA projection, originate from the Locus coeruleus (LC) or A6 region (7) and are distributed to all regions of the allo- and isocortex of the adult rat (2,20). This innervation is seemingly sparse and ubiquitous in the neocortex, and for the rat frontoparietal cortex there is a certain predominance in the outer molecular layer, as well as in layers II and III (8). There is at present no quantitative data on the distribution of NA endings in rat visual cortex, but in the kitten CA nerve endings are also distributed to all the layers of the visual cortex, and are more dense in layers II and III (21), in agreement with biochemical determinations of endogenous CA (22). The rat visual cortex (occipital, areas 17 & 18) also receives an important noradrenergic input, as reflected by the endogenous levels of NA measured by fluorometric (23), radioenzymatic (5,24) and HPLC procedures (25). Recent studies
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suggest that NA may interact with alpha- and beta-adrenoceptors in the cerebral cortex, and the stimulation of these receptors can regulate the enzymatic activity of adenylate cyclase (26,27). Moreover, the existence of both alpha and beta-adrenoceptors has been demonstrated by direct receptor labelling experiments in the neocortex in general, as well as in the occipital cortex (25). These biochemical findings would imply that NA could act as a specific neurotransmitter on adrenergic receptors of the target neuroms in the visual cortex. The present study shows that NA affects both the spontaneous (i.e.: unrelated to the presented stimulus) and evoked activities of neurons in rat visual cortex. For the majority of non-VD (88%) and VD (82%) cells recorded, the microiontophoretic application of NA induced long-lasting inhibitions of cortical neuronal firing, and in only few cases excitations could be documented. These findings are in line with the depressant responses of NA on cortical neurons of adult rats (12), kittens (14), adult cats (13,14,28) and guinea pigs (29). One of the proposed actions of NA, mediated by specific receptor subtypes is to modulate the excitability of non-VD and VD neurons, through the regulation of the enzymatic activity of the adenylate cyclase which synthesizes cAMP (12,30). Such a mechanism of action requires biochemical changes in cortical cells, compatible with the long duration of effects of NA on the firing rate, and which could reflect the phosphorylation of postsynaptic proteins involved in synpaptic transmission (31). In the present study the doses of NA used (10-30 nA) were lower than those previously employed (80-100 nA) and which usually produced very pronounced suppressions of neuronal firing (12,13). Therefore, the effects on VD cells were not maximal and the analysis of the PSH showed greater effects on the "noise"; i.e.: the firing unrelated to the stimulus ( > 400 msec), than on the specific evoked response. At least two regulations in the activity of VD units can explain this enhancement of the S/N ratio: i] the neuromodulator can increase the specific evoked response without changing background discharge, 2] the evoked response remains constant but the background discharge decreases. It has been shown that NA can enhance the evoked firing of neurons without change of background firing (32,33) in support of a modulatory role for NA in the cerebral cortex. In contrast, Videen and collaborators working in the cat and kitten visual cortex (14) reported that NA enhanced the S/N ratio and this was a consequence of the reduction in the spontaneous activity of visual neurons. In the present study and for some of the VD cells, NA increased the S/N ratio by reducing the late phase of excitation, thus supporting the hypothesis that this neuromodulator promotes the selective response of a neuron to a particular stimulus. This modulation induced by NA could involve pre- or postsynaptic adrenergic receptors which could participate in spontaneous discharges unrelated to the photic stimulation. In order to determine the possible involvement of adrenergic receptors of the alpha-2 subtype in the modulation of the activity in cortic~1 neurons, we applied by microiontophoresis the specific agonists CLO and OXY. Previous studies had shown that CLO inhibits the firing of spontaneously active neurons in the LC and raphe nucleus in anesthetized (15,34) as well as unanesthetized animals (35), and this effect can be blocked by the specific antagonist IDA (15). These findings not only demonstrated the involvement of alpha-2 adrenoceptors in the control of firing rate of brain-stem neurons, but enabled the authors to conclude that IDA is the first alpha-2 adrenergic antagonist that allows the resolution of electro-
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physiological responses of this type (15). Numerous pharmacological (36,37) and biochemical (38-40) studies have shown the specific interactions of the adrenergic agonists CLO and OXY with specific receptors of the alpha-2 subtype in the cerebral cortex. In addition pharmacological studies have shown that OXY is more potent than CLO, in line with the long duration of inhibitions here obtained by microiontophoresis of this agonist. In the present investigation the microiontophoretic ejections of CLO and OXY produced long-lasting inhibitions of the firing of both non-VD and VD neurons, as was the case for the natural agonist NA. For VD cells, CLO and OXY also enhanced the S/N ratio by reducing more the background ~iming than the specific evoked discharge. Similar responses obtained wit~i alpha-2 adrenergic agonists and the natural catecholamine strengthen the hypoDhesis that this regulation could be mediated by alpha-2 adrenergic receptors. Finally, and in support of the specificity of the receptor subtype involved we were able to show that the inhibitory effects of adrenergic agents could be blocked or reduced in duration by microiontophoresis of the selective antagonist IDA (15,41). However the implication of other types of adrenergic receptors cannot be totally excluded in the modulatory effects of NA. We were unable to obtain pharmacological blockades with the beta antagonist PRO, mainly due to the fact that this drug produced a response by itself. On the other hand, the transsynaptically mediated depressions evoked in the visual cortex by stimulation of the LC could be blocked by practolol (a beta antagonist), but not by the alpha antagonists piperoxan or WB4101 (42). This issue will be settled with the use of specific betablocking drugs that do not affect the neuronal membrane properties. In conclusion, the role of NA as a neuromodulator of neuronal activity, affecting visual information transfer by modifying the S/N ratio is a valid hypothesis for the visual cortex (12,14). Such a regulatory mechanism calls upon the participation of alpha-2 adrenoceptors, since NA effects are mimicked with alpha-2 agonists. Although the present demonstration of the involvement of alpha-2 adrenoceptors contributes to the understanding of the mechanism of action of NA in the visual cortex, further work is needed to investigate a possible role of alpha-1 and beta-adrenergic receptors in the mediation of NA responses. Acknowledsements This work was supported by the Medical Research Council of Canada (Grant MT-6967) and the University of Montreal. Personal support was provided by the Fonds de la recherche en sant@ du Qu@bec (T.A. Reader; Chercheur-boursier Senior) and the Centre de recherche en sciences neurologiques (L. Diop; Herbert H. Jasper Postdoctoral Fellow). The authors also thank Messrs. G. Battista-Filosi and D. Cyr for the graphic work.
References I. A. CARLSSON, B. FALCK and N.-A. HILLARP. Acta Physiol. Scand. suppl. 196, 1-28 (1962). 2. K. FUXE, B. HAMBERGER and T. HOKFELT. Brain Res. ~, 125-131. (1968). 3. T. HOKFELT, K. FUXE, O. JOHANSSON and A. LJUNGDAHL. Eur. J. Pharmacol. 25, 108-112 (1974). 4. O. LINDVALL and A. BJORKLUND. Acta Physiol. Scand. suppl. 412, 1-48 (1974). 5. T.A. READER. J. Neural Trans. 50, 13-27 (1981). 6. J. VAN DER GUGTEN, M. PALKOWITZ, H.L.J.M. WIJNEN and D.H.G. VERSTEEG. Brain Res. 107, 171-175 (1976).
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7. U. UNGERSTEDT. Acta Physiol. Scand. 82, suppl. 367, 1-48 (1971). 8. Y. LAPIERRE, A. BEAUDET, N. DEMIANCZUK and L. DESCARRIES. Brain Res. 63, 174-182 (1975). 9. B. BERGER, C. VERNEY, C. ALVAREZ, A. VIGNY and K.B. HELLE. Neuros¢i. 15, 983-998 (1985). i0. A. BEAUDET and L. DESCARRIES. Brain Res. 111, 301-309 (1976). 11. K. FUXE. Acta Physiol. Scand. 64, suppl. 247, 37-85 (1965). 12. T.A. READER, A. FERRON, L. DESCARRIES and H.H. JASPER. Brain Res. 160, 217-229 (1979). 13. T.A. READER. Experientia. 34, 1586-1588 (1978). 14. T.O. VIDEEN, N.W. DAW and R.K. RADER. J. Neurosci. ~, 1607-1617 (1984). 15. J.E. FREEDMAN and G.K. AGHAJANIAN. Eur. J. Pharmacol. 105, 265-272 (1984). 16. K. KRNJEVlC. Int. Rev. Neurobiol. ~, 41-98 (1964). 17. T.A. READER. Brain Res. Bull. ~, 719-720 (1978). 18. G.C. SALMOIRAGHI and F.F. WEIGHT. Anesthesiology 28, 54-64 (1967). 19. H.M. GELLER and D.J. WOODWARD. Electroenceph. Clin. Neurophysiol. 33, 430-432 (1972). 20. P. LEVITT and R.Y. MOORE. Brain Res. 139, 219-232 (1978). 21. T. ITAKURA, T. KASAMATSU and J.D. PETTIGREW. Neurosci. ~, 159-175
(1981). 22. T.A. READER, P. MASSE and J. DE CHAMPLAIN. Brain Res. 177, 499-513 (1979). 23. W. KEHR, M. LINDQVIST and A. CARLSSON. J. Neural Transm. 38, 173-180 (1976). 24. M. PALKOVITZ, L. ZABORSKY, M.J. BROWNSTEIN, M.I.K. FEKETE, J.P. HERMAN and B. KANYICSKA. Brain Res. Bull. ~, 593-601. (1979). 25. J. SUTIN and K.P. MINNEMAN. Neurosci. ~, 973-980 (1985). 26. R.J. LEFKOWITZ, J.M. STADEL and M.G. CARON. Ann. Rev. Biochem. 52, 159-186 (1983). 27. S. SABOL and M. NIREMBERG, J. Biol. Chem. 254, 1913-1920 (1979). 28. K. KRNJEVIC and J.W. PHILLIS. Br. J. Pharmacol. Chemother. 20, 471-490 (1963). 29. T.W. STONE and D.A. TAYLOR. Can. J. Physiol. Pharmacol. 55, 1400-1404 (1977). 30. P. GREENGARD and J.W. KEBABIAN. Fed. Proc. 33, 1057-1059 (1974). 31. A.C. DOLPHIN and P. GREENGARD. J. Neurosci. i, 192-203 (1981). 32. B.D. WATERHOUSE, H.C. MOISES and D.J. WOODWARD. Exp. Neurol. 69, 30-49 (1980). 33. D.J. WOODWARD, H.C. MOISES, B.D. WATERHOUSE, B.J. HOFFER and R. FREEDMAN. Fed. Proc. 38, 2109-2116 (1979). 34. J.M. CEDERBAUM and G.K. AGHAJANIAN. Brain Res. 112, 413-419 (1976). 35. P.B. REINER. Eur. J. Pharmacol. 115, 249-257 (1985). 36. K. STARKE. Rev. Physiol. Biochem. Pharmacol. 88, 199-236 (1981). 37. P.B.M.W.M. TIMMERMANS and P.A. VAN ZWIETEN. Eur. J. Pharmacol. 55, 57-66 (1979). 38. D.B. BYLUND and D.C. U'PRICHARD. Int. Rev. Neurobiol. 24, 343-431 (1983). 39. C.L. WOOD, C.D. ARNETT, W.R. CLARKE, B.S. TSAI and R.J. LEFKOWITZ. Biochem. Pharmacol. 28, 1277-1282 (1979). 40. D.C. U'PRICHARD, D.A. GREENBERG and S.H. SNYDER. Mol. Pharmacol. 13, 454-473 (1977). 41. C.B. CHAPLEO, J.C. DOXEY, P.L. MYERS and A.G. ROACH, Br. J. Pharmacol. 74, 942P (1981). 42. H.-R. OLPE, A. GLATT, J. LASZLO and A. SCHELLENBERG, Brain Res. 186, 9-19 (1980).
Pergamon Journal
NORADRENERGIC EFFECTS ON RAT VISUAL CORTEX: SINGLE-CELL MICROIONTOPHORETIC STUDIES OF ALPHA-2 ADRENERGIC RECEPTORS
Arlette Kolta, Laurent Diop and Tomas A. Reader
Centre de recherche en sciences neurologiques, D@partement de physiologie, Facult~ de M~decine, Universit~ de Montreal, C.P. 6128, Succursale A, Montr@al, Quebec H3C 3J7, Canada. (Received in final form May 7, 1987) Summary The catecholamine noradrenaline has been proposed to modulate the excitability of cortical neurons, and such a regulation may be mediated by specific adrenergic receptors. We characterized, using electrophysiological recordings, the types of responses of single cells in the rat visual cortex (areas 17 and 18) to the iontophoretic application of adrenergic agents. For the majority of spontaneous and visually-driven cells sampled, noradrenaline decreased the firing frequency, and in some cases of visuallydriven cells could increase the signal/noise ratio. These effects were also documented after the application of the alpha-2 adrenergic agonists clonidine and oxymetazoline, and could be reduced or blocked by a previous ejection of the specific alpha-2 antagonist idazoxan. The present study supports a role for alpha-2 adrenoceptors in the modulation of sensory inputs to the visual cortex.
The presence of chemically-defined monoamine-containing neurons in the mammalian central nervous system (CNS) has been well established; the existence of central catecholamine (CA) neurons has been thoroughly documented in histof|uorescent and biochemical surveys, including the determination of the endogenous levels and/or the enzymatic activities. At least two types of CA afferent systems have been shown to project to the cerebral cortex: dopamine (DA) and noradrenaline (NA) containing fibers, while only traces of adrenaline (AD) have been measured (1-7). In addition, radioautographic and histofluorescence observations have demonstrated the presence in the cerebral cortex of terminals containing NA (1,8) and DA (9), as well as the indoleamine serotonin (10,11). It has also been proposed that NA plays a role in the modulation of neuronal response properties of spontaneouslyactive cortical units (12). In adult cat, iontophoretic injections of NA in the visual cortex reduced the firing of a majority of cells stimulated by flashes of light (13). These findings were confirmed by Videen and coworkers (14), who found that NA iontophoresed induced an inhibition mainly of the background ("noise") firing rate of visually-driven (VD) neurons in
Copyright
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.
282
Alpha-2 Adrenoceptors
in Visual Cortex
Vol. 41, No. 3, 1987
kittens and adult cats, thus enhancing the signal to noise (S/N) ratio. If NA modulates the excitability and background firing of cortical neurons, the types of adrenergic receptors involved have yet to be demonstrated. In a different type of electrophysiologlcal paradigm, the microiontophoresis of NA inhibited the firing of Locus coeruleus and raphe neurons (15), and these effects could be mimicked by the alpha-2 adrenergic agonist clonidine. Furthermore, these effects were receptor-specific, since they could be antagonized by alpha-2 antagonists. These observations suggest the involvement of alpha-2 adrenoceptors in the modulation of the excitability and/or firing of these brain-stem neurons. Since there is much less information regarding the adrenergic receptors of cortical neurons, the aims of the present investigation were: i) to characterize the responses to the microiontophoretic application of NA by means of electrophysiological unitary recordings in the occipital (visual) cortex of the rat, and 2) to determine the possible participation of the alpha-2 subtype of adrenoceptors in the mediation of the effects of noradrenaline on synaptically activated (VD; visually-driven) cortical neurons by microiontophoresis of clonidine (CLO), oxymetazoline (OXY) and idazoxan (IDA).
Material
and Methods
Adult male Sprague-Dawley rats (250-300 g) were used in this study. For the microiontophoretic recordings, the animals were anesthetized with urethane (1.25-1.5 g/kg, i.p.) and placed in a stereotaxic frame. The bone overlying the occipital cortex was removed, the dura mater retracted and the surface of the cerebral cortex covered with 2% agar in 0.9% NaCI. Standard microiontophoretic and extracelullar recording techniques were employed (16-18). Seven-barrel micropipettes, having an overall tip diameter 5-8 microns were filled with the following drugs dissolved in 0.1% ascorbic acid and at pH 4.0: noradrenaline HCI (0.5 M); clonidine HCI (0.1 M); idazoxan HCI (0.01 M); oxymetazoline HCI (0.I M); isoproterenol HCI (0.2 M) and propranolol HCI (0.2 M). The resistance of the recording barrel was 2-6 megohms. The central barrel, filled with 2-3 M NaC1 was used for recording, and one of the sides barrels (2 M NaCI) used as a balancing channel in conjunction with the operational amplifier (19) of the microiontophoresis programmer (BH-2 Neurophore system, Medical Systems, NY). Backing currents of -10 nA were used when not ejecting drugs. Extracellular unitary activity was amplified (P~511;Grass, MA), displayed on an oscilloscope, and the spikes discriminated by a voltage amplitude discriminator. The neuronal firing rates, integrated with a linear ratemeter over lO-sec intervals, were recorded on a six-channel recorder. Only neurons with a relatlvely stable rate of discharge were studied. On-line analysis of spontaneously-active (non-visually-driven; non-VD) and of visually-driven (VD) cells were also performed with an averager, digitized with an A/D converter (Adalab; Interactive Microware, PA) so that peri-stimulus histograms (PSH) could be generated using 128 bins of 0.5 to i0 msec/bin. VD cortical units were characterized by the PSH in response to a Grass P-20 photostimulator.
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Results
Throughout this study two populations of cells were sampled in the visual cortex of the rat; i.e.: neurons that could be synaptically activated by using a visual stimulation (VD; visually-driven cells) and spontaneously active (non-VD) units. In order to differentiate VD from non-VD neurons, we used the PSH in response to the photostimulator. However, it can not be ruled out that non-VD units were not visual neurons, since the stimulus used may have not been the most adequate to drive them specifically. Furthermore, non-VD units non-responsive to visual cues could also be participating in information processing by altering the excitability of synapticallydriven VD cells. For the majority of cortical neurons (non-VD and VD) sampled throughout this study NA and alpha-2 agonists exerted an inhibitory effect, and in only a few cases excitations could be documented. These results are summarized in TABLE I. As previously described for the rat frontoparietal cortex (somatosensory area), only in very few instances were biphasic responses obtained (12).
TABLE 1
Effects of adrenergic drugs on occipital cortex neurons. Drug
VD neurons n %
non-VD neurons n %
NORADRENALINE Inhibited Excited Biphasic* Unaffected
82 67 5 3 7
82 5 2 9
52 46 3 0 3
88 6 0 6
CLONIDINE Inhibited Excited Unaffected
42 26 3 13
62 7 31
31 27 2 2
87 6 6
OXYMETAZOLINE Inhibited Excited Unaffected
i0 7 I 2
70 I0 20
6 6 0 0
100 0 0
* These cells showed an inhibition followed by an excitation.
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The microiontophoresis of NA (ejection currents of 10-30 nA for 30-60 sec) on VD neurons induced a long-lasting inhibition of firing in 67 (82%) of the 82 VD cells recorded. This effect started at 15 ± 1.7 sec (Mean ±
NA 25 •
CLO 35 am
IDA 5O •
IDA 5O HI CLO 35 m
IDA 50 NA 25 IN
20t ~ ~o v
0
- 2 rain
FIG. 1 Ratemeter record (integrated over 10-sec intervals) of the firing rate of a cortical neuron in response to the photostimulator. The drugs noradrenaline (NA), clonidine (CLO) and idazoxan (IDA) were applied for the time and the dosage (in nA) indicated by the bars. The PSH are illustrated in Fig. 2.
S.E.M.), attained a maximum effect at 61 ± 5 sec and had an average duration of 199 ± 14 sec (See Fig. i for an example). Analysis of the PSH showed thnt NA inhibitory effects would firstly affect the late phase of excitation following the stimulus ( > 400 msec), and to a lesser degree the specific (earlier components) evoked response (Fig. 2). Since the effects of NA clearly outlasted not only the ejection and the period of maximum NA effect, there was an increase in the S/N ratio of the majority of VD cells, especially evident during the recovery or immediately after recovery to control levels of discharge. In the present study and using the same ejection parameters only 5 VD cells (5%) were excited while in 7 VD cells (9%) the firing frequency did not change. The excitations appeared during or after NA ejections, reached a maximum at 60 ± 30 sec and returned progressively to control levels (average duration 133 • 62 sec). In these cases there were no significant changes in the S/N ratio. Only three cells (2%) in this survey showed biphasic responses; i.e.: NA produced a first short period of inhibition, rapidly followed by an increase in firing. Responses to microiontophoretic applications of NA in visual cortex were also evaluated on the firing rate of non-VD neurons when encountered, and the effects were similar to the responses documented for the VD units. Indeed, NA ejections inhibited the spontaneous firing rate of 46 (88%) of 52 non-VD cells tested, and excited only 3 non-VD cells (6%). The durations of the inhibitions obtained for non-VD units were of the same order of
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magnitude as those documented for VD cells (non-VD: 158 ± 17 sec; VD: 14 sec). In only 3 non-VD cells (6%), the firing did not change application of NA.
N ~ R ~ [ N R L
C O N T R ~
x NE
E~
285
199 after
nR
~,,~,L~,,II,,,,,,l~i~,,..~ ,,,II,~,~,, CONTROL
RECOVeRy
I
~ON"t'mCR-
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~ ~1~
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FIG. 2 Peri-stimulus histograms (PSH) generated by 60 successive sweeps of a duration of 1,024 msec each, of the cell shown in Fig.l. The PSH represent the firing of the neuron before, during the effect of the drugs and the recovery period for: A = NA, B = CLO, C = IDA + CLO and D = IDA + NA.
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The ejection of the alpha-2 adrenergic agonists (CLO and OXY; ejection currents of 10-50 nA for 10-30 sec) produced long-lasting inhibitions of firing in the majority of the neurons recorded from the visual cortex. For VD cells CLO inhibited 62% of these neurons (TABLE i); the responses started at 52 ± 13 sec, attained a maximum at I13 ± 21 sec and lasted 194 ± 38 sec. Only 3 VD neurons were excited by CLO and the responses had a duration of 111 ± 50 sec. In i0 VD cells the agonist OXY was tested, and the inhibitory responses were gradual but of a much longer duration ( > 12 min) than those produced by NA or CLO. As was the case for the natural agonist NA, the effects of CLO and OXY seemed to be preferentially exerted on the late components of the PSH (Fig. 2). For non-VD neurons the main effects of the alpha-2 agonists was again a reduction in firing. After the microiontophoretic application of CLO, this inhibition of firing rate started at 15 ± 3 sec, attained a maximum at 82 ± 14 see, and had an average duration of 155 ± 27 sec. The pattern of the inhibitory effects of selective alpha-2 agonists was similar to that induced by NA; i.e.: a reduction of the firing frequency of discharge. Indeed, CLO and OXY mimicked the effects of the neurotransmitter on both non-VD and VD cortical neurons. These effects of adrenergic agents (NA, CLO and OXY) could be reduced or blocked by a previous ejection of the specific alpha-2 adrenergic antagonist IDA in 50% of cells recorded (Figs. I and 2). In three neurons during the long-lasting inhibition produced by OXY, the administration of IDA proved to be effective in re-establishing the initial discharge rate. In subsequent experiments, we investigated the effects of the betaadrenergic drugs isoproterenol (ISO) and propranolol (PRO). These drugs were applied with ejection currents of 25-50 nA for 30-60 sec on VD and non-VD neurons. For VD neurons (n=26) the agonist ISO inhibited 54% (average duration 131 ± 29 see), excited i1.5% (average duration 67 ± 17 sec) and had no effects in the remaining 34.5%. In the case of non-VD cells (n=17), ISO inhibited 53% (average duration 80 ± 15 see), had no effects on 41% and excited 6%. Although the inhibitory responses were of a similar duration as those observed with NA or CLO, their magnitude was of a lesser degree. The antagonist PRO inhibited 74% of VD neurons (average duration 130 ± 18 sec), excited 5% and had no effects on 21% (n=19). It was tested on only 6 non-VD units, and inhibited 5 (average duration 144 ± 4 sec). Because of the inhibitory effect of PRO by itself on neuronal firing, probably due to its lipophilic properties, we could not use it as a blocking agent in microiontophoretic experiments.
Discussion The NA nerve terminals (I) which constitute the main CA projection, originate from the Locus coeruleus (LC) or A6 region (7) and are distributed to all regions of the allo- and isocortex of the adult rat (2,20). This innervation is seemingly sparse and ubiquitous in the neocortex, and for the rat frontoparietal cortex there is a certain predominance in the outer molecular layer, as well as in layers II and III (8). There is at present no quantitative data on the distribution of NA endings in rat visual cortex, but in the kitten CA nerve endings are also distributed to all the layers of the visual cortex, and are more dense in layers II and III (21), in agreement with biochemical determinations of endogenous CA (22). The rat visual cortex (occipital, areas 17 & 18) also receives an important noradrenergic input, as reflected by the endogenous levels of NA measured by fluorometric (23), radioenzymatic (5,24) and HPLC procedures (25). Recent studies
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suggest that NA may interact with alpha- and beta-adrenoceptors in the cerebral cortex, and the stimulation of these receptors can regulate the enzymatic activity of adenylate cyclase (26,27). Moreover, the existence of both alpha and beta-adrenoceptors has been demonstrated by direct receptor labelling experiments in the neocortex in general, as well as in the occipital cortex (25). These biochemical findings would imply that NA could act as a specific neurotransmitter on adrenergic receptors of the target neuroms in the visual cortex. The present study shows that NA affects both the spontaneous (i.e.: unrelated to the presented stimulus) and evoked activities of neurons in rat visual cortex. For the majority of non-VD (88%) and VD (82%) cells recorded, the microiontophoretic application of NA induced long-lasting inhibitions of cortical neuronal firing, and in only few cases excitations could be documented. These findings are in line with the depressant responses of NA on cortical neurons of adult rats (12), kittens (14), adult cats (13,14,28) and guinea pigs (29). One of the proposed actions of NA, mediated by specific receptor subtypes is to modulate the excitability of non-VD and VD neurons, through the regulation of the enzymatic activity of the adenylate cyclase which synthesizes cAMP (12,30). Such a mechanism of action requires biochemical changes in cortical cells, compatible with the long duration of effects of NA on the firing rate, and which could reflect the phosphorylation of postsynaptic proteins involved in synpaptic transmission (31). In the present study the doses of NA used (10-30 nA) were lower than those previously employed (80-100 nA) and which usually produced very pronounced suppressions of neuronal firing (12,13). Therefore, the effects on VD cells were not maximal and the analysis of the PSH showed greater effects on the "noise"; i.e.: the firing unrelated to the stimulus ( > 400 msec), than on the specific evoked response. At least two regulations in the activity of VD units can explain this enhancement of the S/N ratio: i] the neuromodulator can increase the specific evoked response without changing background discharge, 2] the evoked response remains constant but the background discharge decreases. It has been shown that NA can enhance the evoked firing of neurons without change of background firing (32,33) in support of a modulatory role for NA in the cerebral cortex. In contrast, Videen and collaborators working in the cat and kitten visual cortex (14) reported that NA enhanced the S/N ratio and this was a consequence of the reduction in the spontaneous activity of visual neurons. In the present study and for some of the VD cells, NA increased the S/N ratio by reducing the late phase of excitation, thus supporting the hypothesis that this neuromodulator promotes the selective response of a neuron to a particular stimulus. This modulation induced by NA could involve pre- or postsynaptic adrenergic receptors which could participate in spontaneous discharges unrelated to the photic stimulation. In order to determine the possible involvement of adrenergic receptors of the alpha-2 subtype in the modulation of the activity in cortic~1 neurons, we applied by microiontophoresis the specific agonists CLO and OXY. Previous studies had shown that CLO inhibits the firing of spontaneously active neurons in the LC and raphe nucleus in anesthetized (15,34) as well as unanesthetized animals (35), and this effect can be blocked by the specific antagonist IDA (15). These findings not only demonstrated the involvement of alpha-2 adrenoceptors in the control of firing rate of brain-stem neurons, but enabled the authors to conclude that IDA is the first alpha-2 adrenergic antagonist that allows the resolution of electro-
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physiological responses of this type (15). Numerous pharmacological (36,37) and biochemical (38-40) studies have shown the specific interactions of the adrenergic agonists CLO and OXY with specific receptors of the alpha-2 subtype in the cerebral cortex. In addition pharmacological studies have shown that OXY is more potent than CLO, in line with the long duration of inhibitions here obtained by microiontophoresis of this agonist. In the present investigation the microiontophoretic ejections of CLO and OXY produced long-lasting inhibitions of the firing of both non-VD and VD neurons, as was the case for the natural agonist NA. For VD cells, CLO and OXY also enhanced the S/N ratio by reducing more the background ~iming than the specific evoked discharge. Similar responses obtained wit~i alpha-2 adrenergic agonists and the natural catecholamine strengthen the hypoDhesis that this regulation could be mediated by alpha-2 adrenergic receptors. Finally, and in support of the specificity of the receptor subtype involved we were able to show that the inhibitory effects of adrenergic agents could be blocked or reduced in duration by microiontophoresis of the selective antagonist IDA (15,41). However the implication of other types of adrenergic receptors cannot be totally excluded in the modulatory effects of NA. We were unable to obtain pharmacological blockades with the beta antagonist PRO, mainly due to the fact that this drug produced a response by itself. On the other hand, the transsynaptically mediated depressions evoked in the visual cortex by stimulation of the LC could be blocked by practolol (a beta antagonist), but not by the alpha antagonists piperoxan or WB4101 (42). This issue will be settled with the use of specific betablocking drugs that do not affect the neuronal membrane properties. In conclusion, the role of NA as a neuromodulator of neuronal activity, affecting visual information transfer by modifying the S/N ratio is a valid hypothesis for the visual cortex (12,14). Such a regulatory mechanism calls upon the participation of alpha-2 adrenoceptors, since NA effects are mimicked with alpha-2 agonists. Although the present demonstration of the involvement of alpha-2 adrenoceptors contributes to the understanding of the mechanism of action of NA in the visual cortex, further work is needed to investigate a possible role of alpha-1 and beta-adrenergic receptors in the mediation of NA responses. Acknowledsements This work was supported by the Medical Research Council of Canada (Grant MT-6967) and the University of Montreal. Personal support was provided by the Fonds de la recherche en sant@ du Qu@bec (T.A. Reader; Chercheur-boursier Senior) and the Centre de recherche en sciences neurologiques (L. Diop; Herbert H. Jasper Postdoctoral Fellow). The authors also thank Messrs. G. Battista-Filosi and D. Cyr for the graphic work.
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