Dose-dependent inhibitory effects of angiotensin II on visual responses of the rat superior colliculus: AT1 and AT2 receptor contributions

Neuropeptides(1997) 31 (5), 469-481 © HarcourtBraceand CompanyLtd 1997

D o s e - d e p e n d e n t inhibitory e f f e c t s of a n g i o t e n s i n II on visual r e s p o n s e s of t h e rat s u p e r i o r colliculus: A T 1 and AT 2 receptor contributions L. Merabet*, M. de Gasparo*, C. Casanova*t *D~partements d'ophtalmologie et de physiologie et biophysique, Faculte de m6decine, Universite de Sherbrooke, Quebec, Canada tCentre de recherche en g6rontologie et g6riatrie de I'h6pital d'Youville, Sherbrooke, Quebec, Canada ~CIBA-GEIGY Ltd. Pharmaceuticals Division, Basel, Switzerland

Summary AngiotensJn II (Ang II) has traditionally been regarded as a peripherally circulating and acting hormone involved in fluid homeostasis and blood pressure regulation. With the rather recent localization of Ang II receptors within the mammalian brain, renewed interest has emerged in the hope of elucidating the central impact and function of this hormone. One region that has been clearly demonstrated to express Ang II receptors is the superior colliculus (SC). This mesencephalic structure plays an important role in sensory visuomotor integration. Receptors for Ang II (of both the AT 1 and AT2 subtypes) have been localized within the superficial layers of this structure, i.e. the areas that are visually responsive. In the hopes of characterizing the role of Ang II in the SC, we have attempted to physiologically activate these receptors in vivo and observe the effects of Ang II on visually evoked responses. In the attempt to identify the receptor subtype(s) responsible in mediating these effects, Ang II was injected concomitantly with selective receptor ligands. Experiments were performed on adult rats prepared in classical fashion for electrophysiological studies. Through microinjection of Ang II, and the simultaneous recording of visually evoked potentials to flash stimulation, we have observed that this peptide yields a strong suppressive effect on visual neuronal activity. By injecting Ang II at various concentrations (10-3-10 -10 M), we have further observed that the effects of this peptide express a dose-related dependency. Injection of Ang II in progressively more ventral layers yielded less pronounced effects, demonstrating physiologically the discrete localization of these receptors in the stratum griseum superficiale. Coinjection of Ang II with Losartan yielded a near complete blockade of Ang II suppressive effects, suggesting that AT1 receptors play a prominent role in mediating these responses. However, coinjection of Ang II with PD 123319 yielded a slight, yet significant partial blockade. Coinjection of Ang II with both the AT t and AT2 receptor antagonists yielded a complete blockade of the Ang II effect. Finally, some of the results suggest that the AT2 receptor ligand CGP 42112 may possess agonist properties. Taken together, these findings suggest that the AT 1 receptor is predominately involved in mediating Ang II responses in the SC and there also appears to be some indication of AT2 receptor involvement. However, the underlying mechanisms (such as receptor interactions), the exact specificity of the ligands used, and the possibility of other receptor subtype implication have yet to be explored fully.

INTRODUCTION Angiotensin II (Ang II) is a powerful vasopressor molecule and the main effector hormone of the renin-angiotensin Received 23 April 1997 Accepted 29 July 1997 Correspondence to: Christian Casanova, E~coled'optom~trie, Universit6 de Montreal, Montreal, Quebec, Canada H3C 3J7, Tel: (514) 343-2407; Fax: (514) 343-2382; e-mail: [email protected]

system (RAS).1 The role of this octapeptide has traditionally been associated with the peripheral maintenance of fluid homeostasis, and it remains one of the most important hormones involved in the regulation of blood pressure. 2 Until fairly recently, Ang II was considered to be exclusively a circulating and peripherally acting hormone. The identification of Ang II as well as all the other components of the R_ASlocalized in the mammalian brain has generated interest in the hopes of localizing a central

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control of cardiovascular function3 and link central peptidergic signaling mechanisms and their peripheral physiological responses. At least two receptor subtypes, termed AT1 and AT2, have been identified4-7 and a third, termed AT4 is also gaining considerable attention.8,9 Presently, the 'classical' physiological functions of Ang II have generally been attributed to the AT1 receptor subtype2 ° There are suggestions that AT2 receptors could be involved in morphogenesis and development) ~-15 but there is now more recent evidence that these receptors show modulatory and interactive effects with AT1 receptors. ~6,~7It is now well established that Ang II receptors are also localized in regions of the brain not related to cardiovascular control (for review see~°), such as the ventral hippocampus containing ATj receptors and the locus coeruleus reported to show specific binding to AT2 receptors. As such, interest has now arisen as to possible novel and uncharacterized neuroregulatory roles for this peptide within the realm of sensory perception, behaviour, memory and learning. 18,19 Autoradiographic studies have shown that one particular sensory structure that expresses receptors for Ang II is the superior colliculus (SC).2,~2,2°-22The SC is believed to play an important role in sensory integration, particularly in the orientation of head and eye movements, visual attention and locomotor activity.23-26Although the relative proportions remain somewhat controversial, 1°,27 Ang II receptors of both the AT1 and AT2 subtypes appear to be solely localized within the superficial layers of the SC, and more particularly within the stratum zonale (SZ) and stratum griseum superficiale (SGS) layers. The localization of these receptors is somewhat interesting as these superficial layers correspond to regions that receive and integrate direct visual input from the retina and visual cortex, suggesting that Ang II may perhaps have a modulatory role in the visual system. The main purpose of our study was to investigate and characterize the physiological effects of Ang II in the mesencephalon. The first objective was to study the effects of local injection of Ang II on visual evoked potentials (VEPs) in the superficial layers of the SC and to determine if these effects vary in a dose-related manner. Secondly, we wished to determine the contribution of AT~ and AT2 receptors in the mediation of Ang II-related effects. Parts of these results have been published elsewhere. 28

METHODS AND MATERIALS Animal preparation

Experiments were performed on normotensive adult Long Evans rats (n = 90) weighing between 250 and 400 g (13-15 weeks of age). All animals were treated according to the guidelines of the Canadian Council on Animal Neuropeptides (1997) 31(5), 469-481

Care. Anaesthesia was induced by intramuscular injection of ketamine (80 mg/kg) in conjunction with atropine (0.04mg/kg) and acepromazin (1 mg/kg) to reduce tracheal secretions and muscular rigidity, respectively. Anaesthesia was maintained by the continuous subcutaneous infusion (0.4 ml/h) of ketamine in a lactated Ringer's solution (5%). The level of anaesthesia was monitored throughout the experiment (i.e. using a leg stretch or pinch reflex) and supplemental doses of ketamine were administered as needed. The animal was then placed in a stereotaxic head holder. The electrocardiogram (ECG) was continuously monitored and the core temperature was maintained at 37°C (+0.5°C) by a heating pad placed under the animal. Atropine sulfate (1%) eye drops were used to dilate the pupils, and the corneas were protected by application of artificial tears. Lidocaine hydrochloride (2%) was administered at all points of pressure and incision. A bilateral craniotomy was performed to expose the cerebral cortex overlying the SC of each hemisphere. The dura was incised and reflected and a glass microelectrode was lowered until the tip just penetrated the cortex. The cortex was then protected from desiccation with agar. Using a micro manipulator, the microelectrode was advanced vertically through the cortex and into the superficial layers of the SC according to stereotaxic coordinates 29 and visual response cues. Recording and visual stimulation

VEP recording has long been an established technique used extensively in the physiological characterization of various agents and toxins in vivo. 3° The advantage of this technique stems particularly from the fact that VEPs represent a stable and reproducible mass response of a population of neurones. Thus, altering these responses represents an effect on a neuronal cell pool or population as a whole. Glass microelectrodes with a tip opening of 20-27 ~tm were used to record VEP in the SC. The electrode was fashioned using thin-wall (1 ram) borosflicate capillaries (WPI) heated and pulled with a vertical micropipette puller (Kopf). The same microelectrode was used to deliver the peptide or its ligand (see below). The signals were amplified and bandpass filtered between 10 and 1000 Hz? ~,32The recorded signals were also passed through an audio monitor and an oscilloscope. They were then fed to a PC-compatible computer via an analogue/digital interface (CED 1401). Triggered evoked potentials were averaged over 25 successive presentations. Visual stimulation was provided by a diffuse flash (Grass photostimulator, intensity level 4) placed 30 cm away from the eye contralateral to the hemisphere being recorded from. The stimulus frequency was controlled by an event generator and set to 1 Hz. In a few experiments, a light or dark moving edge (displayed on a cathode ray © Harcourt Brace and Company Ltd 1997

Effects of angiotensin II 471

tube) was swept across the receptive field at optimal velocity and presented along three axes of movement (in both directions), i.e. vertical, horizontal and oblique. In these experiments, the VEP was also recorded simultaneously in order to confirm the success of the injection.

Delivery of pharmacological agents As previously mentioned, the recording electrode was also used to inject Ang II or its ligands. More precisely, the glass microelectrode filled with the drug was inserted in the head of a nanopump (WPI A1400 nanoliter injector) modified to allow the simukaneous recording of neuronal activity from the region immediately beneath the tip of the electrode. 33 Ang II (Val5 or Ile 5 Ang II; CibaGeigy; dissolved in physiological saline) was injected by micropressure. Similar effects were observed whether the sequence injected was Val5 or lle ~ and thus the data were pooled (t-test, P = 0.115z8). The peptide was administered at concentrations varying from 10-3 M to 10-1° M at a constant rate of 10 nl/min for a total of 40 nl for 4 min. In most cases, solutions injected were stained with Chicago Sky Blue (0.5%) for subsequent histological confirmation (see below). Studies of the effect of the specific A T 1 receptor antagonist Losartan (DUP 753) were carried out at 10-~ M and injected in conjunction with Ang II at equal concentrations and volume ratios (1:1). Further, injections were also carried out at an increased antagonist ratio (1:10 Ang II:Losartan) in order to augment the presence of the antagonist relative to the agonist. Similarly, the AT2 receptor ligand PD 123319 was also injected with Ang II at equal concentrations (10 -~ M) and at equal and increased antagonist ratios (1:1 and 1:10, respectively). The effects of the AT2 receptor ligand CGP 42112 were also studied. CGP 42112 was injected alone at different concentrations (varying between 10-6 M and 10-22 M) and also in conjunction with Ang II in order to test the possibility of any interactive effects when both ligands were present. In this scenario, a fixed concentration of CGP 42112 (10 -~1 M) was injected in combination with Ang II (ratio 1:1) at 10-6 M, 10-z M and 10-9 M. Finally, CGP 42112 (10-" M) was also injected with either of the two receptor antagonists Losartan (10 -6 M and equal ratio) and PD 123319 (10 -6 M and equal ratio) in order to clarify the receptor specificity of this ligand.

Experimental protocol and data analysis The electrode was lowered and positioned at the surface of the colliculus (determined by the first appearance of a visual response to the flash presentation). The electrode was then advanced slowly and positioned into the superficial layers on the basis of the VEP waveform. Recordings of the averaged VEPs (1 Hz, 25 presentations) © Harcourt Brace and Company Ltd 1997

were taken at approximately 1 to 2 min intervals until the waveform and amplitude appeared stable between recordings. Several control recordings were taken and then the pharmacological agent was injected (note that the injection onset represents time at zero). The VEP was recorded every minute throughout the period of injection. In order to compare the effects of injection of the various pharmacological agents, identical injection procedures were used throughout. Based on the observations of preliminary tests, an injection regime of 40 nl for a period of 4 rain (i.e. 10 nl/min) was used (see2S). An injection was determined to be 'successful' once the following criteria were met. Firstly, partial or total recovery of the VEP amplitude could be observed after injection had terminated. Secondly, confirmation of the site of injection could be identified during histological observation and was determined to be located within the superficial layers. Once injection had been stopped, the VEP was allowed to recover, during which recordings were taken roughly every 10 min. The amplitude and latency of the evoked potential waveform were calculated before, during and after injection of the peptide. The main measurement for this potential was the amplitude of the highest peak to peak distance that was deterlnined to be the most stable (see Fig. 2). Using the same peaks as points of reference, the latency of the responses was similarly measured. The calculated amplitudes were all normalized and the magnitude of the effect was expressed as a percentage (of the initial control amplitudes) to allow comparison between experiments. Overall results are expressed as means _+ SEM and statistical significance of the observed effects was determined using unpaired Student's t-test (*P( 0.05, **P < 0.01). Calculation of the ECs0 value was performed using Microcal Origin scientific software.

Histology At the end of each experiment, the animal was given an overdose of halothane by inhalation. The brain was removed and fixed in buffered formalin (10%) for a period of 1 week. The fixed brain was then cut in coronal sections and stained to reveal acetylcholinesterase (ACHE) activity. Histological observation was carried out to confirm the position of the recording site and the extent of the injection. Figure 1 depicts a photograph of a coronal plane through the rat SC showing the injection of Ang II in the superficial layers of the SC. It could be noted that the injection prevented the appropriate staining of AChE at the injection site. However, this observation has no functional significance since no differences were ever observed between coloured and non-coloured solutions with respect to the magnitude of effect on the VEP amplitude (see also28,33). Neuropeptides (1997) 31(5), 469-481

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Fig. 1 Photograph of a coronal section of the rat brain stained to reveal AChE activity. The arrow indicates the site of injection and recording, which appears pale when compared to neighbouring regioas. SGS: stratum griseum superficiale (superficial grey layer), SO: stratum opticum. Bar scale = 1 mm.

B Injection

RESULTS General observations

A total of 52 successful injections (out of 69) of Ang II were made in the SC. Histological observation indicated that the peptide always reached the upper and lower parts of the SGS (Fig. 1). The remaining injections were discarded due to a lack of a tendency for recovery or lack of histological confirmation. In the greater majority of the cases, injection of Ang II led to a decrease in the amplitude of the VEP in response to flash stimulation. The maximal effect occurred generally between 2 and 4 min after injection had commenced (mean time for maximal effect: 3.75 + 0.23 min). Generally, a tendency for recovery was evident shortly after injection termination and would then reach its maximum level 20-50 min thereafter. Recovery of the signal was at least partial (defined as > 75% of the original signal amplitude) in 86.3% of the cases and complete (i.e. > 100% of original signal amplitude) in 52.30/0 of the cases. The latency of the VEP was not affected due to injection of the peptide. Inhibitory effects of angiotensin II

Evoked potentials recorded from the superficial layers of the SC displayed the general waveform as described in other studies (e.gg). A representative example of the inhibitory effect of injection of Ang II (10-6 M) in the superior colliculus is shown in Figure 2. Figure 2A illustrates the field potential evoked by a flash stimulation. The waveform complex is characterized by a large positive (descending) deflection followed by another large negative deflection. There was a marked reduction (52.1%) in the amplitude of the evoked potential without any change in its latency after injection of the peptide (trace B). Approximately 40 min after injection Neuropeptides (1997) 31(5), 469-481

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Fig. 2 Representative example showing the effect of injection of Ang II (10-6 M) on VEP recorded from the SC. Note the marked reduction in the amplitude of the VEP after injection. An almost complete recovery of the potential was evident approximately 40 min after injection. S: stimulus.

termination, recovery of most components of the potential was evident (trace C). Figure 3 shows representative examples of the profile of effect of Ang II delivered at two different concentrations. Figure 3A depicts the normalized amplitude of the VEP as a function of time for an Ang II concentration of 10-3 M. One observes a reduction in the amplitude of the VEP shortly after the onset of injection. The maximal effect (82.5%) occurred around 4 rain, corresponding to the moment of injection termination. © Harcourt Brace and Company Ltd 1997

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This example further illustrates that the time course for the effect to ensue was much shorter and more abrupt compared with the recovery period. In this example, a partial recovery (-81.5% of the original signal amplitude) was evident 30 min after the end of injection. As a global tendency, we found that the effect due to injection of the peptide was rather rapid and the maximal effect would generally occur during the period of drug application (i.e. ___4 rain). Recovery of the potential was comparatively much slower and would generally commence immediately after injection of Ang II had stopped or shortly thereafter. Figure 3B shows another profile of action of Ang II at a concentration of 10-6 M. Note in this example there was a 42.4% reduction in the VEP amplitude and a complete recovery in the amplitude of the signal at the 40-rain mark. Many studies have demonstrated previously that neurons in the SC respond not only to stationary stimuli but are also sensitive to movement stimulation.34,35 In order to study the possibility that the effects of Ang II may depend on the visual stimulus used, we observed the action of Ang II (10 -6 M) on responses in the visual layers of the SC evoked by the movement of an edge. After injection of Ang II, there was a very strong and global suppression of the responses to the moving edge regardless of its direction of motion (data not shown). Overall, these results indicate that qualitatively, Ang II has a strong suppressive effect that is non-specific with respect to the stimulus used (i.e. stationary vs moving).

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Time (min) Fig. 3 Representative examples of the profile of action of Ang II injected at three different concentrations. Normalized amplitude is plotted as a function of time, Shaded region represents period of injection. (A) Injection of Ang II at 10-a M yielded a 82.5% reduction of the VEP amplitude after which only a partial recovery ensued• (B) Injection of Ang II at 104 M. At this concentration, injection of the peptide yielded a 42.4% reduction in the VEP amplitude with a complete recovery of the VEP amplitude evident at the 40-min mark. (C) Injection of saline showed no change in the overall amplitude of the VEP.

© Harcourt Brace and Company Ltd 1997

In order to reveal any concentration-dependent effect of Ang II, the peptide was injected at varying concentrations and the effects on the VEP amplitude were compared. Our results showed that the strength of the inhibitory effect of Ang II decreased as the concentration of the peptide was lowered. Half-maximal inhibition was observed at peptide concentrations between 10-5 M and 10-6 M (ECs0= 3.0 x 10-6 M). Figure 4 illustrates an in vivo dose-response curve for Ang II injected at varying concentrations. Concentrations ranging between 10-3 M and 10-~° M are plotted as a function of the mean reduction in amplitude of the VEP (%). The decrease in the amplitude of the potential by Ang II at 10-]° M was less pronounced (12.9 + 12.7%) compared with higher concentrations tested. As the concentration of Ang II administered increased, the magnitude of inhibition progressively became more pronounced and reached a maximum at 10-3 M and 10-4 M (reduction of 68.1 + 5.8% and 72.3 _+ 6.4%, respectively). Statistical analysis indicated that all mean reductions observed above the concentration of 10-9 M were significant at the P K 0.05 level with respect to control injections with physiological saline. Neuropeptides (1997) 31(5), 469-481

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Observation of a dose-responsive effect of Ang II in vivo at the level of the CNS has been reported elsewhere. 36 Control injections

As a means of ensuring that the effects observed were indeed due to the physiological activation of Ang II receptors and not the injection per se, a number of experiments (n = 22) were carried out to study the effect of injecting the vehicle following the same protocol as that for Ang II. In general, injection of NaC1 failed to modify the amplitude and the components of the VEP waveform. In a few cases, however, injection of saline produced a slight reduction in the VEP amplitude (mean 6 . 5 +_ 2.63%). These reductions were, however, transitory in nature, and a full recovery of the VEP amplitude was evident either during the injection period itself or immediately thereafter. An example of the effect of a saline injection is shown in Figure 3C. As was the case with Ang II, no differences in the magnitude of effect on the VEP were observed between coloured and non-coloured saline injections. We were confident that the injection itself did not produce any deleterious effects and that the observed reductions on the amplitude of the visual responses in the colliculus were indeed physiological and due to the activation of Ang II receptors. Depth profile: localization of effect

in order to investigate physiologically the laminar localization of the Ang II receptors in the SC, the peptide was Neuropeptides (1997) 31(5), 469-481

injected at progressively deeper layers in this mesencephalic structure. Aug II did n o t modify the evoked potential amplitude when delivered in collicular layers ventral to the SZ and SGS. A representative example of this experiment is shown in Figure 5. Figure 5A illustrates the approximate location of three consecutive sites where Aug II was injected over a depth of 1 mm. Figure 5B depicts the VEP recordings prior to ('control'), and immediately after injection of Aug II ('injection': 20 nl/ 2 min) at each laminar position indicated in Figure 5A. Note that there was a reduction in the amplitude of the VEP (74.4%, trace 1) when Aug II was injected in the most superficial site (SZ/SGS). As the microelectrode was advanced 500 ~tm and the same volume of Aug II was injected, the effect on the VEP signal was much less pronounced ( 3 6 0 trace 2). As the electrode was further advanced by 500 ~tm, injection of Aug II yielded very little reduction on the VEP amplitude (140/0, trace 3). This finding is in agreement with autoradiographic studies which indicate that the receptors for Ang II are most densely localized in the superficial layers of the SC and more precisely in the SZ and SGS.2,~ Identification of receptor subtypes

On the basis of autoradiographical findings, the Aug II receptors located within the superficial layers of the SC have been shown to be of both the AT1 and AT2 subtypes. 2,12In order to delineate between the receptor subtypes involved in mediating the inhibitory effects of Aug II, we injected the peptide along with selective receptor ligands. AT~ receptor identification

We injected the peptide Aug II with the AT1 receptor antagonist Losartan (]:)UP 753). Coinjection of Losartan and Ang II at equal concentrations (10 -6 M) and ratio (1:1) yielded a slight blockade of the inhibitory effect of Aug II. Although this result did not attain overall significance, it preliminarily suggested that AT1 receptor activation could be involved in mediating the suppressive effects of Ang II. In order to further confirm this hypothesis, we injected Aug II concomitantly with Losartan at equal concentrations but at a ratio in which the antagonist was increased (i.e. 1:10)} z-39 In this case, only a slight reduction in amplitude of the evoked potential ensued, i.e. the suppressive effects of Aug II were almost completely abolished by the Losartan. Figure 6A represents a summary of the effects on the VEP amplitude of Aug II in combination with kosartan (ratio 1:10). The effect of Ang II alone at 10-6 M (value taken from the Aug II dose-response curve) is also indicated for comparison. As previously reported by our group, 2s coinjection of Aug II:Losartan at a ratio of 1:10 led to a blockade of the effect © Harcourt Brace and Company Ltd 1997

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of Ang II. Indeed, the mean decrease of amplitude was only 7.2 +_ 8.1%, and statistical analysis had confirmed these results to be highly significant (P< 0.01).

the overall degree of blockade when both antagonists were present was not significantly greater.

AT2 receptor identification

The AT2 receptor ligand CGP 42112 was also injected in order to further investigate the possible role of the AT2 receptor in the Ang II-related effects observed in the SC. CGP 42112 was injected alone and it was found that this ligand also led to a decrease in the amplitude of the VEP in the superficial layers of the SC. These effects were particularly evident at very low concentrations of the ligand (e.g. 10-9 M and 10-11 M). Figure 7 shows examples of the effects of CGP 42112 injected at different concentrations (10 -6 M, 10-9 M and 10-13 M). Injection of the ligand at these concentrations all led to a decrease in the amplitude of the VEP (respective mean reductions of 20.8 + 6.6%, 2Z7 + 8.7%, and 2Z0 + 7.3%). The time course required for the appearance of the maximal effect for CGP 42112 (3.8 + 0.3 rain SEM) was similar to that for Ang II. Recovery of the amplitude generally ensued 20-50 min after the termination of injection, as was the case for Ang II. The action of CGP 42112 was also restricted to the superficial layers, as no reduction was observed when injections were carried out within the more ventral layers (Fig. 7B).

CGP 42112

The lack of effect of Ang II in the presence of Losartan strongly suggested that the suppressive effects of Ang II on the amplitude of the VEP mainly involved the activation of the AT1receptor. In order to verify whether or not AT2 receptors also contributed in the mediation of the Ang II effects, we coinjected Ang II with PD 123319, a ligand which is generally established as a selective AT2 receptor antagonist in in vitro and in vivo studies. 4°-42 A reduction in the amplitude of the VEP was present when Ang II and PD 123319 were injected at equal concentrations and at equal ratios (23.7 +_ 5.3%). These results show that there was a small but significant blockade of the Ang II effect on the VEP in the presence of this ATa receptor antagonist (P < 0.05) and further suggest that the AT2 receptor may also be involved in mediating the effects of Ang II. A summary of these results is presented in Figure 6B. For comparison purposes with the Losartan experiments, we also changed the relative proportions between the peptide and its antagonist (1:10 in favour of PD 123319)? 7-39 In this case, however, the resuks observed were different: PD 123319 did not block the effect of Ang II on the VEP (see Discussion). The mean reduction in amplitude was not statistically different from the decrease observed with Ang II alone (32.8 + 5.3% compared to 40.9 _+4.7%; P > 0.05). Ang II with both AT 1 and AT2 antagonists

As a continued test to identify the AT1 and AT2 receptor contributions, we injected Ang II with both Losartan and PD 123319 in order to ascertain if the presence of both these antagonists could block completely the effects of Ang II. The injections were carried out at equal ratios (1:1:1) and at higher antagonist ratios (1:10:10). Injection of Ang II with both AT~ and AT2 receptor antagonists at equal ratios and concentration resulted in a 18.48 + 8.9% reduction in amplitude of the VEP, suggesting that the inhibitory effect of Ang II was blocked in the presence of both antagonist ligands. When Ang II was injected in the presence of Losartan and PD 123319 at increased antagonist ratios (1:10:10), a complete blockade of the inhibitory Ang II effect was observed (overall, a slight increase in amplitude was observed: 4.27 + 9.2O/o).A summary of these findings is presented in Figure 6C. Upon initial observation, it appeared that the blockade of both AT1and AT2 receptor subtypes may be necessary in order to abolish completely the effects of Ang II on the VEP. However, statistical comparison of the injection of Ang II with only one antagonist (Fig. 6A and B) with the injection of Ang II with both antagonists (Fig. 6C) showed that Neuropeptides (1997) 31(5), 469-481

CGP 42112 with Ang II

Since CGP 42112 appeared to act as an Ang II agonist, we investigated the possibility that the injection of both the peptide and CGP 42112 would yield effects stronger then those observed when injected alone. We compared the effects of Ang II injected at three different concentrations (10 -6 M, 10-7 M, 10-9 M) in conjunction with CGP 42112 at 10-11 M. When both ligands were injected together, there was no clear additive effect. For instance, when CGP 42112 was injected with Ang II at 10-6 M, the inhibitory effect (44.2 + 10.7%) was comparable to that of Ang II injected alone at the same concentration (40.9 + 4.7%). Coinjection of the two ligands CGP 42112 with Ang II at 10-7 M yielded a 21.1% reduction in the amplitude of the VEP, which was inferior (by 10.7%) to the reduction yielded by Ang II at 10-7 M injected alone (31.8 + 7.9%). Upon statistical comparison, the differential effects were found to be not significant (P > 0.05), suggesting that the coinjection of both-Ang II and CGP 42112 (at 10-11 M) did not produce additive effects. If a synergy between Ang II and CGP 42112 was not observed, this may be due to the similar affinity of both ligands for the AT2 receptor. 43 CGP 42112 with AT 1 and AT2 receptor antagonists

In a preliminary attempt to investigate further the nature

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PD 123319 were all carried out at 10-6 M and at equal ratios. Injection of CGP 42112 at 10-11 M alone resulted in a 27 + 7.3% reduction in the amplitude of the VEP. When the ligand was injected with Losartan, there was no evidence of a blockade of the CGP 42112 inhibitory effect (mean reduction in amplitude: 35.6 _+5.3%). These results would thus be in agreement that first, CGP 42112 acts as an agonist at this concentration, and secondly, this ligand has its effects mediated by the AT2 receptor. However, conflicting results were found when CGP 42112 was injected with the AT2 antagonist, PD 123319. In this scenario, a 25.7 + 6.9% reduction in amplitude resulted, suggesting that PD 123319 was not able to block the effects of CGP 42112 either. Thus, our preliminary data do not allow the determination of which receptor subtype is involved in mediating the effects of CGP 42112 at a concentration of 10-1' M. Although rather preliminary, these findings do not identify dearly the receptor implicated (i.e. AT~ or AT2) in the observed effects of CGP 42112 in our in vivo model, and this remains to be demonstrated clearly.

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Time (min) Fig. 7 Representative examples of the effect of injection of CGP 42112 at three concentrations, i.e. (A) 10-6 M, (B) 10 -9 M, (C) 10-11 M. In (B), CGP 42112 was also injected in the deep collicular layers (e). Clearly, the effects of CGP 42112 were only observed in the superficial layers where receptors are reported to be located.

antagonists, Losartan and PD 123319. We hypothesized that for a CGP 42112 concentration of 10-~1 M, the AT2 receptor antagonist PD 123319 should be effective in blocking any inhibitory effect of the ligand while Losartan should remain ineffective. Injections with Losartan and

© Harcourt Brace and Company Ltd 1997

While there is growing awareness and understanding as to the Ang II-dependent effects on CNS function, there are very few studies that have illustrated the effects of this peptide at the level of sensory and motor systems. The results of this study indicate that Ang II can modulate the activity of visual neurons in the superficial layers of the SC. Ang II strongly suppressed the VEPs in a manner that was dose-dependent with respect to concentration. These effects were limited to the superficial layers, which is consistent with autoradiographic findings reporting that Ang II receptors are located in the SZ and SGS layers. 2,~2 Our resuks further indicate that the suppressive effects of Aug II appear to be non-specific with regard to the nature of the visual stimulus used. The implication of AT1 receptors was clearly demonstrated in our study, with the near complete blockade of the Aug II action using the selective antagonist, Losartan. However, we have also obtained evidence that AT2 receptors could also be implicated in the Ang II-related effects. Methodological considerations

The effects observed through injection of Aug II are believed to be the direct resuk of activating the receptors present in the superficial layers of the SC for the following reasons. Firstly, control injections with physiological saline failed to modify both the amplitude and waveform of the VEP. This suggests that the suppressive effects

Neuropeptides (1997) 31(5), 469-481

478

Merabet et al

observed were not due to mechanical displacement of tissue or tortuosity. Secondly, depth profile studies demonstrated a lack of effect on the VEP when pharmacological agents were injected at sites ventral to the presumed location of Ang II receptors. Importantly, this last observation indicated not only that the action of Ang II was restricted to the superficial layers but also that the effects were due solely to the central actions of this peptide. At the same time, this observation rules out the possibility that the inhibitory effect of Ang II resulted from action of this peptide upon blood vessels (i.e. localized systemic effects or ischaemia). In this context, it is worth pointing out that in the central nervous system, ischaemia has been reported to induce a hypersensitivity of neuronal responses 44 which is contrary to the observations reported here. The fact that the Ang II effects observed showed a dose-dependent relation with respect to concentration is further indicative of the actual physiological activation of Ang II receptors. Finally, the successful pharmacological blockade of the action of Ang II (by ligands such as Losartan) exemplifies that the effects we reported were not related to the injection per se. A point of concern may be raised regarding the type of anaesthesia used in this study. Ketamine is known to be an NMDA receptor antagonist,45,46 and this anaesthetic agent may have effects on the generation of the VEP and/or possibly have interactive effects with Ang II itself. Despite the fact that ketamine anaesthetic preparations have been used in other studies to investigate the effect of Ang II at the level of the CNS (in rodents 47,48and in cats49), one can raise the question as to whether or not the resuks could be influenced by the nature of the anaesthetic used. While we cannot totally rule out this possibility, it is unlikely for the following reasons: (1) to our knowledge, studies on the interactive effects of ketamine and Ang II receptors have yielded only negative results at both the molecular46 and systemic levels,45 (2) While all known anaesthetics can have, to a certain extent, effects on various cellular mechanisms 5° and thus may have repercussions on the generation of the VEP (e.g. pentobarbital, a commonly used anaesthetic in rodent preparations has been shown to alter VEPs in ratsS~), it is important to note that the VEPs recorded served as their own controls in each case of injection of the peptide and/or its ligand; and (3) preliminary results from our laboratory have shown that Ang II also shows inhibitory effects on the VEP of the SC in urethane-anaesthetized rats, ~2 suggesting that the Ang II effects observed do not depend on the nature of the anaesthetic used. Receptor subtypes Pharmacological ligands were used in order to investigate the receptor subtype(s) implicated in mediating the Neuropeptides (1997) 31(5), 469--481

inhibitory effects of Ang II observed in the SC. Injection of Ang II in the presence of Losartan showed an almost complete blockade of the inhibitory effects of the peptide. The blockade of Ang II effects were evident only at an increased antagonist relative to Ang II ratio (i.e. 10 Losartan: 1 Ang II) as reported in other physiological studies. 37 This latter observation may be related to the relatively lower receptor affinity of Losartan compared with Ang II? °,53 It is also possible that the diffusion and/or other pharmacokinetic factors may be different between the two compounds. Nevertheless, these resuks suggest that AT~ receptor activation is implicated in mediating the Ang II effects observed in this study. It may be worth noting that the physiological effects of Ang II have been generally associated with the AT~ receptor (see 6,~° for reviews). However, recent studies have illustrated that AT2 receptors also regulate central nervous system functions, including behaviour. 4s,$4,55In conjunction with the autoradiographic identification of AT2 receptors being present in such a large relative quantity in the SC, 2,12 the contribution of this receptor was also investigated. Using the AT2 receptor antagonist, PD 123319, a partial blockade was also evident when in the presence of Ang II. Curiously,this partial blockade effect was observed only in the case where Ang II and PD 123319 were present in equal ratios. It may be possible that at a higher ratio, PD 123319 would be acting as a partial agonist. This possibility has been raised by others at the level of the CNS? 6 The involvement of AT2 receptors is also supported by our observation that CGP 42112 can alter collicular activity ff one considers CGP 42112 as a specific AT2 ligand. Specificity of CGP 42112 When injected alone, CGP 42112 resulted in a reduction in the VEP amplitude, and its profile of action resembled that of Ang II. The fact that the agonistic action of this ligand was localized within the superficial layers suggests that CGP 42112-sensitive receptors are needed to obtain these results. It is likely that the latter correspond to the abundant Ang II receptors present and putatively the AT2 receptors. 3s,53,Sz However, our data do not provide clear evidence of the level of involvement of Ang II receptors. Nevertheless, the effects observed following the administration of CGP 42112 do raise interesting possibilities and further questions regarding the specificity of this ligand. For example, whether or not these effects are the result of direct mediation of AT2 receptors or an indirect mechanism via AT]-AT2 receptor interactions remains an open question. Such functional cross-talk between AT~ and AT2 receptors has already been observed, 42,57and AT] receptor-mediated effects have been shown to be modulated upon AT2 receptor implication in the brain. ]6 The © Harcourt Brace and Company Ltd 1997

Effects of angiotensin II 479

further possibility of the activation of another receptor site also cannot be ruled out. For instance, there have been recent reports that the ligand CGP 42112 recognizes novel non-Ang II CGP 42112 binding sites, particularly in the brain after injury. 5s,59Brechler et al53 have also reported that the profile of action of this ligand varies according to the concentration used. It is also worth pointing out that the reported agonist properties of CGP 42112 are the result of studies performed in cultured cells expressing only AT2 receptors? 3 Formal conclusions in vivo cannot be extrapolated easily since the SC is a structure that expresses both receptor subtypes. Comparison with other studies

The neuromodulatory effects of Ang II have been investigated in a variety of structures in the central nervous system and have been reviewed extensively. ~°,1z6° There is growing evidence that Ang II exerts a modulatory action in sensory and motor control at the level of the mammalian brain. 61,62 Perhaps more relevant to this study is the work of Mooney and co-workers. 63 This group studled the effects of microiontophoretic application ofAng II on single-unit activity of neurons in the SC of the hamster. In agreement with our data, they reported that application of Ang II decreased the visual responses of most SC cells as well as the discharges evoked by stimulation of the optic chiasm and visual cortex. Their data further suggested that the site of action of Ang II was postsynaptic. They have reported that both classes of antagonists (as used in this study, i.e. Losartan and PD 123319) were equally effective in antagonizing the suppressive action of Ang II. Thus, in contrast to our findings, these authors suggest that both AT~ and AT2 receptors may independently and equally mediate the inhibitory effects of Ang II. This discrepancy may be related to the nature of the technique used (i.e. singlecell vs VEP recordings). Akhough this study has provided valuable insight into the effects of Ang II in the SC, the experiments were carried out at only one concentration (10-5 M). This may be of relative importance when one considers that different effects with respect to concentration and ratios used can influence results as reported in the present study. Recently, AlbrechP 4 has reported that Ang II also has neuromodulatory effects on the visual responses of cells in the rat dorsal lateral geniculate nucleus (dLGN). In this structure, mainly AT2 and AT4 receptors have been shown to be present. Iontophoretic application of Ang II provoked an increase in the responses of dLGN neurons. It is worth commenting at this point that Ang II seems to have an opposite modulatory effect upon two different sensory structures and visual pathways. Further, these two structures are part of two distinct and parallel routes of visual processing: the © Harcourt Brace and Company Ltd 1997

retino-geniculo-cortical route is responsible for form discrimination, while the retino-collicular pathway is involved in visuomotor function. Functional considerations

Recent reviews ~°,65have stressed the wide distribution of Ang II in the central nervous system and its effects on behaviour and neuronal activity. It has been suggested that neuropeptides play integrative roles in a given response alongside other peptides such as vasopressin and oxytocin,66 excitatory amino acid n e u r o t r a n s m i t t e r s ~7 and with more 'classical' neurotransmitters such as serotonin, noradrenaline (seP 6 for review) and dopamineP 8 Thus, it has been proposed that neuropeptides such as Ang II may not be implicated in the moment by moment regulation of information processing per se but rather modify it through their global implication upon motivation, attention and arousal states, as Since most behavioural acts involve three major functional systems in the brain, i.e. sensory, motor and motivational systems, 65 perhaps it is not surprising that receptors for a globally acting peptide like Ang II be located in a sensorimotor structure such as the SC. We can speculate that levels of Ang II, either blood-borne or locally synthesized, would reflect the internal state of the organism onto its activity and this could be manifested in terms of changes in exploratory or avoidance behaviour: a role that has been attributed to the SC.69-72

ACKNOWLEDGEMENTS

Supported by an MRC-Industry operating grant and CIBA-GEIGY Ltd in collaboration with N. Gallo-Payet and D. Payer. We thank F. J. Jolicoeur for his helpful comments on the manuscript.

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