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Excitatory action of angiotensins II and IV on hippocampal neuronal activity in urethane anesthetized rats
Regulatory Peptides 70 (1997) 105–109
Excitatory action of angiotensins II and IV on hippocampal neuronal activity in urethane anesthetized rats ¨ Doris Albrecht*, Michael Broser, Hagen Kruger Institute of Physiology, Faculty of Medicine ( Charite´ ), Humboldt University, Tucholskystr. 2, D-10117 Berlin, Germany Received 15 November 1996; received in revised form 10 March 1997; accepted 11 March 1997
Abstract The renin–angiotensin system of the mammalian brain seems to interfere with the process of cognition and has been associated with the hippocampal function in relation to mechanisms of learning and memory. In our investigation, the effects of angiotensin II (Ang II) and angiotensin IV (Ang II) on neuronal activity have been studied in the hippocampus of adult rats anesthetized with urethane. Excitatory effects of both angiotensins predominated over inhibitory effects. Angiotensins also induced an enhancement of burst discharges. These angiotensin-induced effects were blocked by the specific angiotensin antagonists. Our findings showed that the different effects of Ang II and Ang IV in behavioral studies are not similarly reflected in a different change of the discharge rate and / or pattern of hippocampal neurons after microiontophoretic administration of both substances. 1997 Elsevier Science B.V. Keywords: Hippocampus; Angiotensin II; Angiotensin IV; Single unit activity; Iontophoresis
1. Introduction A renin–angiotensin system is known to be present in the mammalian brain, complete with the precursors and enzymes required for the formation and metabolism of the biologically active forms of angiotensin as angiotensin II and IV (Ang IV) [1–4]. Ang II acts on two different angiotensin receptors, the AT1 and the AT2 receptor, respectively, while Ang IV (Ang II 3–8) selectively binds to the AT4-receptor site. In addition to the classic cardiovascular functions of angiotensins several reports indicate that Ang II and / or Ang IV may participate in mechanisms of learning and memory and interfere with cognitive function. Behavioral investigations have shown that losartan, a highly specific AT1-receptor antagonist, possessed cognitive enhancing and anxiolytic-like potential, whilst PD 123177, a specific AT2-antagonist, lacks any anxiolytic effect but showed a cognitive enhancing *Corresponding author. Tel.: 1 49 30 28026153; fax: 1 49 30 28026669; e-mail: [email protected] 0167-0115 / 97 / $17.00 PII S0167-0115( 97 )00015-3
potential [5]. Studies investigating the prominent role of the hippocampus in memory acquisition and retention revealed that Ang II administered directly to the dentate gyrus impaired the retention of an inhibitory shock avoidance response [6]. This effect was blocked by the selective AT1 antagonist losartan. Furthermore, an activation of the AT1 receptor via Ang II inhibit the induction of long term potentiation in medial perforant path-stimulated dentate granule cells, if Ang II is injected into the hippocampus [7,8]. In contrast, Ang IV may contribute to an enhancement of memory retrieval in a passive avoidance conditioned response in rats. In addition to enhancing memory retrieval, Wright et al. [9] have observed a facilitation of retention of passive avoidance training with Ang IV on days 2 and 3 after conditioning. Several other studies [10,11] provide support of these findings. The different angiotensin receptors in the rat hippocampus [12,13] vary in their distribution pattern. In this area a high density of AT4 receptors have been found, whereas the AT1 and especially the AT2 receptor occur with lower density [14]. Recently it has been shown that AT1 B as well
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as AT1A receptor mRNA is present in the hippocampus [15]. However, analyses investigating the effects of Ang II and Ang IV on hippocampal neurons are lacking. Therefore, we were interested in comparing the influences of iontophoretically administered Ang II and Ang IV on the discharge rate of hippocampal neurons.
2. Materials and methods All procedures were carried out according to the standards of animal welfare and approved by the regional Berlin animal ethics committee. The experiments were performed on male adult albino rats. The rats were kept four to a cage with food and water available ad libitum and on a diurnal light–dark cycle. All testing took place during daylight hours. The rats were anesthetized with urethane (1.2 g / kg, i.p.) and placed in a stereotaxic instrument. Subsequent injections of urethane were administered as needed (for detail see [16]). Rectal temperature was maintained at 37–388C by a heating pad. The electrocardiogram and the cortical EEG were monitored. A small hole was drilled into the skull at a site 3.5 mm lateral to the midline suture and 5.0 mm anterior to the lambdoid suture. An electrode was lowered 2.5 to 4 mm with a microdrive through the hole to the level of the hippocampus. Glass microelectrodes for extracellular recording were filled with saturated Trypan blue solution (tip resistance 10–30 MV). The recorded action potentials were amplified and displayed on an oscilloscope and were, after passing a window discriminator (World Precision Instruments, USA), analyzed with custom-made software (spike 2 from Cambridge Electronic Design, UK) running on a personal computer. Standardized pulses corresponding to individual action potentials were used for computing frequency time histograms which were displayed on-line during sampling. Data were stored on disc for subsequent analysis. Either five or seven barrel glass micropipettes (tip diameter 5–7 mm) were glued to the recording electrode so that their tips were separated vertically by 20–40 mm. The following drugs were used: angiotensin II (Ang II; 100 mM, pH 4.5; Research Biochemicals International (RBI) or synthesized by Dr. P. Henklein, Institute of Biochemistry, ´ and angiotensin IV (Ang IV; 100 mM, pH 4.5; Charite) synthesized by Dr. P. Henklein, Institute of Biochemistry), losartan (AT1-antagonist; 100 mM, pH 8.0; a gift of Dr. R.D. Smith [Dupont Merck, Pharmaceutical Company, Wilmington, Delaware, USA]), PD 123,319 ditrifluoroacetate (AT2-antagonist; 100 mM, pH 4.5; RBI) and divalanal-Ang IV (AT4-antagonist; 100 mM, pH 4.5; Pacific Northwest Biotechnology, Pullman, USA). Retaining currents (4–10 nA) were applied to the pipettes between drug ejections. In a number of experiments a barrel filled with sodium chloride (165 mM, pH 4.5 or pH 8) was used for
current balance. In control experiments no significant contribution from current or pH was detected. The discharge rates were analyzed off-line for periods prior to, during and after termination of drug administration. From the continuously recorded rate meter counts, the average discharge rate of each neuron was evaluated for 120 s prior to the iontophoresis. This value (referred to as ‘‘control’’) was subtracted from all subsequent changes in firing rate and the results were expressed as ‘‘% change of control’’. If the average change of discharge rate during the entire response time was larger than 640%, the neuron was considered as being sensitive to the applied substance. In addition, we analyzed the discharge pattern of the spontaneous activity in pre-drug conditions, during and after drug effects (at a time of 120 s). We determined the number of spikes in this period (expressed in the discharge rate, imp. / s), and analyzed whether the spikes singularly occurred or in groups. Sequences of spikes with an interspike interval # 4 ms were regarded as bursts as recommended in the literature for thalamic units [17,18]. Similar to our previous investigation [19] several parameters were determined: the absolute number of bursts / 120 s, the percentage of spikes involved in bursts, and the number of spikes in a burst. Since not all neurons could be studied in a full experimental program, the numbers of cells involved differ in the Results section. To judge whether there was a statistically significant change for the population studied, the Wilcoxon paired rank sum test was applied. Differences were considered to be statistically significant at p , 0.05 (two-tailed). The blocking action of the antagonists was determined by comparing the differences between discharge rates obtained during various recording conditions. At the end of recording, a small amount of Trypan blue was iontophoretically deposited in the brain by passing a 10 mA negative current through the recording electrode for approximately 10 min. The rat was killed with an overdose of urethane, decapitated and the brain fixed with 10% formaldehyde. Frontal frozen sections were stained with nuclear red. The location of blue spots within the hippocampus was determined.
3. Results A total of eighty two histologically verified hippocampal neurons were recorded. Sixty one of them were located in the CA3 region and twenty one in the gyrus dentate. Ang II iontophoretically administered mainly induced an increase of the discharge rate in hippocampal neurons ( p , 0.0035, Wilcoxon test, N 5 72). An example is shown in Fig. 1 Fig. 2. An increase in the firing frequency by more than 40% was observed in twenty one out of the seventy two neurons tested (29%). In eight out of the seventy two neurons (11%) an Ang II-induced decrease by more than 40% of
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Fig. 3. Effects of angiotensin II (Ang II) on the mean (6SEM) of firing frequency of hippocampal neurons. No effect: neurons whose firing frequency was not influenced by Ang II and was changed less than 40%, respectively; excitation: increases of discharge rates by more than 40%; inhibition: decreases of discharge rates by more than 40%.
Fig. 1. Excitatory effects of angiotensin IV (AIV; figure A) and angiotensin II (AII; figure B) on spontaneous activity of a hippocampal neuron. The y-axes indicate the number of spikes per second, and the x-axes the time in seconds (bin width: 5 s). The bars represent time and duration of the ejection of angiotensins, the numbers the current in nA.
led to a reduced responsiveness of the neurons to the second Ang II-ejection. The effect of Ang IV could be tested in forty three neurons. Ang IV also predominantly caused an increase in the hippocampal activity ( p , 0.002, N 5 43; Figs. 4 and 5). The firing frequency was increased by more than 40% in sixteen out of forty three neurons (37%). Ang IV mainly increased the frequency of slowly discharging neurons, too, as did Ang II. The mean duration of the Ang IV-
Fig. 2. Excitatory effects of angiotensin II (Ang II) on spontaneous activity of a hippocampal neuron. Losartan (LOS) did block the Ang II-induced response (A), whereas divalanal was not able to modify the Ang II-induced excitation (B). Sodium chloride (NaCl, pH 8.0) applied with the same current as that used for the co-administration of losartan and Ang II did not change the neuronal activity. The ejections of antagonists preceded the onset of the ejection of angiotensin II and lasted longer.
Fig. 4. Angiotensin IV (Ang IV) induced a decrease in the discharge rate (A) which could be blocked by divalanal (DIV, B). Twenty five min later losartan (LOS) did not antagonize the Ang IV-caused inhibition of activity. Neither sodium chloride (NaCl, pH 4.5) nor angiotensin II (Ang II) did change the discharge rate.
the discharge rate was found. Excitatory effects of Ang II were mainly induced in slowly discharging units, whereas inhibitory effects were more frequently obtained in neurons with higher spontaneous discharge rates (Fig. 3). The effects induced by Ang II lasted frequently several minutes (mean: 550682 s). In a few cases long-lasting effects in the range of half an hour occurred (see Fig. 1). After a recovery period of at least 20 min the effect could be repeated. Shorter time intervals between Ang II-ejections
Fig. 5. The effect of angiotensin IV (Ang IV) on the hippocampal discharge rate and on the discharge pattern (bursts) could be blocked by the AT4-antagonist (DIV).
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induced effects amounted to 438662 s. A decrease in the hippocampal activity was only found in three neurons (7%, Fig. 4). A cross-desensitization between Ang II and Ang IV was never observed. Seven neurons responded to both angiotensins (N 5 34), five of them with an increase of the discharge frequency induced by Ang II as well as by Ang IV. Burst discharges spontaneously occurred in twenty five of the eighty two (30%) hippocampal neurons. The percentage of spikes involved in bursts was 10% to 20%. Ang II produced an increase of the percentage of spikes which were involved in bursts ( p , 0.01) as well as an increase of the absolute number of bursts (p , 0.02), while Ang IV only caused a significant augmentation of the absolute number of bursts ( p , 0.005, Fig. 5). The number of spikes within a burst was not significantly changed, neither during the Ang II ejection nor during the Ang IV ejection. The bursts consisted of 2 or 3 spikes. As shown in Fig. 2, the Ang II-induced change in the discharge rate could be blocked by the AT1-antagonist. When co-administered with Ang II, losartan was found to antagonize the Ang II action in eight out of ten neurons tested. Divalanal-Ang IV was not able to block the Ang II-induced effects (N 5 2). PD 123,319 effectively blocked the Ang II-induced effects only in one neuron. Except in two neurons angiotensin antagonists given alone did not influence the discharge rate of hippocampal neurons. To be sure that the Ang II-receptors are not desensitized by the preceding Ang II-ejection, the testing of angiotensin antagonists was performed not earlier than 20–40 min after the first administration of Ang II. Therefore, the blocking potency of the antagonists could only be tested in a small sample of neurons. The specific AT4-antagonist was administered in nine neurons, in six of them it blocked the Ang IV-induced increase of the discharge rate (Figs. 4 and 5). Losartan was ineffective to antagonize the Ang IVinduced effects (N 5 2).
4. Discussion The results showed that Ang II as well as Ang IV iontophoretically ejected caused an increase of the firing frequency in hippocampal neurons. Besides this exciting effect on the discharge rate, the discharge pattern was also influenced by angiotensins in some neurons. Both angiotensins produced an increase of burst discharges. Comparing the overall responsiveness to both the angiotensins, no significant differences between Ang II and Ang IV were obtained. Different excitatory effects induced by Ang IV and Ang II at the same neuron could be a consequence of a different distribution of specific receptors at this neuron or of a different transport number of both peptides [20]. Other investigations performed under in vivo as well as under in vitro conditions also mainly observed an increase of the discharge rate of hippocampal neurons induced by the
action of Ang II [21–23]. Inhibitory effects occurred in a similar frequency as in our investigation [21]. As far as we know the effects of Ang IV on the hippocampal neuronal activity have not been investigated until now. The Ang II-induced increase of the discharge rate is most probably mediated by the angiotensin AT1 receptor. AT1 receptors are coupled to numerous types of ion channels, including Ca 21 , Cl - , and nonselective cationic ion channels [24–28]. The resulting depolarization induced by these different mechanisms may be involved in determining the level of cellular activation. These results show that at least AT1mediated effects of Ang II should cause a depolarization and therefore an increase in the discharge rate. This suggestion is supported by the density of the different angiotensin receptor subtypes as well as by our results with the Ang II antagonists. However, concerning the angiotensin-induced inhibitory effects the involvement of interneurons in the mediation of angiotensin-induced effects has to be considered. Concerning the effects of Ang IV on hippocampal activity, the signal transducing mechanisms induced by stimulation of the AT4-receptor are unclear until now. In contrast to the frequent appearance of burst discharges in the thalamus [17,18,29], we observed bursts only in few hippocampal neurons. Moreover, the percentage of spikes involved in bursts is lower in the hippocampus than in the thalamus [30]. In the hippocampus high threshold as well as low threshold calcium channels have been found [31,32]. As at least the stimulation of AT1 receptors could increase the calcium current, an influence on the appearance of burst discharges could be expected. Although Ang IV seems to have some affinity to the AT1 receptor [14], losartan did neither modify the angiotensin IV-induced response in this investigation nor in the thalamus (unpublished observations). Similarly, divalanal-Ang IV was not able to block Ang II-induced responses, although a low affinity of Ang II for the AT4 receptor has been found [4]. Ang II has long been shown to downregulate its own receptor. Moreover, the recovery of the surface receptor after removal of the angiotensin agonist occurs with a half-life of 15 min [33]. We used angiotensin concentrations which were 10 times lower than usually administered in vivo experiments. Nevertheless, the repeated ejection of Ang II within short time intervals caused a reduction or a loss of the neuronal response, although the release rate should be lower than 17,9 fmol / min / nA in our experiments. This release rate was determined for 1 mM solutions of Ang II (pH of 4.5) ejected from 5-barrel glass micropipettes with tip diameters of 4 mm [34]. In conclusion, the absence of a cross-desensitization between Ang II and Ang IV and the presumed co-localization of different angiotensin receptor subtypes on the same neuron support the hypothesis that both angiotensins, Ang II and Ang IV, influence the unit activity of the hippocampus. But in contrast to the different influence of Ang II and
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Ang IV on mechanisms of learning and memory shown in some behavioral studies, in this electrophysiological study both angiotensins had a similar exciting influence on the hippocampal neurons.
[14]
[15]
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (INK-21 / B7; Al 342 / 5-3). We wish to thank Dr. R.D. Smith (Dupont Merck, Pharmaceutical Company, Wilmington, Delaware, USA) for providing losartan and Dr. John W. Wright (Washington State University, USA) for support in providing divalanal-Ang IV from the Pacific Northwest Biotechnology. The authors thank Mrs. Ursula Seider for her excellent technical assistance.
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Excitatory action of angiotensins II and IV on hippocampal neuronal activity in urethane anesthetized rats ¨ Doris Albrecht*, Michael Broser, Hagen Kruger Institute of Physiology, Faculty of Medicine ( Charite´ ), Humboldt University, Tucholskystr. 2, D-10117 Berlin, Germany Received 15 November 1996; received in revised form 10 March 1997; accepted 11 March 1997
Abstract The renin–angiotensin system of the mammalian brain seems to interfere with the process of cognition and has been associated with the hippocampal function in relation to mechanisms of learning and memory. In our investigation, the effects of angiotensin II (Ang II) and angiotensin IV (Ang II) on neuronal activity have been studied in the hippocampus of adult rats anesthetized with urethane. Excitatory effects of both angiotensins predominated over inhibitory effects. Angiotensins also induced an enhancement of burst discharges. These angiotensin-induced effects were blocked by the specific angiotensin antagonists. Our findings showed that the different effects of Ang II and Ang IV in behavioral studies are not similarly reflected in a different change of the discharge rate and / or pattern of hippocampal neurons after microiontophoretic administration of both substances. 1997 Elsevier Science B.V. Keywords: Hippocampus; Angiotensin II; Angiotensin IV; Single unit activity; Iontophoresis
1. Introduction A renin–angiotensin system is known to be present in the mammalian brain, complete with the precursors and enzymes required for the formation and metabolism of the biologically active forms of angiotensin as angiotensin II and IV (Ang IV) [1–4]. Ang II acts on two different angiotensin receptors, the AT1 and the AT2 receptor, respectively, while Ang IV (Ang II 3–8) selectively binds to the AT4-receptor site. In addition to the classic cardiovascular functions of angiotensins several reports indicate that Ang II and / or Ang IV may participate in mechanisms of learning and memory and interfere with cognitive function. Behavioral investigations have shown that losartan, a highly specific AT1-receptor antagonist, possessed cognitive enhancing and anxiolytic-like potential, whilst PD 123177, a specific AT2-antagonist, lacks any anxiolytic effect but showed a cognitive enhancing *Corresponding author. Tel.: 1 49 30 28026153; fax: 1 49 30 28026669; e-mail: [email protected] 0167-0115 / 97 / $17.00 PII S0167-0115( 97 )00015-3
potential [5]. Studies investigating the prominent role of the hippocampus in memory acquisition and retention revealed that Ang II administered directly to the dentate gyrus impaired the retention of an inhibitory shock avoidance response [6]. This effect was blocked by the selective AT1 antagonist losartan. Furthermore, an activation of the AT1 receptor via Ang II inhibit the induction of long term potentiation in medial perforant path-stimulated dentate granule cells, if Ang II is injected into the hippocampus [7,8]. In contrast, Ang IV may contribute to an enhancement of memory retrieval in a passive avoidance conditioned response in rats. In addition to enhancing memory retrieval, Wright et al. [9] have observed a facilitation of retention of passive avoidance training with Ang IV on days 2 and 3 after conditioning. Several other studies [10,11] provide support of these findings. The different angiotensin receptors in the rat hippocampus [12,13] vary in their distribution pattern. In this area a high density of AT4 receptors have been found, whereas the AT1 and especially the AT2 receptor occur with lower density [14]. Recently it has been shown that AT1 B as well
106
D. Albrecht et al. / Regulatory Peptides 70 (1997) 105 – 109
as AT1A receptor mRNA is present in the hippocampus [15]. However, analyses investigating the effects of Ang II and Ang IV on hippocampal neurons are lacking. Therefore, we were interested in comparing the influences of iontophoretically administered Ang II and Ang IV on the discharge rate of hippocampal neurons.
2. Materials and methods All procedures were carried out according to the standards of animal welfare and approved by the regional Berlin animal ethics committee. The experiments were performed on male adult albino rats. The rats were kept four to a cage with food and water available ad libitum and on a diurnal light–dark cycle. All testing took place during daylight hours. The rats were anesthetized with urethane (1.2 g / kg, i.p.) and placed in a stereotaxic instrument. Subsequent injections of urethane were administered as needed (for detail see [16]). Rectal temperature was maintained at 37–388C by a heating pad. The electrocardiogram and the cortical EEG were monitored. A small hole was drilled into the skull at a site 3.5 mm lateral to the midline suture and 5.0 mm anterior to the lambdoid suture. An electrode was lowered 2.5 to 4 mm with a microdrive through the hole to the level of the hippocampus. Glass microelectrodes for extracellular recording were filled with saturated Trypan blue solution (tip resistance 10–30 MV). The recorded action potentials were amplified and displayed on an oscilloscope and were, after passing a window discriminator (World Precision Instruments, USA), analyzed with custom-made software (spike 2 from Cambridge Electronic Design, UK) running on a personal computer. Standardized pulses corresponding to individual action potentials were used for computing frequency time histograms which were displayed on-line during sampling. Data were stored on disc for subsequent analysis. Either five or seven barrel glass micropipettes (tip diameter 5–7 mm) were glued to the recording electrode so that their tips were separated vertically by 20–40 mm. The following drugs were used: angiotensin II (Ang II; 100 mM, pH 4.5; Research Biochemicals International (RBI) or synthesized by Dr. P. Henklein, Institute of Biochemistry, ´ and angiotensin IV (Ang IV; 100 mM, pH 4.5; Charite) synthesized by Dr. P. Henklein, Institute of Biochemistry), losartan (AT1-antagonist; 100 mM, pH 8.0; a gift of Dr. R.D. Smith [Dupont Merck, Pharmaceutical Company, Wilmington, Delaware, USA]), PD 123,319 ditrifluoroacetate (AT2-antagonist; 100 mM, pH 4.5; RBI) and divalanal-Ang IV (AT4-antagonist; 100 mM, pH 4.5; Pacific Northwest Biotechnology, Pullman, USA). Retaining currents (4–10 nA) were applied to the pipettes between drug ejections. In a number of experiments a barrel filled with sodium chloride (165 mM, pH 4.5 or pH 8) was used for
current balance. In control experiments no significant contribution from current or pH was detected. The discharge rates were analyzed off-line for periods prior to, during and after termination of drug administration. From the continuously recorded rate meter counts, the average discharge rate of each neuron was evaluated for 120 s prior to the iontophoresis. This value (referred to as ‘‘control’’) was subtracted from all subsequent changes in firing rate and the results were expressed as ‘‘% change of control’’. If the average change of discharge rate during the entire response time was larger than 640%, the neuron was considered as being sensitive to the applied substance. In addition, we analyzed the discharge pattern of the spontaneous activity in pre-drug conditions, during and after drug effects (at a time of 120 s). We determined the number of spikes in this period (expressed in the discharge rate, imp. / s), and analyzed whether the spikes singularly occurred or in groups. Sequences of spikes with an interspike interval # 4 ms were regarded as bursts as recommended in the literature for thalamic units [17,18]. Similar to our previous investigation [19] several parameters were determined: the absolute number of bursts / 120 s, the percentage of spikes involved in bursts, and the number of spikes in a burst. Since not all neurons could be studied in a full experimental program, the numbers of cells involved differ in the Results section. To judge whether there was a statistically significant change for the population studied, the Wilcoxon paired rank sum test was applied. Differences were considered to be statistically significant at p , 0.05 (two-tailed). The blocking action of the antagonists was determined by comparing the differences between discharge rates obtained during various recording conditions. At the end of recording, a small amount of Trypan blue was iontophoretically deposited in the brain by passing a 10 mA negative current through the recording electrode for approximately 10 min. The rat was killed with an overdose of urethane, decapitated and the brain fixed with 10% formaldehyde. Frontal frozen sections were stained with nuclear red. The location of blue spots within the hippocampus was determined.
3. Results A total of eighty two histologically verified hippocampal neurons were recorded. Sixty one of them were located in the CA3 region and twenty one in the gyrus dentate. Ang II iontophoretically administered mainly induced an increase of the discharge rate in hippocampal neurons ( p , 0.0035, Wilcoxon test, N 5 72). An example is shown in Fig. 1 Fig. 2. An increase in the firing frequency by more than 40% was observed in twenty one out of the seventy two neurons tested (29%). In eight out of the seventy two neurons (11%) an Ang II-induced decrease by more than 40% of
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Fig. 3. Effects of angiotensin II (Ang II) on the mean (6SEM) of firing frequency of hippocampal neurons. No effect: neurons whose firing frequency was not influenced by Ang II and was changed less than 40%, respectively; excitation: increases of discharge rates by more than 40%; inhibition: decreases of discharge rates by more than 40%.
Fig. 1. Excitatory effects of angiotensin IV (AIV; figure A) and angiotensin II (AII; figure B) on spontaneous activity of a hippocampal neuron. The y-axes indicate the number of spikes per second, and the x-axes the time in seconds (bin width: 5 s). The bars represent time and duration of the ejection of angiotensins, the numbers the current in nA.
led to a reduced responsiveness of the neurons to the second Ang II-ejection. The effect of Ang IV could be tested in forty three neurons. Ang IV also predominantly caused an increase in the hippocampal activity ( p , 0.002, N 5 43; Figs. 4 and 5). The firing frequency was increased by more than 40% in sixteen out of forty three neurons (37%). Ang IV mainly increased the frequency of slowly discharging neurons, too, as did Ang II. The mean duration of the Ang IV-
Fig. 2. Excitatory effects of angiotensin II (Ang II) on spontaneous activity of a hippocampal neuron. Losartan (LOS) did block the Ang II-induced response (A), whereas divalanal was not able to modify the Ang II-induced excitation (B). Sodium chloride (NaCl, pH 8.0) applied with the same current as that used for the co-administration of losartan and Ang II did not change the neuronal activity. The ejections of antagonists preceded the onset of the ejection of angiotensin II and lasted longer.
Fig. 4. Angiotensin IV (Ang IV) induced a decrease in the discharge rate (A) which could be blocked by divalanal (DIV, B). Twenty five min later losartan (LOS) did not antagonize the Ang IV-caused inhibition of activity. Neither sodium chloride (NaCl, pH 4.5) nor angiotensin II (Ang II) did change the discharge rate.
the discharge rate was found. Excitatory effects of Ang II were mainly induced in slowly discharging units, whereas inhibitory effects were more frequently obtained in neurons with higher spontaneous discharge rates (Fig. 3). The effects induced by Ang II lasted frequently several minutes (mean: 550682 s). In a few cases long-lasting effects in the range of half an hour occurred (see Fig. 1). After a recovery period of at least 20 min the effect could be repeated. Shorter time intervals between Ang II-ejections
Fig. 5. The effect of angiotensin IV (Ang IV) on the hippocampal discharge rate and on the discharge pattern (bursts) could be blocked by the AT4-antagonist (DIV).
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induced effects amounted to 438662 s. A decrease in the hippocampal activity was only found in three neurons (7%, Fig. 4). A cross-desensitization between Ang II and Ang IV was never observed. Seven neurons responded to both angiotensins (N 5 34), five of them with an increase of the discharge frequency induced by Ang II as well as by Ang IV. Burst discharges spontaneously occurred in twenty five of the eighty two (30%) hippocampal neurons. The percentage of spikes involved in bursts was 10% to 20%. Ang II produced an increase of the percentage of spikes which were involved in bursts ( p , 0.01) as well as an increase of the absolute number of bursts (p , 0.02), while Ang IV only caused a significant augmentation of the absolute number of bursts ( p , 0.005, Fig. 5). The number of spikes within a burst was not significantly changed, neither during the Ang II ejection nor during the Ang IV ejection. The bursts consisted of 2 or 3 spikes. As shown in Fig. 2, the Ang II-induced change in the discharge rate could be blocked by the AT1-antagonist. When co-administered with Ang II, losartan was found to antagonize the Ang II action in eight out of ten neurons tested. Divalanal-Ang IV was not able to block the Ang II-induced effects (N 5 2). PD 123,319 effectively blocked the Ang II-induced effects only in one neuron. Except in two neurons angiotensin antagonists given alone did not influence the discharge rate of hippocampal neurons. To be sure that the Ang II-receptors are not desensitized by the preceding Ang II-ejection, the testing of angiotensin antagonists was performed not earlier than 20–40 min after the first administration of Ang II. Therefore, the blocking potency of the antagonists could only be tested in a small sample of neurons. The specific AT4-antagonist was administered in nine neurons, in six of them it blocked the Ang IV-induced increase of the discharge rate (Figs. 4 and 5). Losartan was ineffective to antagonize the Ang IVinduced effects (N 5 2).
4. Discussion The results showed that Ang II as well as Ang IV iontophoretically ejected caused an increase of the firing frequency in hippocampal neurons. Besides this exciting effect on the discharge rate, the discharge pattern was also influenced by angiotensins in some neurons. Both angiotensins produced an increase of burst discharges. Comparing the overall responsiveness to both the angiotensins, no significant differences between Ang II and Ang IV were obtained. Different excitatory effects induced by Ang IV and Ang II at the same neuron could be a consequence of a different distribution of specific receptors at this neuron or of a different transport number of both peptides [20]. Other investigations performed under in vivo as well as under in vitro conditions also mainly observed an increase of the discharge rate of hippocampal neurons induced by the
action of Ang II [21–23]. Inhibitory effects occurred in a similar frequency as in our investigation [21]. As far as we know the effects of Ang IV on the hippocampal neuronal activity have not been investigated until now. The Ang II-induced increase of the discharge rate is most probably mediated by the angiotensin AT1 receptor. AT1 receptors are coupled to numerous types of ion channels, including Ca 21 , Cl - , and nonselective cationic ion channels [24–28]. The resulting depolarization induced by these different mechanisms may be involved in determining the level of cellular activation. These results show that at least AT1mediated effects of Ang II should cause a depolarization and therefore an increase in the discharge rate. This suggestion is supported by the density of the different angiotensin receptor subtypes as well as by our results with the Ang II antagonists. However, concerning the angiotensin-induced inhibitory effects the involvement of interneurons in the mediation of angiotensin-induced effects has to be considered. Concerning the effects of Ang IV on hippocampal activity, the signal transducing mechanisms induced by stimulation of the AT4-receptor are unclear until now. In contrast to the frequent appearance of burst discharges in the thalamus [17,18,29], we observed bursts only in few hippocampal neurons. Moreover, the percentage of spikes involved in bursts is lower in the hippocampus than in the thalamus [30]. In the hippocampus high threshold as well as low threshold calcium channels have been found [31,32]. As at least the stimulation of AT1 receptors could increase the calcium current, an influence on the appearance of burst discharges could be expected. Although Ang IV seems to have some affinity to the AT1 receptor [14], losartan did neither modify the angiotensin IV-induced response in this investigation nor in the thalamus (unpublished observations). Similarly, divalanal-Ang IV was not able to block Ang II-induced responses, although a low affinity of Ang II for the AT4 receptor has been found [4]. Ang II has long been shown to downregulate its own receptor. Moreover, the recovery of the surface receptor after removal of the angiotensin agonist occurs with a half-life of 15 min [33]. We used angiotensin concentrations which were 10 times lower than usually administered in vivo experiments. Nevertheless, the repeated ejection of Ang II within short time intervals caused a reduction or a loss of the neuronal response, although the release rate should be lower than 17,9 fmol / min / nA in our experiments. This release rate was determined for 1 mM solutions of Ang II (pH of 4.5) ejected from 5-barrel glass micropipettes with tip diameters of 4 mm [34]. In conclusion, the absence of a cross-desensitization between Ang II and Ang IV and the presumed co-localization of different angiotensin receptor subtypes on the same neuron support the hypothesis that both angiotensins, Ang II and Ang IV, influence the unit activity of the hippocampus. But in contrast to the different influence of Ang II and
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Ang IV on mechanisms of learning and memory shown in some behavioral studies, in this electrophysiological study both angiotensins had a similar exciting influence on the hippocampal neurons.
[14]
[15]
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (INK-21 / B7; Al 342 / 5-3). We wish to thank Dr. R.D. Smith (Dupont Merck, Pharmaceutical Company, Wilmington, Delaware, USA) for providing losartan and Dr. John W. Wright (Washington State University, USA) for support in providing divalanal-Ang IV from the Pacific Northwest Biotechnology. The authors thank Mrs. Ursula Seider for her excellent technical assistance.
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