Angiotensin IV enhances LTP in rat dentate gyrus in vivo

Abstract

Angiotensins have been shown to play a significant role in a variety of physiological functions including learning and memory processes. Relatively recent evidence supports the increasing importance of angiotensin IV (Ang IV), in many of these functions previously associated only with Ang II, including learning and memory. An interesting hypothesis generated by these results has been that Ang II is a precursor for the production of a more active peptide fragment, Ang IV. Since Ang II impairs learning and memory, when administered directly or released into the hippocampal dentate gyrus, and inhibits long term potentiation (LTP) in medial perforant path-dentate granule cell synapses, as well; it remained to be seen what effects Ang IV had on LTP in these same synapses. Results of this study show clearly that Ang IV significantly enhances LTP, and the enhancement is both dose and time dependent. The following solutions of Ang IV were administered over a five min period, at the end of baseline and before the first tetanus was applied: 2.39, 4.78, and 9.56 nM. An inverted U-type dose related effect was observed. A complex time related effect was observed with a maximum at 5 min, a return to normal LTP at 30 min and a minimum below normal at 90 min, and a return to normal LTP at 120 min. The effects of the 4.78 nM solution were determined at the following intervals between administration and the first tetanus: 5, 15, 30, 60, 90, and 120 min. The enhancement of LTP can be prevented by pretreatment with Divalinal, an Ang IV antagonist, without any effect on normal LTP. Two solutions of Divalinal were used; 5 nM and 5 μM, and the 5 μM was more effective and completely blocked the enhancement of normal LTP. Results were also obtained with 4.78 nM Nle¹-Ang IV (Norleucine), an Ang IV agonist. Norleucine was less effective than Ang IV in the enhancement of normal LTP and displayed a similar time course of activity. Both Ang IV and Norleucine produced a significant suppression of normal LTP at 90 min; that remains to be explained. However, the inhibition by Ang IV was dose dependent and was blocked by Divalinal. The fact that the Ang IV enhancement of normal LTP was blocked by losartan, an Ang II AT₁ receptor antagonist, is puzzling since Divalinal had no effect on the inhibition of LTP by Ang II.

Introduction

In 1991 we reported that angiotensin II (Ang II) when administered to the hippocampus inhibited long term potentiation (LTP) induction in medial perforant path-dentate granule cell synapses [6] and that the inhibition is mediated by the AT₁ receptor because it can be blocked by pretreatment with losartan, an AT₁ antagonist [2], [29]. Inhibition of LTP induction by Ang II is time dependent and begins slowly over one hour, when administered directly to the hippocampus. The inhibition is complete and continues for another 30 min and then fully recovers by the end of the next 30 min [30]. Also, Ang III administered in the same way in the rat is not as effective as Ang II and 40–50 times as much Ang III is required to inhibit granule cell LTP induction [6]. We have also shown that ethanol and diazepam inhibit dentate granule cell LTP and that the inhibition can be blocked by pretreatment with losartan [28]. Ang II, ethanol, and diazepam all impair learning and the memory process; and, in the rat, the impairments can be prevented or reduced, dependent upon dose, by pretreating the animals with losartan [13], [21], [22], [23].

Some of these results might have been expected because brain angiotensins had been shown to play a significant role in a variety of physiological functions involving Ang II, Ang III, and shorter fragments [31] and their receptor subtypes [3], [32]. Most of these functions are involved in cardiovascular and body fluid regulation and to an increasing extent in cognitive processes. Results of an early study in 1984 [37] showed that captopril, an angiotensin converting enzyme inhibitor, had the unexpected ability to elevate mood and reduce depression. Since then a considerable amount of research on possible cognitive effects of angiotensin synthesis inhibitors and specific receptor antagonists has been carried out [15], [29], [32] on both humans and animals. In general, increased angiotensin brain activity tends to be closely associated with impaired cognitive processes, particularly in dementias characteristic of aging and Alzheimer’s disease. Most of these data have been obtained on humans.

Results of behavioral studies on rats have been difficult to interpret because of methodological differences between investigations. Major differences can occur particularly between intracerebroventricular (ICV) application of Ang II and Ang II injected directly into the brain parenchyma. For example, when Ang II is injected directly into the dorsal neostriatum [18], retention of a step-down shock avoidance response was impaired; whereas, retrieval in a similar passive avoidance conditioning task was improved following ICV administration of Ang II [5]. More recently, we found that Ang II administered to the hippocampus impaired retention of a single trial step through shock avoidance response [13]. Previously, it had been shown that renin applied directly into the lateral brain ventricles, not only increased cerebrospinal fluid Ang II significantly; but disrupted passive avoidance learning and the impairment was blocked by pretreatment with SQ 14225, an angiotensin converting enzyme inhibitor [11]. Some of these behavioral effects of the angiotensins might be interpreted in terms of the reports of relatively high concentrations of angiotensinogen [14] and Ang II [20] found in the striatum and hippocampus; and the low to moderate binding levels of Ang II observed in the striatum and hippocampus [8], [9], [16]. These results have been difficult to reconcile with more recent data in support of Ang II as a precursor molecule to Ang III, Ang II [1], [2], [3], [4], [5], [6], [7], and Ang II [3], [4], [5], [6], [7], [8] (Ang IV) and the fact that Ang IV receptor density in the hippocampus is very high in comparison to the distribution of Ang II receptors [33]. However, a recent study utilizing fluorescein-coupled Ang II demonstrated widespread high densities of stained cells throughout the hippocampus and several limbic structures [25]. The highest cell densities were observed in the piriform cortex and dentate gyrus.

Early work on a comparison of Ang II and Ang III revealed electrophysiological differences, in rats of neuronal firing and peptide stimulation thresholds, indicating that conversion of Ang II to Ang III had to occur for receptor activation. These data provide additional support for the role of Ang II as a precursor in the production of more active angiotensin fragments. Very few electrophysiological studies have been done on Ang IV. Recently a comparison of Ang II and Ang IV on single neuronal activity recorded in hippocampal dentate gyrus and CA3 [1] in vitro showed similar predominantly excitatory effects. These similar effects of Ang II and Ang IV on CA3 and dentate granule cells are in contrast to what would be expected in terms of the opposite effects observed with these two peptides on avoidance conditioning and retention. In the lateral nucleus of the amygdala, in brain slices, Ang II increased field potential amplitude; whereas Nle¹-Ang IV, an Ang IV analog, decreased amplitudes of field potentials [24]. Recently, some preliminary data indicated that Ang IV enhances LTP induction in medial perforant path-dentate granule cell synapses and the enhancement was blocked by Divalinal-Ang IV (Divalinal), an AT₄ receptor antagonist in vivo [10]. These results were confirmed in hippocampal slices using the Ang IV analog, Nle¹-Ang IV [12]. Facilitated hippocampal LTP by Ang IV would provide a basis at the cellular level for expecting enhanced performance in learning and memory tasks by Ang IV.

Intracerebral administration of Ang IV enhances retention of passive avoidance and acquisition of conditioned avoidance [4] and memory retrieval [34]. Deficits in water maze and conditioned avoidance performance produced by either transections of the perforant path or ibotenic acid lesions in CA1 are also significantly reduced by ICV administration of the Ang IV analog, Nle¹-Ang IV (L.A. Stubley, J.W. Harding, and J.W. Wright, unpublished observations). Chronic ICV infusion of Nle¹-Ang IV facilitates spatial learning and Divalinal, an Ang IV antagonist, impaired acquisition of successful learning strategies, as did knife cuts of the perforant paths [35]. These performance deficits due to Divalinal and knife cuts could be reversed by further treatment with Norleucinal, another Ang IV agonist. Since Ang II and Ang IV when administered ICV potentiate conditioned avoidance learning and later retrieval of memory; whereas, Ang II when administered to the dorsal neostriatum and hippocampus impair the retention of conditioned avoidance responses; it seemed reasonable to carry out a thorough examination of the effects of Ang IV on hippocampal LTP, when administered directly into the hippocampus in vivo. Because Ang IV has a short half life we modified the procedure we have been using to deliver Ang II to the hippocampus, in order to assure a more direct application to the granule cell dendrites, by reducing the distance between the recording glass capillary electrode tip and the delivery of the peptide.

The general purpose of the present study was to examine the effects of Ang IV and the analog Nle¹-Ang IV applied directly into the hippocampal dentate gyrus on LTP induction in medial perforant path-dentate granule cell synapses. Specifically to determine (a) a dose and time related effect of Ang IV (b) a dose and time related effect of Nle¹-Ang IV and (c) the effectiveness of Divalinal, an antagonist of Ang IV, in blocking these effects. Results show that both Ang IV and Nle¹-Ang IV are similar in producing an immediate short term but significant enhancement of LTP that can be blocked effectively by Divalinal.