Contributions of the brain angiotensin IV-AT4 receptor subtype system to spatial learning

Abstract

The development of navigational strategies to solve spatial problems appears to be dependent on an intact hippocampal formation. The circular water maze task requires the animal to use extramaze spatial cues to locate a pedestal positioned just below the surface of the water. Presently, we investigated the role of a recently discovered brain angiotensin receptor subtype (AT4) in the acquisition of this spatial learning task. The AT4 receptor subtype is activated by angiotensin IV (AngIV) rather than angiotensins II or III, as documented for the AT1 and AT2 receptor subtypes, and is heavily distributed in the CA1-CA3 fields of the hippocampus. Chronic intracerebroventricular infusion of a newly synthesized AT4 agonist (Norleucine1-AngIV) via osmotic pump facilitated the rate of acquisition to solve this task, whereas treatment with an AT4 receptor antagonist (Divalinal) significantly interfered with the acquisition of successful search strategies. Animals prepared with bilateral knife cuts of the perforant path, a major afferent hippocampal fiber bundle originating in the entorhinal cortex, displayed deficits in solving this task. This performance deficit could be reversed with acute intracerebroventricular infusion of a second AT4 receptor agonist (Norleucinal). These results suggest that the brain AngIV-AT4 system plays a role in the formation of spatial search strategies and memories. Further, application of an AT4 receptor agonist compensated for spatial memory deficits in performance accompanying perforant path knife cuts. Possible mechanisms underlying this compensatory effect are discussed.

Fig. 1.

Representative photomicrographs of perforant path knife cuts (A) and control knife cuts to the neocortex (B) from animals treated with Norleucinal. Horizontal sections of the right hemisphere were taken at the level of 4.1 mm ventral to bregma according to Paxinos and Watson (1986). These knife cuts transsected the perforant pathway at the anteroposterior level of the dorsal hippocampus commissure (dhc), forceps major of the corpus collosum (fmj), and subiculum (Sub). These cuts isolated the entorhinal cortex (Ent) from the dentate gyrus (DG) and other anterior brain structures, such as the CA₁–CA₃ fields. Scale bar, 1 mm.

Fig. 2.

Mean ± SEM group changes in latencies (A) and swim distances (B) to locate the submerged pedestal in a circular water maze task during 6 acquisition days with osmotic pumps in place, followed by 6 additional acquisition days with the pumps removed. All treatments were intracerebroventricularly delivered via osmotic pump at the indicated doses in a volume of 1 μl aCSF/hr. Those animals treated with 0.5 nmol of Nle¹-AngIV performed better during days 1 and 2 of acquisition with respect to latency to find the pedestal (p < 0.005) and swim distance (p < 0.05) than members of the control group (0.0 nmol of Nle¹-AngIV) or those rats infused with AngII(4–8) (Penta). Pentapeptide binds at the AT₄ receptor with low affinity. These groups did not differ during days 3–6 of acquisition. After pump removal, the location of the submerged pedestal was shifted to the opposite quadrant for each animal. Although those animals treated with pentapeptide revealed longer swim distances to find the pedestal on days 9–11, by days 12 and 13, the groups did not differ. Each group consisted of six rats surgically prepared with a 7 d osmotic pump and were given 1 d to recover before the initiation of acquisition trials. Five trials were administered per day with entry points randomly assigned (N, S, E, W), although the location of the submerged pedestal was fixed for each rat.

Fig. 3.

Representative examples of search patterns in the circular water maze during a 2 min trial by one member of the group treated intracerebroventricularly with 0.5 nmol/hr Nle¹-AngIV during days 1 (A) and 6 (B) of acquisition training and one member of the group treated with 0.5 nmol/hr pentapeptide during days 1 (C) and 6 (D) of acquisition. Latency (Lat) in seconds to find the submerged pedestal and distance swam (Dist) in meters are indicated for each animal. Those rats treated with Nle¹-AngIV displayed a superior search strategy compared with animals treated with AngII(4–8) (Pentapeptide) on day 1 of acquisition, as evidenced by significantly shorter latencies (p < 0.05) and swim distances (p < 0.05) to find the submerged pedestal. By day 6, all animals had acquired efficient search strategies and did not differ. Each group consisted of six rats prepared with 7 d osmotic pumps that infused at a rate of 1 μl/hr aCSF.

Fig. 4.

Mean ± SEM group changes in latency (A) and swim distance (B) to find the submerged pedestal in a circular water maze task during 6 d of acquisition training with osmotic pumps in place and 6 additional acquisition days with the pumps removed. All treatments were intracerebroventricularly delivered via osmotic pump at the indicated doses in a volume of 1 μl/hr aCSF. Those animals treated with 5.0 and 0.5 nmol/hr Divalinal (Dival) revealed significant deficits in performance compared with members of the control group (0 nmol Dival) and nontreated controls on days 4–6 (p < 0.05). After pump removal, the location of the submerged pedestal was shifted to the opposite quadrant for each animal. By day 13 of training, there were no differences in latencies to find the pedestal among the groups; however, those rats that had been treated with 5.0 nmol of Divalinal continued to reveal significantly longer swim distances to find the pedestal than members of the other three groups (p < 0.05). Each group consisted of six rats surgically prepared with 7 d osmotic pumps and were provided 1 d of recovery before initiation of acquisition training.

Fig. 5.

Representative examples of search patterns in the circular water maze by one member of the group treated with 5.0 (A) and one treated with 0.5 (B) nmol/hr Divalinal during day 6 of acquisition; also, one member from the control group (C) infused with aCSF (0 nmol Divalinal) and a member of the nontreated control group (D). All treatments were intracerebroventricularly delivered via osmotic pump in a volume of 1 μl/hr aCSF. Those animals treated with Divalinal performed poorly compared with members of the groups infused with aCSF or nontreated controls. Specifically, the search pattern strategies of the Divalinal-treated rats were not as sophisticated as the control animals and often included positive thigmotaxis (persistent swimming near the walls of the maze), as evidenced by the animal from the group treated with 5.0 nmol/hr Divalinal (A). Each group consisted of six rats prepared with 7 d osmotic pumps that infused at a rate of 1 μl/hr aCSF. Members of the fourth group served as nontreated controls.

Fig. 6.

Mean ± SEM group changes and time spent within the correct (target) quadrant (A) and the number of entries into the quadrant (B) during one probe trial conducted at the conclusion of training trials on day 6 of acquisition for animals continuously intracerebroventricularly treated with 5.0, 0.5, or 0 nmol/hr Divalinal via osmotic pump for 6 d and nontreated controls. Those animals treated with 5.0 and 0.5 nmol/hr doses of Divalinal indicated significantly less time spent within the target quadrant compared with those rats that received intracerebroventricular infusion of aCSF or the nontreated control animals (p < 0.05). The groups did not differ with respect to number of entries into the target quadrant.

Fig. 7.

Mean ± SEM group changes in latencies (A) and swim distances (B) to find the submerged pedestal in the circular water maze tasks during 8 d of acquisition training by two groups of animals surgically prepared with bilateral PP knife cuts and intracerebroventricular guide cannulas and treated intracerebroventricularly with bolus injections, 2 μl of aCSF or 1.0 nmol of Norleucinal in 2 μl of aCSF, 5 min before the initiation of training trials on each day of acquisition. Two additional groups of animals served as controls and received bilateral knife cuts to the neocortex immediately superior to the PP and were also treated with aCSF or Norleucinal 5 min before training trials each day. Those animals prepared with PP knife cuts and treated with Norleucinal displayed an acquisition curve not different from the control groups prepared with neocortex knife cuts and treated with Norleucinal or aCSF. In contrast, those rats that received PP knife cuts and were infused with aCSF displayed significant impairment in acquisition of the spatial memory task with respect to latencies (p < 0.001) and swim distances (p < 0.001) to find the submerged pedestal. These differences became evident by day 3 of acquisition training and persisted during subsequent days. Each group consisted of eight rats.

Fig. 8.

Representative examples of search patterns in the circular water maze by two members of the group prepared with bilateral PP knife cuts. One animal received intracerebroventricular administration of aCSF (A), and one member was treated with Norleucinal (B). For comparison purposes, two rats from the group prepared with bilateral neocortex knife cuts are also presented. One of these animals was from the group that was administered aCSF (C) and one that received Norleucinal (D). The animal that was prepared with PP knife cuts and was subsequently treated with Norleucinal displayed a search pattern that was equivalent with those by animals prepared with neocortex knife cuts and administered aCSF or Norleucinal. In contrast, those animals prepared with PP knife cuts and administered aCSF displayed significantly impaired search strategies (p < 0.001). Each group consisted of eight rats.