1 De Bundel, D.

Angiotensin IV and LVV-Haemorphin 7 Enhance Spatial

Working Memory in Rats: Effects on Hippocampal Glucose

Levels and Blood Flow.

Dimitri De Bundel1, Ilse Smolders1, Rui Yang1, Anthony L. Albiston2, Yvette

Michotte1, Siew Yeen Chai2,3

1 Research Group Experimental Pharmacology: Department of Pharmaceutical

Chemistry, Drug Analysis and Drug Information, Vrije Universiteit Brussel,

Laarbeeklaan 103, 1090 Brussels, Belgium

2Neuropeptides Group, Howard Florey Institute, 3Centre for Neuroscience, University of

Melbourne, Parkville, Victoria 3010 Australia

Corresponding author:

Siew Yeen Chai

Fax : +61 3 9348 1707

Phone: +61 3 8344 7782

E-mail: sychai@florey.edu.au

2 De Bundel, D.

Abstract

The IRAP ligands Angiotensin IV (Ang IV) and LVV-haemorphin 7 (LVV-H7) enhance

performance in a range of memory paradigms in normal rats and ameliorate memory

deficits in rat models for amnesia. The mechanism by which these peptides facilitate

memory remains to be elucidated. In recent in vitro experiments, we demonstrated that

Ang IV and LVV-H7 potentiate activity-evoked glucose uptake into hippocampal

neurons. This raises the possibility that IRAP ligands may facilitate memory in

hippocampus-dependent tasks through enhancement of hippocampal glucose uptake.

Acute intracerebroventricular (i.c.v.) administration of 1 nmol Ang IV or 0.1 nmol LVV-

H7 in 3 months-old Sprague Dawley rats enhanced spatial working memory in the plus

maze spontaneous alternation task. Extracellular hippocampal glucose levels were

monitored before, during and after behavioral testing using in vivo microdialysis.

Extracellular hippocampal glucose levels decreased significantly to about 70% of baseline

when the animals explored the plus maze, but remained constant when the animals were

placed into a novel control chamber. Ang IV and LVV-H7 did not significantly alter

hippocampal glucose levels compared to control animals in the plus maze or control

chamber. Both peptides had no effect on hippocampal blood flow as determined by laser

Doppler flowmetry, excluding that either peptide increased the hippocampal supply of

glucose. We demonstrated for the first time that Ang IV and LVV-H7 enhance spatial

working memory in the plus maze spontaneous alternation task but no in vivo evidence

was found for enhanced hippocampal glucose uptake or blood flow.

3 De Bundel, D.

Key Words: Angiotensin IV; LVV-haemorphin 7; glucose; memory; hippocampus;

IRAP

4 De Bundel, D.

Introduction

Insulin-regulated aminopeptidase (IRAP) was initially characterized in adipocytes as a

marker protein of specialized vesicles containing the insulin-regulated glucose

transporter-4 (GLUT4) (Keller, Scott, Mastick, Aebersold & Lienhard, 1995; Ross, Scott,

Morris, Leung, Mao, Lienhard & Keller, 1996). IRAP is a membrane bound

aminopeptidase with a large extracellular catalytic domain, a single transmembrane

domain and an extended cytoplasmic domain containing two dileucine motifs preceded by

acidic clusters which are associated with trafficking (Johnson, Lampson & McGraw,

2001; Keller et al., 1995). In adipocytes, IRAP redistributes with GLUT4 to the plasma

membrane in response to insulin. IRAP is also found in other tissues such as the brain

(Keller et al., 1995), where it is predominantly expressed in neurons, with high

concentrations in areas involved in cognitive functions such as the hippocampus, the

amygdala, the basal forebrain and the cerebral cortex (Fernando, Larm, Albiston & Chai,

2005). Intriguingly, in these brain areas, IRAP and GLUT4 are co-localized in secretory

vesicles, similarly to the insulin-regulated vesicles of adipocytes, suggesting that IRAP

may be involved in the regulation of GLUT4 dependent neuronal glucose uptake

(Fernando, Luff, Albiston & Chai, 2007).

The role of IRAP in the brain remained elusive until it was identified as a high affinity

binding site for two endogenous peptides, angiotensin IV (Ang IV) and LVV-haemorphin

7 (LVV-H7) (Albiston, McDowall, Matsacos, Sim, Clune, Mustafa, Lee, Mendelsohn,

Simpson, Connolly & Chai, 2001). Acute or chronic administration of these IRAP ligands

into the cerebral ventricles of rats enhances performance in a range of memory paradigms

including the passive avoidance (Braszko, Kupryszewski, Witczuk & Wisniewski, 1988;

5 De Bundel, D.

Wright, Miller-Wing, Shaffer, Higginson, Wright, Hanesworth & Harding, 1993), Barnes

circular maze (Lee, Albiston, Allen, Mendelsohn, Ping, Barrett, Murphy, Morris,

McDowall & Chai, 2004) and Morris water maze (Wright, Stubley, Pederson, Kramar,

Hanesworth & Harding, 1999) tasks. Moreover, IRAP ligands ameliorate memory deficits

induced by chronic alcohol exposure (Wisniewski, Borawska & Car, 1993), global

ischemia (Wright, Clemens, Panetta, Smalstig, Weatherly, Kramar, Pederson, Mungall &

Harding, 1996), bilateral perforant pathway lesion (Wright et al., 1999), or chemical

perturbations of the septo-hippocampal cholinergic pathways (Albiston, Pederson, Burns,

Purcell, Wright, Harding, Mendelsohn, Weisinger & Chai, 2004; Olson, Olson, Qualls,

Stratton, Harding & Wright, 2004; Pederson, Krishnan, Harding & Wright, 2001).

Whereas several brain areas may be involved in the different memory effects of IRAP

ligands, many studies have focused on the hippocampus because of the robust effects of

these peptides on the hippocampal-dependent spatial learning. The observation that i.c.v.

administration of Ang IV predominantly evokes c-FOS expression in the hippocampus

(Roberts, Krebs, Kramar, Shaffer, Harding & Wright, 1995) indeed suggests that this

brain area is a major target site for the memory effects of IRAP ligands. In support of

their spatial memory enhancing effects, IRAP ligands were demonstrated to facilitate

long-term potentiation in the CA1 region of the hippocampus in vitro (Kramar,

Armstrong, Ikeda, Wayner, Harding & Wright, 2001) and in the dentate gyrus in vivo

(Wayner, Armstrong, Phelix, Wright & Harding, 2001). Furthermore, Ang IV and LVV-

H7 facilitated potassium-evoked acetylcholine release in rat hippocampal slices (Lee,

Chai, Mendelsohn, Morris & Allen, 2001).

6 De Bundel, D.

The ability of IRAP ligands to facilitate memory in both normal animals and a range of

memory deficit models makes IRAP a potential therapeutic target. However, the

mechanism by which IRAP ligands facilitate memory is not fully understood. Several

candidate mechanisms have been proposed including facilitation of neuronal glucose

uptake (Albiston, Mustafa, McDowall, Mendelsohn, Lee & Chai, 2003; De Bundel,

Smolders, Vanderheyden & Michotte, 2008). In recent in vitro experiments, we

demonstrated that Ang IV and LVV-H7 potentiate activity-evoked glucose uptake into

mice hippocampal neurons (Fernando, Albiston & Chai, 2008). This effect was absent in

IRAP knockout mice, strongly suggesting a role for IRAP in hippocampal glucose uptake

(Fernando et al., 2008). IRAP ligands may therefore enhance spatial memory through

potentiation of glucose uptake into hippocampal neurons. Indeed, it is well established

that glucose is a potent modulator of memory. Systemic or intrahippocampal glucose

administration facilitates spatial memory in rats (Dash, Orsi & Moore, 2006; McNay &

Gold, 1999). Furthermore, a previous microdialysis study elegantly demonstrated a

decrease in extracellular glucose levels in the hippocampus of rats performing a plus

maze spontaneous alternation task (McNay, Fries & Gold, 2000). This decrease in

extracellular glucose is indicative of enhanced glucose uptake by hippocampal cells

during spatial memory processing. We hypothesized that under these conditions, Ang IV

and LVV-H7 may facilitate hippocampal glucose uptake similarly to our observations in

vitro.

The present study uses the in vivo microdialysis technique to test this hypothesis. We

investigated the effects of Ang IV and LVV-H7 on hippocampal glucose levels during

performance of a spatial working memory task. Since the hippocampal extracellular

7 De Bundel, D.

glucose concentration not only depends on the local usage but also on the supply from the

blood, we further determined the effects of Ang IV and LVV-H7 on hippocampal blood

flow. Identification of the mechanism by which IRAP ligands facilitate memory may

provide a novel strategy for the treatment of cognitive impairment.

Materials and methods

Subjects

Male Sprague-Dawley rats (Animal Resource Centre, Canning Vale, WA, Australia and

Charles-River Laboratories, L’Arbresle, Cedex, France ), 3 months old at the time of

surgery, were housed individually in an animal housing facility maintained at a 12/12

hours light/dark cycle initiated at 7:00 h. All animals were given free access to water and

standard rat chow. The experiments were carried out in accordance with the Prevention of

Cruelty to Animals Act and the National Health and Medical Research Council Code of

Practice for the Use of Animals for Scientific Purposes in Australia and according to the

European directive 86/609/EEC with the corresponding national guidelines as approved

by the Ethical Committee for Animal Experimentation of the Faculty of Medicine and

Pharmacy of the Vrije Universiteit Brussel.

Surgery

Behavior and microdialysis experiments

Rats were anesthetized with 5% (v/v) isoflurane (Fluothane; ICI, Melbourne, Australia),

placed on a stereotaxic frame and maintained on 2% (v/v). Rectal temperature was

maintained at 37.5°C using a heating pad. The rats were stereotaxically implanted with a

microdialysis guide cannula (CMA/12; CMA Microdialysis, Solna, Sweden) above the

8 De Bundel, D.

left or right hippocampus and an injection guide cannula (22 g; Plastics One, Roanoke,

USA) above the ipsilateral ventricle. The location of both the microdialysis guide cannula

and injection guide cannula were randomized between left and right hemisphere for

different animals within each experimental group. The flat skull coordinates were 4.6 mm

lateral from midline, 5.6 mm posterior from bregma and 3.6 mm ventral from dura for the

microdialysis guide cannula aimed at the hippocampus and 1.5 mm lateral from midline,

0.8 mm posterior to bregma and 3.5 mm ventral from dura for the injection guide cannula

aimed at the lateral ventricle. After stereotaxic positioning, the guide cannulae were

secured to the skull with stainless steel screws and dental cement. Rats received an

intramuscular injection of 0.05 mg/kg buprenorphine (Temgesic; Reckitt Benckiser, West

Ryde, Australia) and a subcutaneous injection of 10 mg/kg enrofloxacin (Baytril; Bayer,

Pymble, Australia). Dummy cannulae were introduced into the guide cannulae to prevent

occlusion. Rats were allowed to recover for a minimum of 6 days and were handled daily

for at least 1 minute during this time. On completion of testing, the rats received an

overdose of Lethobarb (Virbac, Peakhurst, Australia) and the brains were removed for

verification of the guide cannula localizations.

Hippocampal blood flow experiments

Rats were anesthetized with an intraperitoneal injection of 60 mg/kg pentobarbital

(Nembutal; Ceva Sante Animal, Brussels, Belgium) and maintained with an

intraperitoneal infusion of 0.06 mg/kg pentobarbital at a flow rate of 1 µl/min. Tracheal

intubation was performed to facilitate breathing throughout the experiment and the right

jugular vein of some of the rats was cannulated for supplementary drug administration.

The animals were placed on a stereotaxic frame and rectal temperature was maintained at

9 De Bundel, D.

37.5°C using a heating pad. The rats were stereotaxically implanted with an injection

guide cannula (22 g; Plastics One, Roanoke, USA) above the left or right ventricle and a

circular craniotomy with 2 mm diameter was performed above the ipsilateral

hippocampus. The location of both the injection guide cannula and the craniotomy were

randomized between left and right hemisphere for different animals within each

experimental group. The flat skull coordinates were 1.5 mm lateral from midline, 0.8 mm

posterior to bregma and 3.5 mm ventral from dura for the injection guide cannula aimed

at the lateral ventricle. The injection guide cannula was secured to the scull with a screw

and dental cement. The craniotomy above the hippocampus was performed at flat skull

coordinates 4.6 mm lateral from midline and 5.6 mm posterior from bregma. After

careful removal of the dura, a laser Doppler recording probe (0.8 mm diameter; Vasamed,

Eden Prairie, USA) was lowered 3.6 mm ventral from dura to measure blood flow in the

hippocampus. On completion of testing, the rats received an overdose of pentobarbital

and the brains were removed for verification of the guide cannula and laser Doppler

recording probe localizations.

Peptide administration

In all experiments, the peptides were administered into the lateral ventricle with a 10 µl

Hamilton syringe (Scientific Glass Engineering, Ringwood, Australia) via a FEP tubing

(CMA Microdialysis, Sweden), which was connected to the injection needle (30 g;

Plastics One, Roanoke, USA). The injection needle protruded 0.5 mm beyond the tip of

the guide cannula, thus perforating the wall of the lateral ventricle. The injector was left

in the lateral ventricle for 1 min after the injection to avoid reflux. The animals were not

restrained during the i.c.v. injections and showed no signs of stress or discomfort. All

10 De Bundel, D.

peptides were dissolved in artificial cerebrospinal fluid (aCSF; 128 mM NaCl, 3.0 mM

KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 21.0 mM NaHCO3, 1.3 mM NaH2PO4, 1.0 mM D-

glucose, pH 7.0) and injected in a volume of 2 µl at a rate of 2 µl/min. Ang IV and LVV-

H7 were obtained from Auspep (Parkville, Australia). All other chemicals were obtained

from Sigma-Aldrich (Castle-Hill, Australia).

The effects of IRAP ligands on spatial working memory and hippocampal glucose levels

were investigated by placing the rats in a plus maze with four closed arms, 5 min after

receiving an injection of either aCSF (n = 12), 1 nmol Ang IV (n = 7), 10 nmol Ang IV (n

= 6), 0.1 nmol LVV-H7 (n = 7) or 1 nmol LVV-H7 (n = 6). The timing of peptide

administration was based on previous results (Lee et al., 2004). The effects of IRAP

ligands on hippocampal glucose levels under control conditions were tested by placing

the animals in the control chamber, 5 min after receiving an injection of either aCSF, 1

nmol Ang IV, or 0.1 nmol LVV-H7 (n = 6 for each experimental group). The effects of

IRAP ligands on hippocampal bloodflow was monitored in anaesthetized rats after

injection of aCSF, 1 nmol Ang IV, or 0.1 nmol LVV-H7 (n = 4 for each experimental

group).

Behavioral testing

All rats were habituated overnight to the testing room, where a 12/12 hours light/dark

cycle was maintained. The effects of IRAP ligands in spatial working memory and the

associated hippocampal glucose concentration was assessed by placing the rats into a

novel control chamber or a plus maze with closed arms for 20 mins, after injection of

either aCSF, Ang IV or LVV-H7. The plus maze and control chamber were thoroughly

cleansed before each experiment with 70% ethanol. The control chamber was a black

11 De Bundel, D.

Perspex microdialysis bowl with a 40 cm diameter and 40 cm high walls which was

placed on top of a table in the testing room. The plus maze was composed of four closed

arms with each arm measuring 75 x 10 x 20 cm. The floor and the walls of the central

platform and the arms were made of black Perspex. The plus maze was elevated 1 m

above the floor to discourage the rats from leaving the maze. Visual cues were located

around the room, including a door and posters with black on white geometric patterns.

Spontaneous alternation testing was conducted by placing the rats in the centre of the plus

maze while allowing for unimpeded exploration for 20 min. The number and the

sequence of arm entries were recorded for calculation of an alternation score. An

alternation was counted when a rat visited 4 different arms during 5 consecutive arm

entries. Immediate re-entries were not counted. A 4/5 alternation score was calculated by

dividing the number of observed alternations in overlapping quintuplets by the number of

possible alternations and expressed as a percentage.

Microdialysis procedures

Microdialysis probes (CMA/12; CMA Microdialysis, Solna, Sweden), were inserted into

the microdialysis guide cannulae 16-18 hours before the start of the experiment. The

microdialysis probes were 3 mm long with an outer diameter of 0.5 mm and sampled

across the CA1 to CA3 regions of the hippocampal formation. Each animal was used

only once. Probe insertion was timed to give optimal measurement conditions and to

avoid glial scarring at the probe site. The microdialysis probes were perfused via FEP

tubings (1.2 µl/100 mm; CMA Microdialysis, Sweden) with an aCSF at a flow rate of 1.0

µl/min . The aCSF contained 1.0 mM D-glucose to avoid excessive supply or drainage of

glucose from the extracellular fluid (McNay et al., 2000). To establish a dynamic

12 De Bundel, D.

equilibrium between the extracellular fluid and the perfusion liquid, the perfusion

commenced 30 min prior to the experiment. Stable glucose levels in the baseline samples

indicate that this equilibration period was sufficient. Hippocampal dialysate samples

were collected every 5 min for 90 min before during and after behavioral testing.

Dialysate samples were then stored at -20°C for glucose analysis.

The microdialysis tubings were connected to the microdialysis probe 30 min before

collecting baseline samples. A swivel system (Instech, Playmouth, USA) was used to

provide optimal freedom of movement of the rats while avoiding coiling of the

microdialysis tubings. The swivel system was attached to a pivoting arm for

microdialysis sampling in the home cage or the control chamber and was supported by

hand using a clear Perspex rod for microdialysis sampling in the plus maze. The

experimenter always remained at a fixed location to support the swivel system following

the movements of the rats during plus maze testing. The microdialysis tubings were

further supported to the ceiling using nylon wires and swiveling loopholes (Fig. 1).

Glucose analysis

Microdialysis samples were analyzed for glucose content according to the method of

Lowry and Passoneau (1972). Briefly, 5 µl samples were diluted to 400 µl in a 1.0 mM

Tris buffer (pH 7.4) containing 1.0 mM MgCl2, 4 U/ml glucose-6-phosphate

dehydrogenase (EC 1.1.1.49), 0.03 mM ATP and 0.03 mM NADP+. All samples were

analysed in duplicate. Fluorescence was measured with a multi-well plate reader at

excitation wavelength of 355 nm and emission wavelength of 460 nm. A blank reading

was taken, 10 µl of a hexokinase (EC 2.7.1.1.) solution (200 U/ml) was added to each

well and fluorescence was measured after 20 min reaction at 30°C. Hexokinase and

13 De Bundel, D.

glucose-6-phosphate dehydrogenase convert glucose to 6-phosphogluconolactone with

simultaneous reduction of NADP+ to NADPH, which is detected fluorimetrically. All

chemicals were obtained from Sigma-Aldrich (Castle-Hill, Australia).

Blood flow monitoring

The laser Doppler probe was connected to a flow meter (MD-1001; Bioanalytical

Systems, West Lafayette, Indiana, USA). The hippocampal blood flow was monitored

continuously at a scan rate of 1 Hz starting 30 min after implantation of the probe. The

basal hippocampal blood flow was monitored for 30 min. After i.c.v. injection of either

aCSF, Ang IV or LVV-H7, the hippocampal blood flow was further monitored for

another 30 min. The blood flow was averaged over 1 min intervals. The average basal

hippocampal blood flow was set as 100% and the readout after cardiac arrest at the end of

the experiment was set as 0%. The sensitivity of the blood flow measurements at the

surface of the hippocampus were evaluated by measuring the vascular response injection

of 300 µg nicotine/kg in 0.3 ml saline over 1 min into the jugular vein as previously

described (Kagitani, Uchida, Hotta & Sato, 2000). Nicotine was obtained from Sigma-

Aldrich (Bornem, Belgium).

Statistical analysis

All data are presented as the mean ± S.E.M. for replicate measurements. The effects of

peptide treatment on the spontaneous alternation score and the number of arm entries

were determined by Mann-Whitney. Glucose measurements were corrected for probe

recovery. Basal glucose levels in the different experimental groups were compared by

one way analysis of variance. The glucose concentrations in the dialysate samples were

14 De Bundel, D.

expressed as the percentages of the basal levels of glucose and further analyzed by two

way analysis of variance for repeated measures followed by Bonferroni’s post-hoc test.

The hippocampal blood flow was expressed as the percentage of the basal relative blood

flow. Hippocampal blood flow measurements were analyzed by two way analysis of

variance for repeated measures followed by Bonferroni’s post-hoc test. All statistical tests

were performed with GraphPad Prism 4.0 (GraphPad Prism Software Inc, San Diego,

USA) at the 5% level of significance.

Results

Effects of Ang IV and LVV-H7 on spatial working memory

A significant increase of the alternation score was observed after i.c.v. injection of either

1 nmol Ang IV (Mann-Whitney, p=0.005) or 0.1 nmol LVV-H7 (Mann-Whitney,

p=0.005) compared to vehicle-treated control rats (Fig. 2A and 2B). The mean alternation

score (± S.E.M.) for vehicle-treated controls was 62.2 ± 2.2% compared to 78.0 ± 4.0%

and 78.4 ± 3.8% for 1 nmol Ang IV and 0.1 nmol LVV-H7 respectively. The higher

doses of IRAP inhibitors (10 nmol Ang IV, 1 nmol LVV-H7) exhibited mean alternation

scores of 70.1 ± 5.3% and 69.3 ± 7.4% respectively which did not differ significantly

(Mann-Whitney, p > 0.05) from vehicle-treated control (Fig. 2A and 2B). This is in

keeping with previous studies demonstrating the lack of efficacy of IRAP inhibitors at

higher concentrations (Lee et al., 2004; Lee et al., 2001). Analysis of the number of arm

choices did not reveal a significant difference (Mann-Whitney, p > 0.05) between rats

treated with Ang IV and LVV-H7 compared to controls, confirming that the observed

15 De Bundel, D.

effect of the IRAP inhibitors is due to improved working memory and not to differences

in locomotor activity (Fig. 2C and 2D).

Effect of spatial working memory testing on hippocampal glucose levels

The in vivo microdialysis probe recovery was previously determined under identical

experimental conditions as 46% using the zero net-flux technique (McNay & Gold,

1999). In the present study, the mean ± S.E.M. basal hippocampal glucose concentration

from 3 month-old Sprague Dawley rats was 1.36 ± 0.04 mM (n = 56). This is in

agreement with the previously reported extracellular hippocampal glucose concentrations

of 1.24 ± 0.04 in 3 month-old Fisher 344 rats (n = 15) (McNay & Gold, 1999). The basal

hippocampal glucose concentrations of the different experimental groups were not

significantly different (F7,48 = 1,134, p = 0,36). The effect of spontaneous alternation

testing was compared to the effect of exposure to a novel environment by placing the rats

in either the plus maze or the control chamber for 20 minutes, after receiving a sham

i.c.v. injection. The animals placed in the control chamber actively explored the novel

environment but despite their increased locomotor activity, no effect was observed on

hippocampal glucose. In animals placed in the plus maze, hippocampal glucose levels

dropped to approximately 70% of basal levels. This observation corresponds to previous

findings (McNay et al., 2000). A significant time versus treatment interaction was

observed between the hippocampal glucose levels of both groups, F(16,256) = 2.00, p =

0.01 (Fig. 3). Post-hoc analysis revealed that 5 min after the transfer, the hippocampal

glucose concentration of animals placed in the plus maze was significantly lower (p <

0.05) compared to animals placed in the control chamber.

Effects of Ang IV and LVV-H7 on hippocampal glucose levels

16 De Bundel, D.

Administration of Ang IV or LVV-H7 into the lateral ventricle did not further enhance

the decrease of hippocampal glucose in animals performing the plus maze task. There

was no significant time versus treatment effect of Ang IV or LVV-H7 on the

hippocampal glucose concentration in animals performing the plus maze task compared

to sham treated controls, F(32,368) = 0.63 and 0.53, p = 0.94 and 0.98 for Ang IV (Fig. 4)

and LVV-H7 (Fig. 5) respectively. Similarly, in the control environment, i.c.v.

administration of memory-enhancing doses of Ang IV (1 nmol) or LVV-H7 (0.1 nmol)

had no effect on hippocampal glucose concentrations compared to sham-treated controls,

F(32,240) = 0.57, p = 0.97 (Fig. 6).

Effects of Ang IV and LVV-H7 on hippocampal blood flow

The extracellular hippocampal concentration of glucose is dependent on local usage and

supply from the blood. Since a further decrease in extracellular glucose may be masked

by an increased glucose flux from the bloodstream to the brain we investigated whether

i.c.v. administration of IRAP ligands enhances hippocampal blood flow. However,

hippocampal bloodflow remained unchanged after administration of aCSF, 1 nmol Ang

IV or 0.1 nmol LVV-H7 into the lateral ventricle (Fig. 7A). No time versus treatment

interaction was observed (F(118,531) = 0.75, p = 0.97). The sensitivity of the system for

hippocampal blood flow measurement was assessed by measuring the response to

administration of 300 µg/kg into the carotid vein. Corresponding to previous findings

(Kagitani et al., 2000), we observed an increase hippocampal blood flow to

approximately 125% of basal levels (Fig. 7B).

17 De Bundel, D.

Discussion

The present manuscript investigated a potential mechanism underlying the memory

enhancing effects of Ang IV and LVV-H7 in the hippocampus during spatial memory

testing. Acute or chronic i.c.v. administration of IRAP ligands enhances spatial learning

in Morris water maze (Wright et al., 1999) and Barnes maze (Lee et al., 2004) tasks. The

detection of high levels of IRAP expression in the hippocampal formation (Fernando et

al., 2005) provides anatomical support for a role for this enzyme in spatial memory.

However, the mechanism by which IRAP ligands facilitate spatial memory remains

unknown. Our previous anatomical studies revealed that in certain regions of the brain,

including the hippocampus, there is an almost complete co-localization of IRAP with

GLUT4 in intracellular compartments in neurons (Fernando et al., 2007). This finding

strongly suggests that in these neurons, IRAP plays a role in regulating the trafficking of

GLUT4, as is the case in insulin-responsive tissues. We recently found in hippocampal

slices that IRAP inhibitors facilitated activity-evoked glucose uptake into pyramidal

neurons through a GLUT4 mediated mechanism (Fernando et al., 2008). Therefore, the

hypothesis that Ang IV and LVV-H7 facilitate spatial working memory through

facilitation of hippocampal glucose uptake was investigated in the present in vivo study.

Ang IV and LVV-H7 facilitate spatial working memory in the plus maze

spontaneous alternation task

In this study, we demonstrated that acute i.c.v. administration of Ang IV or LVV-H7

enhanced spatial working memory as detected by increased spontaneous alternation in the

plus maze, in addition to the known effects of these peptides on hippocampus-dependent

spatial reference memory. The observed effects of these IRAP ligands on spatial working

18 De Bundel, D.

memory performance were not secondary to changes in locomotor activity or exploratory

behavior since no significant effects between the treatment groups were observed in the

number of arm entries during spontaneous alternation testing.

The percent alternation score is equal to the ratio of actual alternations/possible

alternations multiplied by 100. Chance performance on this task is 44.4% (Lennartz,

2008). The mean ± S.E.M. alternation score for vehicle-treated controls was 62.5 ± 2.4%

compared to 78.0 ± 4.0% and 78.4 ± 3.9% respectively for 1 nmol Ang IV or 0.1 nmol

LVV-H7 treated animals. In a previous study, we demonstrated the facilitatory effects of

Nle1-Ang IV and LVV-H7 on spatial reference memory in Barnes circular maze

experiments (Lee et al., 2004). A rapid, significant effect on the acquisition of spatial

reference memory was observed after a single injection of Nle1-Ang IV or LVV-H7 into

the lateral ventricle in rats 5 min before the first trial on the first day (Lee et al., 2004).

Both spatial working and reference memory are hippocampus-dependent, but require

distinct processing mechanisms and molecular pathways (Bannerman, Niewoehner,

Lyon, Romberg, Schmitt, Taylor, Sanderson, Cottam, Sprengel, Seeburg, Kohr &

Rawlins, 2008). Our findings in this study provide further evidence that IRAP ligands are

effective at improving performance in a wide range of memory paradigms, affecting both

short and longer term memories.

Spatial working memory testing decreases hippocampal extracellular glucose levels

The present data confirm previous observations of a decrease in hippocampal

extracellular glucose during spatial working memory testing in rats (McNay et al., 2000).

Spatial working memory testing leads to functional activation of hippocampal neurons

(Puryear, King & Mizumori, 2006) which correlates strongly with increases in local

19 De Bundel, D.

cerebral blood flow (Cox, Woolsey & Rovainen, 1993) and in energy metabolism

(Shulman, Hyder & Rothman, 2002). The observed decrease in extracellular glucose to

30% below baseline is therefore indicative of an increase in local glucose uptake and

utilization.

Both neurons and astroglia are capable of taking up glucose under basal conditions

through the glucose transporters, GLUT3 for neurons and GLUT1 for astroglia (Abe,

Takahashi & Suzuki, 2006; Itoh, Abe, Takaoka & Tanahashi, 2004). A recent model of

cerebral glucose utilization predicts approximately 30% glucose utlilization by neurons

and 70% glucose utilization by astroglia under basal conditions (Hyder, Patel, Gjedde,

Rothman, Behar & Shulman, 2006). However, neurons metabolize a combination of

glucose and lactate (Erlichman, Hewitt, Damon, Hart, Kurascz, Li & Leiter, 2008).

Whether glucose or lactate is the major source of energy for neurons during synaptic

activity is currently subject to debate (Simpson, Carruthers & Vannucci, 2007).

According to the lactate-shunt hypothesis, glucose is predominantly metabolized by glia

into lactate, which is subsequently transported to neurons, forming their major energy

source (Magistretti, 2006). In contrast, recent studies demonstrated that enhanced activity

of neurons of the brain stem and the cerebellum is associated with an increase in neuronal

glucose utilization rather than lactate metabolism (Bak, Schousboe, Sonnewald &

Waagepetersen, 2006).

We previously demonstrated that activation of mouse hippocampal slices by K+

depolarization or cAMP treatment, enhanced 3H-2-deoxyglucose uptake into the

pyramidal neurons (Fernando et al., 2008). This activity-dependent glucose uptake was

attenuated by indinavir, which preferentially blocks the inducible glucose transporter

20 De Bundel, D.

GLUT4 (Fernando et al., 2008). In the hippocampus, GLUT4 is exclusively expressed in

neurons (Apelt, Mehlhorn & Schliebs, 1999; Fernando et al., 2005) and is co-localized

with IRAP in cytoplasmic vesicles under basal conditions (Fernando et al., 2005;

Fernando et al., 2007). This suggests that the enhanced glucose uptake in hippocampal

slices requires recruitment of GLUT4 to the cell surface. We hypothesized that activation

of hippocampal neurons by increased cognitive demand such as in spatial memory testing

may provide a stimulus for such a mobilization of GLUT4, which could enhance

neuronal glucose uptake above basal levels provided by the constitutively expressed

glucose transporter GLUT3.

Ang IV and LVV-H7 did not alter hippocampal glucose levels or blood flow

The observation that exogenous administration of glucose improves spatial working

memory in rats and attenuates the associated decrease in extracellular glucose in the

hippocampus, suggests that spatial memory processing is limited by the rate of local

glucose uptake and/or usage and is susceptible to improvement (McNay et al., 2000).

Since Ang IV and LVV-H7 enhanced activity-evoked glucose uptake in hippocampal

slices (Fernando et al., 2008), we investigated whether these IRAP inhibitors improved

spatial working memory through facilitation of glucose transport into hippocampal

neurons in vivo. Surprisingly, Ang IV and LVV-H7 did not reduce further the

extracellular glucose concentration during performance of spatial working memory task,

despite eliciting an improvement in the alternation scores.

A potential explanation for present findings could be the competition of neurons and glia

for the available energy substrates and the competition of glucose and lactate for

cytosolic nicotinamide-adenine-dinucleotide, which results in the redox switch

21 De Bundel, D.

phenomenon where utilization of one of the two substrates limits the utilization of the

other (Bouzier-Sore, Voisin, Bouchaud, Bezancon, Franconi & Pellerin, 2006). In this

context it was previously demonstrated that disruption of the lactate shuttle from astroglia

to neurons increases the neuronal glucose uptake (Erlichman et al., 2008). Conversely, a

recent model for glucose uptake predicts that under conditions of increased neuronal

glucose uptake, glia may take up lactate rather than glucose as an energy substrate

(Simpson et al., 2007). Therefore, it is conceivable that an increase of neuronal glucose

uptake, induced by IRAP inhibitors, may result in a decrease of glial glucose uptake.

Under these conditions, the overall extracellular glucose would remain constant.

Alternatively, a further decrease in extracellular glucose could be masked by an increase

in glucose flux from the bloodstream to the brain. In this context, Ang IV was

demonstrated to increase cerebral microcirculation when administered into the carotid

artery of anesthetized rats in a dose of 0.1 nmol (Kramar, Harding & Wright, 1997).

Similarly, administration of 1 nmol Ang IV into the carotid artery protected rats against

ischemic damage by inducing a redistribution of blood flow to the ischemic areas (Faure,

Bureau, Oudart, Javellaud, Fournier & Achard, 2008; Faure, Oudart, Javellaud, Fournier,

Warnock & Achard, 2006). These effects were not associated with an increase in arterial

blood pressure but were blocked by the nitric oxide synthase inhibitor L-NAME (Faure et

al., 2006; Kramar, Krishnan, Harding & Wright, 1998). Ang IV was therefore suggested

to increase cerebral blood flow through stimulation of endothelial nitric oxide production.

In the present setup, acute i.c.v. administration of Ang IV or LVV-H7 had no effect on

hippocampal blood flow in anaesthetized rats. This lack of vasodilatatory effects on the

hippocampal vasculature could be due to the difference in site of administration. Indeed,

22 De Bundel, D.

the present data suggest that the access of Ang IV or LVV-H7 to the endothelial layer of

the hippocampal microvasculature after i.c.v. injection may be limited.

It should also be considered that IRAP is a functional aminopeptidase and that Ang IV

and LVV-H7 bind competitively and with relatively high affinity to the catalytic site of

IRAP (Lew, Mustafa, Ye, McDowall, Chai & Albiston, 2003). IRAP is capable of

cleaving a number of peptides associated with memory in vitro (Matsumoto, Nagasaka,

Hattori, Rogi, Tsuruoka, Mizutani & Tsujimoto, 2001). These substrates include arginine-

vasopressin, oxytocin, met-enkephalin, somatostatin and dynorphin A (1-8). Therefore an

alternative mechanism of these to peptides could be the inhibition of the enzymatic

activity of IRAP resulting in the extension of the half-life of its neuropeptide substrates.

Our recent finding in support of this hypothesis is the demonstration that one of our newly

developed, highly selective, small molecule IRAP inhibitors has memory-enhancing

properties in two distinct memory paradigms (Albiston, Morton, Ng, Pham, Yeatman, Ye,

Fernando, De Bundel, Ascher, Mendelsohn, Parker & Chai, 2008).

Conclusion

The current study shows that a single injection of an IRAP ligand, Ang IV or LVV-H7,

into the lateral ventricle of normal rats enhances spatial working memory in the

spontaneous alternation task. Extracellular hippocampal glucose concentrations and

hippocampal blood flow were unaffected by i.c.v. administration of Ang IV or LVV-H7,

suggesting that facilitation of glucose uptake or enhancement of local blood flow in the

hippocampus were not involved in the observed memory effects. IRAP inhibitors have

been found to facilitate other types of memory such as spatial reference memory, object

recognition memory, passive avoidance memory and fear conditioning. Elucidation of the

23 De Bundel, D.

mechanism underlying these cognitive effects, including possible tests of additional brain

areas, may therefore provide a novel strategy for repair of cognitive function.

Acknowledgements

This research was supported by a project grant from the National Health and Medical

Research Council of Australia (NHMRC) and the Robert J. Kleberg Jr. and Helen C.

Kleberg, and CASS Foundations (Australia), the Queen Elisabeth Medical Foundation

(Geneeskundige Stichting Koningin Elisabeth, Belgium); the Research Foundation

Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen, Belgium) and the Vrije

Universiteit Brussel (GOA-2007 and OZR 895, Belgium). S.Y.C. is an Australian

NHMRC Senior Research Fellow. D.D.B. is a Belgian Doctoral Research Fellow of the

Research Foundation Flanders.

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Figure Legends

Figure 1. Overview of the setup for microdialysis sampling during plus maze

spontaneous alternation testing. The microdialysis tubings were supported through

loopholes which were attached to the ceiling with nylon wire. The microdialysis swivel

system could easily be moved from a static arm above the home cage or control chamber

to a loophole above the plus maze which was attached to the ceiling with an elastic band.

A transparent rod was used to manually guide the movement of the microdialysis swivel

following the movements of the rat in the plus maze, while the experimenter stayed at a

fixed position relative to the maze.

Figure 2. (A and C) Spatial working memory performance and the corresponding

locomotor activity after i.c.v. injection of aCSF (n=12), 1 nmol Ang IV (n = 7) or 10

nmol Ang IV (n = 6). (B and D) Spatial working memory performance and the

corresponding locomotor activity after i.c.v. injection of aCSF (n=12), 0.1 nmol LVV-H7

(n = 7) or 1 nmol LVV-H7 (n = 6). The mean ± S.E.M. 4/5 alternation score on the plus

maze spontaneous alternation task is expressed as the percentage of possible

alternations.** p < 0.01 compared to aCSF

Figure 3. Mean ± S.E.M. extracellular hippocampal glucose levels during control

chamber ● (n = 6) and plus maze ○ (n = 12) experiments after i.c.v. injection of aCSF,

expressed as the percentage of the baseline concentration (A). Baseline samples were

collected with the rats in the homecage from 0-30 min. The rats received the i.c.v.

32 De Bundel, D.

injection of aCSF at 30 min (arrow) and were placed in the control box or the plus maze

(shaded area) from 35-55 min. Between 55-85 min the rats were placed back in the

homecage. * p < 0.05 compared to control chamber.

Figure 4. Mean ± S.E.M. extracellular hippocampal glucose levels during plus maze

spontaneous alternation experiments after i.c.v. injection of aCSF ○ (n = 12), 1 nmol Ang

IV ▲ (n = 7) or 10 nmol Ang IV ▲ (n = 6) expressed as the percentage of the baseline

concentration. Baseline samples were collected with the rats in the homecage from 0-30

min. The rats received the i.c.v. injection of aCSF, Ang IV or LVV-H7 at 30 min (arrow)

and were placed in the plus maze (shaded area) between 35-55 min. From 55-85 min the

rats were placed back in the homecage.

Figure 5. Mean ± S.E.M. extracellular hippocampal glucose levels during plus maze

spontaneous alternation experiments after i.c.v. injection of aCSF ○ (n = 12), 0.1 nmol

LVV-H7 (n = 7) or 1 nmol LVV-H7 (n = 6) expressed as the percentage of the

baseline concentration. Baseline samples were collected with the rats in the homecage

from 0-30 min. The rats received the i.c.v. injection of aCSF, Ang IV or LVV-H7 at 30

min (arrow) and were placed in the plus maze (shaded area) between 35-55 min. From

55-85 min the rats were placed back in the homecage.

Figure 6. Mean ± S.E.M. extracellular hippocampal glucose levels during control box

experiments after i.c.v. injection of aCSF ● (n = 6), 1 nmol Ang IV ▲ (n = 6) or 0.1 nmol

LVV-H7 (n = 6) expressed as the percentage of the baseline concentration. Baseline

33 De Bundel, D.

samples were collected with the rats in the homecage from 0-30 min. The rats received

the i.c.v. injection of aCSF, Ang IV or LVV-H7 at 30 min (arrow) and were placed in the

control chamber (shaded area) between 35-55 min. From 55-85 min the rats were placed

back in the homecage.

Figure 7 Mean ± S.E.M. hippocampal bloodflow after (A) i.c.v. injection (arrow) of

aCSF ● (n = 4), 1 nmol Ang IV ▲ (n = 4) or 1 nmol LVV-H7 (n = 4) and (B) after i.v.

infusion (arrow) of 300 µg/kg nicotine as a positive control (n = 4). The relative blood

flow is expressed as the percentage of the baseline blood flow.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7