Hippocampal effects of neuronostatin on memory, anxiety-like behavior and food intake in rats

Neuroscience 197 (2011) 145–152

HIPPOCAMPAL EFFECTS OF NEURONOSTATIN ON MEMORY, ANXIETY-LIKE BEHAVIOR AND FOOD INTAKE IN RATS V. P. CARLINI,a1* M. GHERSI,b2 L. GABACH,b3 H. B. SCHIÖTH,c M. F. PÉREZ,b1 O. A. RAMIREZ,b1 M. FIOL DE CUNEOa1 AND S. R. DE BARIOGLIOb1

SOM facilitates memory (Vécsei et al., 1983, 1984; Lamirault et al., 2001) and has a positive correlation between the performance of hippocampus-dependent learning tasks and the endogenous expressed SOM (Nilsson et al., 1993; Nakagawasai et al., 2000, 2003). In addition, SOM reduces anxiety-like behavior (Engin and Treit, 2009) and locomotor activity (Izquierdo-Claros et al., 2001; Tashev et al., 2001, 2004). A 13-amino acid peptide named neuronostatin (NST) encoded in the SOM pro-hormone has been recently reported. It is produced throughout the body, particularly in brain areas that have significant actions over the metabolic and autonomic regulation (Samson et al., 2008). The hypothesis of the central actions of NST and its possible interaction with different neural circuits is supported by evidences that point out that this neuropeptide is involved with neuronal migration in the cerebellum and hypothalamic neuron firing (Samson et al., 2008). Besides, it has also been demonstrated that the peripheral and i.c.v. administration of NST decreases food and water intake in rats in a dose-related manner. Although NST exerts similar effects to SOM in feeding behavior, fails to activate any of the five recognized SOM receptors in vitro (Samson et al., 2008). The fact that NST induces c-Fos expression in the hippocampus suggests the possibility that this brain structure would be the target of some of the NST effects. Recent evidence indicates that the hippocampus may have a distinctive role on anxiety and emotional behavior, independently of its role in learning and memory (Bannerman et al., 2004). Moreover, the hippocampus is involved in processes related to feeding control and eating behavior (Tracy et al., 2001). Considering all the evidence presented above, the aim of the present study was to elucidate the functional role of NST in memory, anxiety-like behavior and food intake, and the hippocampal participation in these effects. To this end, the peptide was administered both, in the third ventricle (i.c.v.) and intra-hippocampally.

a Cátedra de Fisiología Humana, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Santa Rosa 1085, 5000 Córdoba, Argentina b Departamento de Farmacología, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba; IFEC-CONICET, Haya de la Torre y Medina Allende s/n, Ciudad Univesitaria, 5000 Córdoba, Argentina c Uppsala University, Dept. of Neuroscience, Section of Pharmacology, Uppsala, Sweden

Abstract—A 13-amino acid peptide named neuronostatin (NST) encoded in the somatostatin pro-hormone has been recently reported. It is produced throughout the body, particularly in brain areas that have significant actions over the metabolic and autonomic regulation. The present study was performed in order to elucidate the functional role of NST on memory, anxiety-like behavior and food intake and the hippocampal participation in these effects. When the peptide was intra-hippocampally administered at 3.0 nmol/␮l, it impaired memory retention in both, object recognition and stepdown test. Also, this dose blocked the hippocampal longterm potentiation (LTP) generation. When NST was intrahippocampally administered at 0.3 nmol/␮l and 3.0 nmol/␮l, anxiolytic effects were observed. Also, the administration in the third ventricle at the higher dose (3.0 nmol/␮l) induced similar effects, and both doses reduced food intake. The main result of the present study is the relevance of the hippocampal formation in the behavioral effects induced by NST, and these effects could be associated to a reduced hippocampal synaptic plasticity. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: neuronostatin, memory performance, hippocampal excitability, anxiety-like behavior, food intake.

Somatostatin (SOM), a 14-amino acid cyclic polypeptide, originally investigated for its ability to inhibit the release of growth hormone from cultured rat pituitary cells (Brazeau et al., 1973), regulates the feeding behavior and has also been implicated in cognitive functions (Vécsei and Widerlöv, 1990; Schettini et al., 1991; Patel, 1999; Lamirault et al., 2001). The intracerebroventricular administration of

EXPERIMENTAL PROCEDURES Animals

1

Established Investigators from CONICET. 2 Fellow from SECyT-UNC. 3 Fellow from CONICET. *Corresponding author. Fax: ⫹0054-351-4332019. E-mail address: [email protected] (V. P. Carlini). Abbreviations: ACSF, artificial cerebrospinal fluid; fEPSP, field excitatory post synaptic potentials; fO, familiar object; HFS, high frequency stimulation; i.c.v., third ventricle; LTP, long-term potentiation; nO, novel object; NST, neuronostatin; PP, perforant path; PS, population spike; SEM, standard error of the mean; SOM, somatostatin.

Male Wistar rats of 2 months of age, weighing 260 –290 g were kept under controlled temperature at 21⫾1 °C and at a 12:00/ 12:00 light/dark cycle with food and water ad libitum. Animals were handled daily for 7 days before the experiments. All procedures were conducted according to the National Institutes of Health (NIH) Guidelines for Care and Use of Laboratory Animals (Publications No. 80-23, 1996). Every attempt to minimize the number of animals used and their suffering was made. The total number of animals used was 237. Different animals were used for each

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behavioral paradigm (step down, object recognition test, and plus maze). Some animals used in the step-down test were sacrificed for the electrophysiological experiments, and the food intake was measured in animals groups used for the object recognition test. All behavioral tests were performed by direct observation and blind to the observer.

Drugs For administration, rat NST (Bachem, Weil am Rhein, Germany) was resuspended in artificial cerebrospinal fluid (ACSF) to provide final concentrations of 0.03, 0.3, and 3.0 nmol/␮l. To prevent variations due to circadian rhythms, all infusions were administered between 10:00 AM and noon.

Surgery The rats were anesthetized with 55-mg/kg ketamine HCl plus 11-mg/kg xylazine (Kensol König, Argentina) and placed in a stereotaxic gear. Then, the animals were implanted i.c.v. or bilaterally into the hippocampus with steel guide cannula, according to the atlas of Paxinos and Watson (2009). For i.c.v., the coordinates relative to bregma were anterior: ⫺4.0 mm; lateral: 0.0 mm; vertical: ⫺4.2 mm. For the CA1 hippocampus, the coordinates relative to bregma were anterior: ⫺3.6 mm; lateral: ⫾2.0 mm; vertical: ⫺2.8 mm. Cannulas were fixed to the skull surface with dental acrylic cement. Animals were infused with the three different NTS concentrations using a Hamilton syringe connected by polyethylene tubing to a 30-gauge needle. For i.c.v., a 1-␮l infusion was delivered over a 1-min period and for the hippocampus, an infusion of 0.5 ␮l per side was delivered simultaneously over a 1-min period.

Spontaneous object recognition This task was similar to that described by Ennaceur and Aggleton (1997) to assess the ability of rats to recognize a novel object compared with a familiar one in a familiar environment. In this test, the interest of each animal in a novel object compared with a familiar one was measured and compared. Non-amnesic animals usually spent more time exploring the novel object, reflecting the use of learning and memory processes. The absence of any differences in the exploration of the objects can be interpreted as memory deficit. The apparatus consisted in a 50⫻60-cm wood box of 50-cm height, painted in beige. The object recognition task included two trials; the first one was the acquisition phase, also called training phase, and the second one was the testing phase. All rats underwent a habituation session in which they were allowed to explore the device for 5 min (without objects). In the training phase, to get familiar with the objects, the rats were placed into the box with two identical objects (familiar objects, fO) located at a specific distance (15 cm) from each other and allowed to explore them for 3 min (training). The time spent in exploring each object was measured and only animals that explore each object by a 50% of the total time were tested in the next phase. Control animals and subjects treated with NST were infused immediately after the training session and returned to their home cage. Twenty-four hours later, the rats were placed into the apparatus for the retention test (testing phase), and allowed to explore the two objects for 3 min: one object was the same one used for training (familiar object) and the other was a novel object (nO). The time spent in exploring the novel object was measured. It is important to point out that we used three identical familiar objects (brown glass square), two of them were used during the training phase (t1 and t2) and the third one (t3) was used as familiar object during the retention test; the odour cues left during previous exploration were not present in the testing phase. The novel object (tnovel) used in the testing phase was a pink plastic ball.

Object exploration was defined when the rats directed their noses to the object at a distance closer than 2 cm; turning around or sitting on the object were not considered exploratory behaviors. The following parameters were measured: time spent in exploring identical objects during training (fO), time spent to explore the novel (nO) and the familiar objects in the retention test, and total exploration time. The percentage of time spent in the novel object exploration was considered as index of memory retention. The data were expressed as the mean of the percentage familiar object exploration time in training phase (t2-familiar/[t2familiar⫹t1-familiar]⫻100)⫾standard error (SEM) and as mean of percentage of novel object exploration time in the retention phase (tnovel/[tnovel⫹t3-familiar]⫻100)⫾SEM. The results were evaluated with repeated measurements of analysis of variance (times: training and test) and LSD post hoc test was applied; P-valuesⱕ0.05 were considered statistically significant.

Step-down test (inhibitory avoidance) Rats were subjected to one trial in the step-down test. The apparatus consisted in a 50⫻25⫻25 cm3 plastic box with a 2.5-cm high and 7.0-cm wide platform on the left side of the training box. The floor was made of parallel 0.1-cm diameter stainless steel bars spaced 1.0 cm apart from each other. The animals were placed on the platform, and latency to step down by placing the four paws on the grid was measured. In the training session, immediately upon stepping down, the rats received a 0.4 mA, 2-s scrambled shock on the feet and were immediately removed, infused i.c.v. into the hippocampus with ACSF (control) or with the different doses of NST, and returned to their cages. To measure long-term memory, a retention test was carried out 24 h after training. This test session was identical in all the procedures, except that no shock was given. A ceiling of 180 s was imposed on the retention test measurements. Latency time to step down was taken as a measure of memory retention. Since the variables of the step-down inhibitory test did not follow a normal distribution, they were expressed as medians (inter-quartile range) and analyzed by non-parametric tests (Mann–Whitney or Kruskal–Wallis). P-valuesⱕ0.05 were accepted as statistically significant.

Electrophysiological procedures To evaluate if the changes on memory retention induced by intra-hippocampal NST administration could be correlated with the changes in the hippocampal dentate gyrus excitability, some animals were sacrificed for the electrophysiological experiments immediately after the test session in the step-down test (described elsewhere by Carlini et al., 2010). A total of 16 animals were used. Twenty-four hours after the step-down test, electrophysiological experiments were carried out in in-vitro hippocampal slice preparations using two different protocols to generate long-term potentiation (LTP) elsewhere described by Pérez et al. (2002). To prevent variations caused by circadian rhythms or non-specific stressors, rats were sacrificed between 11:00 AM and noon (Teyler and Di Scenna, 1987). The hippocampal formation was dissected, and transverse slices of approximately 400-␮m thick were placed in a recording chamber (BSC-BU Harvard Apparatus), perfused with standard Krebs solution (NaCl 124.3 mM, KCl 4.9 mM, MgSO4·7H2O 1.3 mM, H2KPO4 1.25 mM, HNaCO3 25.6 mM, glucose 10.4 mM, CaCl2·2H2O 2.3 mM; Sigma, St. Louis, MO, USA) saturated with 95% O2 and 5% CO2. The perfusion rate was 1.6 ml/min, and the bathing solution temperature was kept at 28 °C with a Temperature Controller (TC-202A Harvard Apparatus). A stimulating electrode made of two twisted wires, which were insulated except for the cut ends (diameter 50 ␮m), was placed in the perforant path (PP). Then, a recording microelectrode was inserted in the dentate granule cell body layer. Only slices

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showing a stable response were included in the study. Field excitatory postsynaptic potentials (fEPSP) that responded to 0.2-Hz stimuli were sampled for 20 – 40 min until fEPSP stabilization (baseline). Once no further changes were observed in the amplitude of fEPSP or in the amplitude of population spike (PS), one of the two stimulation protocols were applied. In the first protocol, the threshold to generate LTP was determined. The PP was primed with a train of pulses (0.5 ms each) of 2-s length, of increasing variable frequency. These were delivered by an A310 accupulser pulse generator (World Precision Instruments Inc.), at intervals ranging from 10 to 20 min, starting with a stimulus of 5 Hz. Ten minutes after the stimulus, a new average response at 0.2 Hz was recorded, and if no LTP was observed, a new stimulation at the next higher frequency was applied (10, 25, 50, 75, 100, 150, and 200 Hz). Once LTP was achieved, no further tetanus was given. Long-term potentiation was considered to have occurred when the amplitude of the fEPSP or the amplitude of the PS, recorded after the tetanus, had risen at least 30% from baseline and persisted for 60 min. For each animal, another hippocampal slice was used to apply the second protocol to induce LTP. The tetanization paradigm consisting of three 100-Hz high frequency stimulation (HFS) trains (of 1-s duration each) was given at 20-s intervals. LTP was considered to have occurred as described above for the first protocol. The data were expressed as means⫾SEM and analyzed by repeated measurements for ANOVA (time) or unpaired t-test, when the tetanization or threshold protocol were used, respectively.

central platform (10⫻10 cm2); the whole device was elevated 50 cm over the floor. To avoid rats from falling down, the open arms were bordered by transparent plastic railings of 1-cm high. The test was developed under red light. Thirty-five minutes after i.c.v. or hippocampus ACSF or NST administration, rats were placed on top of the central platform and observed for 5 min. The behavioral performance parameters herein recorded included: number of entries into the open arms; number of entries into closed arms; total number of entries; time spent in open arms; percentage of entries in the open arms; percentage of time inside the open arms; rearing, grooming, and risk-assessment (forward elongation of head and shoulders followed by retraction to original position). The test was performed as a single trial per animal. Data were expressed as mean⫾SEM and analyzed by MANOVA. Hotelling’s T2 test was performed as post hoc analysis (P-valuesⱕ0.05). A total of 76 animals were used.

Elevated plus maze

Histology

The plus maze equipment was made of wood and consisted of two open arms and two enclosed arms of 50⫻10 cm2 each; two opposed arms of the same size crossed at a right angle; the latter were enclosed by walls of 40-cm high. The arms extended from a

After behavioral tests, rats were anesthetized with chloral hydrate, cardiacally perfused with paraformaldehyde (4%), and their brains were removed. Frontal sections were cut in a cryostat (Leica), and the injection site was localized. Only results obtained from animals

Food intake To reduce the total number of animals, the same animals used for object recognition were employed again; in this case, they were infused immediately after training for the behavioral test. Food intake was estimated as the difference between food weight (g) placed in the dispenser and the remainder 24-h post infusion; this parameter was measured in a period in which animals were housed in their home cages (period between the training and test of the object recognition task). Data were expressed as mean⫾SEM and analyzed by one way ANOVA.

Fig. 1. Effect of i.c.v. (A) or intra-hippocampal (B) neuronostatin (NST) administration on memory performance in object recognition test. ACSF: control animals infused with artificial cerebrospinal fluid. All treatments were performed immediately post training, and retention test was carried out 24 h later. Results are expressed as mean⫾SEM. N⫽10 animals in each group. fO: familiar object, nO: novel object. * Significant differences comparison with control animals, Pⱕ0.05.

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in which the tips of the cannulae were placed into the hippocampus or i.c.v. were considered.

RESULTS Neuronostatin effects on memory retention To evaluate the effects of the peptide on memory retention, two behavioral paradigms, step-down and object recognition test, were applied. A total of 161 animals were used. Results obtained when NST was administered i.c.v. are illustrated in Fig. 1A; only animals infused with NST 3.0 nmol/␮l exhibited a significant reduction in the percentage of exploration time of the novel object; total exploration time (a measure of animal motivation) was not modified. Repeated measures ANOVA test showed significant interaction between treatment and time (ACSF and NST vs. training and test) (F⫽11.84, df⫽(2, 41), Pⱕ0.05) and significant effects of time (training vs. test) (F⫽19.66, df⫽(1, 41), Pⱕ0.05) and treatment (ACSF vs. NST) (F⫽7.06, df⫽(2, 41), Pⱕ0.05). When NTS was administered intra-hippocampally (Fig. 1B), two NTS concentrations (0.3 and 3.0 nmol/␮l) caused significant decrease in the percentage of novel object exploration time when compared to controls. Repeated measures ANOVA test demonstrated a significant interaction between treatment and time (ACSF and NST vs. training and test) (F⫽13.78, df⫽(3, 30), Pⱕ0.05) and significant effects of time (training vs. test) (F⫽26.65, df⫽(1, 30), Pⱕ0.05) and treatment (ACSF vs. NST) (F⫽21.43, df⫽(3, 30), Pⱕ0.05). No significant effects were observed on the total exploration time (familiar⫹novel object). Results obtained in the step-down test are illustrated in Fig. 2; latency time was significantly reduced when NST 3.0 nmol/␮l was administered i.c.v. (Median Test Kruskal– Wallis, chi-square⫽6.75, df⫽2, P⫽0.03 and Mann–Whitney test, U Control-NST 3.0 nmol/␮l⫽2.00, P⫽0.00) (Fig. 2A). Similar results were obtained when the same dose of NST was intra-hippocampally administered (Median Test Kruskal–Wallis, chi-square⫽15.95, df⫽3, P⫽0.00 and Mann–Whitney test, U ACSF-NST 3.0 nmol/␮l⫽2.00, P⫽0.00) (Fig. 2B). Neuronostatin effects on hippocampal excitability Fig. 3 shows the effects of NST on the hippocampal excitability 24 h postadministration. When 3.0 nmol/␮l of NST were administered in vivo, LTP generation in hippocampal slices was abolished when the tetanization paradigm was applied (Fig. 3C). In this case, HFS was able to induce LTP in slices from control animals, showing increases greater than 30% in fEPSP when compared to basal fEPSP, and with these rises being maintained for at least 60 min (F⫽4.27, df⫽(3, 27), P⬍0.05) (Fig. 3D). Similar results were obtained when the results from PS were analyzed for this concentration (data not shown). When using the threshold protocol, LTP was not generated in slices from animals treated with NST 3.0 nmol/␮l, even when 200 Hz of stimulation frequency were used (data not shown). Similar results were obtained when the results of PS were analyzed for this concentration (data not shown).

Fig. 2. Effect of i.c.v. (A) or intra-hippocampal (B) neuronostatin (NST) administration on latency time in step-down test (inhibitory avoidance). ACSF: control animals infused with artificial cerebrospinal fluid. All treatments were performed immediately post training, and retention test was carried out 24 h later. The results are expressed as median (inter-quartile range). N⫽12–15 animals in each group. * Significant differences comparison with control animals, Pⱕ0.05.

Oppositely, when NST was administered at 0.3 nmol/ ␮l, the tetanization paradigm induced LTP in the two groups (ACSF and NST 0.3) showing a rise greater than 30% in fEPSP compared with basal fEPSP, and an upholding of this rise for at least 60 min (F⫽0.64, df⫽(3, 27), P⬎0.05) (Fig. 3E). Similar results were obtained when the PS data were analyzed for this concentration (data not shown). Furthermore, the threshold to generate LTP in the NST 0.3 group was significantly lower when compared with ACSF group (Fig. 3F). Similar results were observed when PS data were analyzed for this concentration (data not shown). Neuronostatin effects on anxiety-like behavior (plus maze test) The NST 3.0 nmol/␮l i.c.v. administration induced significant increase of the time spent in the open arms (Pⱕ0.05) (data not shown), percentage of entries into the open arms (Pⱕ0.05), and percentage of time inside the open arms (Pⱕ0.05) (Fig. 4A). The MANOVA test revealed a significant effect of NST i.c.v. administration between groups (Wilks⫽0.03, df⫽(3, 34), Pⱕ0.05). No significant differences were observed on the total number of entries (a measure of overall locomotor activity) (Fig. 4A), as well as on grooming behavior (NST 3.0 nmol/␮l⫽2.83⫾0.87 vs. ACSF⫽2.69⫾0.72), rearing (NST 3.0 nmol/␮l⫽9.16⫾0.70

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Fig. 3. Effect of intra-hippocampal neuronostatin (NST) administration on synaptic plasticity. (A) Hippocampal slice cartoon indicating the position of stimulation and recording electrodes. (B) Field excitatory post synaptic potentials (fEPSP) example traces showing how measurements of fEPSP and population spike amplitude were taken before and after high frequency stimulation (HFS). (C) fEPSP sample traces for NST 3.0 nmol/␮l and artificial cerebrospinal fluid (ACSF) groups before (full line) and after (dotted line) tetanization protocol. (D) Time course graph showing increments in fEPSP, as % of basal fEPSP, after tetanization protocol in NST 3.0 nmol/␮l and ACSF groups. Circles represent means⫾SEM. Black arrow indicates time in which tetanization protocol was delivered. * Significant differences compared with ACSF-injected animals, Pⱕ0.05. (E) Time course graph showing increments in fEPSP, as % of basal fEPSP, after tetanization protocol in NST 0.3 nmol/␮l and ACSF groups. Circles represent means⫾SEM. Black arrow indicates time in which tetanization protocol was delivered. (F) Bar graphs showing the threshold to generate LTP in NST 0.3 nmol/␮l and ACSF groups. Bars represent means⫾SEM. * Significant differences compared with ACSF group, Pⱕ0.05.

vs. ACSF⫽6.69⫾0.97) and risk assessment (NST 3.0 nmol/␮l⫽8.16⫾1.85 vs. ACSF⫽5.94⫾0.82). When NST was administrated intra-hippocampally, the doses of 0.3 and 3.0 nmol/␮l induced significant increases in most of the parameters measured: time spent in open arms (Pⱕ0.05) (data not shown), percentage of entries in the open arms (Pⱕ0.05), and percentage of time spent in open arms (Pⱕ0.05) (Fig. 4B). The MANOVA test revealed a significant effect of intra-hippocampal NST administration between groups (Wilks⫽0.06, df⫽(3, 38), Pⱕ0.05). No significant differences were observed in the total number of entries (Fig. 4B), as well as on grooming behavior (NST 3.0 nmol/␮l⫽2.30⫾0.73; NST 0.3 nmol/␮l⫽2.50⫾0.22 vs. ACSF⫽2.57⫾0.75), rearing (NST 3.0 nmol/␮l⫽11.40⫾ 0.84; NST 0.3 nmol/␮l⫽9.20⫾1.47 vs. ACSF⫽8.21⫾ 1.09), and risk assessment (NST 3.0 nmol/␮l⫽6.60⫾1.40; NST 0.3 nmol/␮l⫽3.50⫾0.96 vs. ACSF⫽4.14⫾0.93). Neuronostatin effects in food intake Fig. 5 shows the NST effects upon food intake; when the peptide was i.c.v. administered, both highest doses induced a significant decrease in this parameter (F⫽4.33, df⫽(3, 46), Pⱕ0.05) (Fig. 5A). On the contrary, intra-hippocampal NST administration did not exert any effect in this parameter (Fig. 5B).

DISCUSSION The results presented in this article provide evidence that NST alters hippocampal dependent behavior such as

memory and anxiety. Moreover, food intake was also modified, but only when the peptide was i.c.v. administered. It is well known that rats have a natural tendency to spend more time exploring novel objects than familiar ones, and this preference is used as an index of memory retention. Our results indicate that the i.c.v. administration of NST induced a reduction in memory retention only after the highest NST dose tested (3.0 nmol/␮l). These results related to NST effects upon memory are opposite to those exerted by SOM (Vécsei et al., 1983, 1984; Lamirault et al., 2001). Furthermore, we evaluated the hippocampal dependence of these effects, administering the peptide directly into this structure, and we observed a reduction in memory retention. It is reasonable to consider that the diminished performance of NST-treated groups in the object recognition test could be a consequence of a motivational deficit, that is, rats would not be interested in exploring novel objects. To explore this hypothesis, we quantified the total exploration time during each experiment phase (training and test) and noticed that there were no differences between them, indicating that motivation is normal in all groups. We also demonstrated that the intra-hippocampal administration of NST at 3.0 nmol/␮l impaired LTP generation in the hippocampal dentate gyrus, which is the first synaptic contact of input information that arise the hippocampus from the entorhinal cortex (Jones, 1994; Leranth and Hajszan, 2007; Goodrich-Hunsaker et al., 2008). The effects of NST on hippocampal synaptic plasticity are closely

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Fig. 4. Effects of i.c.v. (A) or intra-hippocampal (B) neuronostatin (NST) administration in plus maze test. ACSF: control animals infused with artificial cerebrospinal fluid. All treatments were performed 15 min before the test. The results are expressed as mean⫾SEM. N⫽9 –11 animals in each group. * Significant differences in comparison with control animals, Pⱕ0.05.

related to the behavioral results observed in the step-down and object recognition tests, since the same animals that showed memory impairment after NST 3.0 nmol/␮l administration also showed impairment of in vitro LTP generation. When the intra-hippocampal NST dose of 0.3 nmol/␮l was administered, the threshold to generate LTP was significantly lower than that in the control group, showing that NST exerts different effects on hippocampal synaptic plasticity, depending on the concentration reached in this brain area. However, this level of synaptic plasticity is not sufficient to improve the animals’ performance on the memory tests used in the present study, probably due to that tests were not enough sensitive. Other scenario could be that these behaviors can be affected by other emotional factors. The elevated plus maze test is commonly used for anxiety measurement. In this test, rats tend to avoid the open arms and spend more time on the enclosed ones. Anxiolytic compounds, which decrease the natural aversion toward open arms, elevate the percentage of time spent in these arms (Cruz et al., 1994). In the present work, we observed that i.c.v. administration of NST induced anxiolytic effects (evaluated by the percentage of

time spent in the open arms) only at the highest dose tested. When NST was administered intra-hippocampally, both doses exerted significant effects on anxiety reduction. Since there were no changes in the total number of entries in the plus maze test the locomotor activity was not modified by NST (i.c.v. or intra-hippocampally administered). Several evidence suggested that the anxiety and memory processes could be related, for instance several drugs, such as benzodiazepines, reduce anxiety and also impair memory (Lister, 1985). Moreover, Bertoglio et al. have corroborated the hypothesis that the dorsal and ventral hippocampus may preferentially regulate memory and anxiety-related processes, respectively (Bertoglio et al., 2006). Considering the evidence mentioned previously, our findings suggest that the hippocampus participates in the anxiolytic and memory impairments induced by NST, supporting the fact that these two effects could be related. It is well known that i.c.v. injection of any drug or peptide reaches numerous brain structures acting on selective receptors, while the direct administration into a particular brain structure affects only the receptors of this area (McGaugh and Izquierdo, 2000). Our results regarding the NST effects on food intake confirmed that this

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REFERENCES

Fig. 5. Effects of i.c.v. (A) or intra-hippocampal (B) neuronostatin (NST) administration on food intake 24 h post injection. ACSF: control animals infused with artificial cerebrospinal fluid. The results are expressed as mean (g)⫾SEM. N⫽10 –13 animals in each group. * Significant differences comparison with control animals, Pⱕ0. 05.

peptide decreased this parameter when it was i.c.v. administered to satiated animals. However, when NST was infused directly into the hippocampus, it did not induce any changes on this behavior. The findings of the present study suggest that the hippocampus does not participate in NSTmediated effects on hunger, at the tested doses, while the effects on anxiety, memory, and synaptic plasticity are dependent on this brain structure. The results reported in this study are, as far as we know, the first ones that associate the NST peptide with anxiety and memory. In conclusion we can speculate that the hippocampus is one of the brain structures involved in mediating the NST effects regarding memory retention and anxiety. The participation of other brain structures such as the amygdale cannot be ruled out, considering their role in anxiety and learning processes involved in the step-down test. Acknowledgments—This work was supported by grants from CONICET (Consejo Nacional de Investigación Científica y Técnica), SECyT-UNC (Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba), and the Swedish Research Council (VR, Medicine). The authors thank Estela Salde (CONICET’s technical) for her technical assistance in animal histology.

Bannerman DM, Rawlins JNP, McHugh SB, Deacon RMJ, Yee BK, Bast T, Zhang WN, Pothuizen HHJ, Feldon J (2004) Regional dissociation within the hippocampus-memory and anxiety. Neurosci Biobehav Rev 28(3):273–283. Bertoglio LJ, Lourenço Joca SR, Guimarães FS (2006) Further evidence that anxiety and memory are regionally dissociated within the hippocampus. Behav Brain Res 175(1):183–188. Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R (1973) Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179(68):77–79. Carlini VP, Perez MF, Salde E, Schiöth HB, Ramirez OA, de Barioglio SR (2010) Ghrelin induced memory facilitation implicates nitric oxide synthase activation and decrease in the threshold to promote LTP in hippocampal dentate gyrus. Physiol Behav 101:117–123. Cruz AP, Frei F, Graeff FG (1994) Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 49:171–176. Engin E, Treit D (2009) Anxiolytic and antidepressant actions of somatostatin: the role of sst2 and sst3 receptors. Psychopharmacology (Berl) 206(2):281–289. Ennaceur A, Aggleton JP (1997) The effects of neurotoxic lesions of the perirhinal cortex combined to fornix transection on object recognition memory in the rat. Behav Brain Res 88:181–193. Goodrich-Hunsaker NJ, Hunsaker MR, Kesner RP (2008) The interactions and dissociations of the dorsal hippocampus subregions: how the dentate gyrus, CA3, and CA1 process spatial information. Behav Neurosci 122:16 –26. Izquierdo-Claros MD, Boyano-Adanez G, Torrecillas M, RodriguezPuyol M, Arilla-Ferreiro E (2001) Acute modulation of somatostatin receptor function by melatonin in the rat frontoparietal cortex. J Pineal Res 31:46 –56. Jones RS (1994) Synaptic and intrinsic properties of neurons of origin of the perforant path in layer II of the rat entorhinal cortex in vitro. Hippocampus 4:335–353. Lamirault L, Guillou JL, Micheau J, Jaffard R (2001) Intrahippocampal injections of somatostatin dissociate acquisition from the flexible use of place responses. Eur J Neurosci 14:567–570. Leranth C, Hajszan T (2007) Extrinsic afferent systems to the dentate gyrus. Prog Brain Res 163:63– 84. Lister RG (1985) The amnesic action of benzodiazepines in man. Neurosci Biobehav Rev 9:87–94. McGaugh JL, Izquierdo I (2000) The contribution of pharmacology to research on the mechanisms of memory formation. Trends Pharmacol Sci 21:208 –210. Nakagawasai O, Hozumi S, Tan-No K, Niijima F, Arai Y, Yasuhara H, Tadano T (2003) Immunohistochemical fluorescence intensity reduction of brain somatostatin in the impairment of learning and memory-related behavior induced by olfactory bulbectomy. Behav Brain Res 142:63– 67. Nakagawasai O, Tadano T, Niijima F, Tan-No K, Kisara K (2000) Immunohistochemical estimation of rat brain somatostatin on avoidance learning impairment induced by thiamine deficiency. Brain Res Bull 51:47–55. Nilsson L, Mohammed AK, Henriksson BG, Folkesson R, Winblad B, Bergström L (1993) Environmental influence on somatostatin levels and gene expression in the rat brain. Brain Res 628:93–98. Patel YC (1999) Somatostatin and its receptor family. Front Neuroendocrinol 20:157–198. Paxinos G, Watson C (2009) The rat brain in stereotaxic coordinates, compact sixth edition. San Diego, CA: Academic Press. Pérez MF, Maglio LE, Marchesini GR, Molina JC, Ramírez OA (2002) Environmental changes modify the expression of Diazepam withdrawal. Behav Brain Res 136:75– 81.

152

V. P. Carlini et al. / Neuroscience 197 (2011) 145–152

Samson WK, Zhang JV, Avsian-Kretchmer O, Cui K, Yosten GL, Klein C, Lyu RM, Wang YX, Chen XQ, Yang J, Price CJ, Hoyda TD, Ferguson AV, Yuan XB, Chang JK, Hsueh AJ (2008) Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions. J Biol Chem 283:31949 –31959. Schettini G (1991) Brain somatostatin: receptor-coupled transducing mechanisms and role in cognitive functions. Pharmacol Res 23(3):203–215. Tashev R, Belcheva S, Belcheva I (2004) Differential effects of somatostatin on exploratory behavior after unilateral injections into rat neostriatum. Peptides 25:123–128. Tashev R, Belcheva S, Milenov K, Belcheva I (2001) Behavioral effects of somatostatin microinjected into caudate putamen. Neuropeptides 35:271–275.

Teyler J, Di Scenna P (1987) Long-term potentiation. Annu Rev Neurosci 10:131–161. Tracy A, Jarrard L, Davidson TL (2001) The hippocampus and motivation revisited: appetite and activity. Behav Brain Res 127: 13–23. Vécsei L, Bollók I, Telegdy G (1983) Intracerebroventricular somatostatin attenuates electroconvulsive shock-induced amnesia in rats. Peptides 4:293–295. Vécsei L, Bollók I, Varga J, Penke B, Telegdy G (1984) The effects of somatostatin, its fragments and an analog on electroconvulsive shock-induced amnesia in rats. Neuropeptides 4:137–143. Vécsei L, Widerlöv E (1990) Preclinical and clinical studies with cysteamine and pantethine related to the central nervous system. Prog Neuropsychopharmacol Biol Psychiatry 14:835– 862.

(Accepted 17 September 2011) (Available online 22 September 2011)