Neonatal serotonin (5-HT) depletion does not affect spatial learning and memory in rats

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Neonatal serotonin (5-HT) depletion does not affect spatial learning and memory in rats

Agnieszka Piechal¹, Kamilla Blecharz-Klin¹, Edyta Wyszogrodzka², Paulina Ko³omañska², Paulina Rok-Bujko², Pawe³ Krz¹œcik¹,

Wojciech Kostowski², Ewa Widy-Tyszkiewicz¹, Ma³gorzata Filip^3,4, Roman Stefañski²

1Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Krakowskie Przedmiecie 26/28, PL 00-927 Warszawa, Poland

2Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Sobieskiego 9, PL 02-957 Warszawa, Poland

3Laboratory of Drug Addiction Pharmacology, Department of Pharmacology, Institute of Pharmacology, Smêtna 12, PL 31-343 Kraków, Poland

4Department of Toxicology, Faculty of Pharmacy, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland

Correspondence:Roman Stefañski, e-mail: stefans@ipin.edu.pl

Abstract:

Background: Extensive previous research has suggested a role for serotonin (5-HT) in learning and memory processes, both in healthy individuals and pathological disorders including depression, autism and schizophrenia, most of which have a developmental onset. Since 5-HT dysfunction in brain development may be involved in disease etiology, the present investigation assessed the effects of neonatal 5-HT depletion on spatial learning and memory in the Morris water maze (MWM).

Methods: Three days old Sprague-Dawley rats were pretreated with desipramine (20 mg/kg) followed by an intraventricular injec- tion of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, 70 µg). Three months later rats were tested in the MWM.

Results: Despite a severe and permanent decrease (80–98%) in hippocampal, prefrontal and striatal 5-HT levels, treatment with 5,7-DHT caused no spatial learning and memory impairment.

Conclusions: Limited involvement of chronic 5-HT depletion on learning and memory does not exclude the possibility that this neu- rotransmitter has an important neuromodulatory role in these functions. Future studies will be needed to identify the nature of the compensatory processes that are able to allow normal proficiency of spatial learning and memory in 5-HT-depleted rats.

Key words:

5,7-DHT, Morris water maze, learning, memory

Abbreviations: 5-HIAA – 5-hydroxyindoleacetic acid, 5-HT – serotonin, 5,7-DHT – 5,7-dihydroxytryptamine, DA – dopamine, DHBA – dihydroxybenzylamine, DMI – desipramine hydrochloride, DOPAC – 3,4-dihydroxy- phenylacetic acid, GABA – g-aminobutyric acid, HPLC –

high pressure liquid chromatography, HVA – homovanillic acid, MDMA – 3,4-methylenedioxymethamphetamine, MWM – Morris water maze, NA – noradrenaline, pCPA – p-chlorophenylalanine, Pet-1 – plasmacytoma-expressed transcript 1

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Introduction

Extensive previous research has suggested a role for serotonin (5-HT) in learning and memory processes [9, 21], both in healthy individuals and pathological disorders including depression, autism and schizophrenia, most of which have a developmental onset [2, 3, 8, 11, 13, 16]. Thus, 5-HT dysfunction in development may be involved in disease etiology.

One of the most frequently employed behavioral tests for assessing the spatial learning and memory function in rodents is the Morris water maze (MWM) [14, 28, 30]. This paradigm requires rats to learn mul- tiple extra maze visual cues, which allow the subject to build a dynamic spatial map of their surroundings.

In addition to the ability of the MWM to dissociate deficits in memory formation from deficits in sensory, motor, motivational and retrieval processes, several components of this task have been used in the valida- tion of rodent models for neurocognitive disorders and the evaluation of possible neurocognitive treatment [14, 28].

Since a large number of brain cognitive disorders that show 5-HT-ergic imbalance have a developmental onset, we examined the effect of neonatal 5-HT depletion on behavioral responses in the spatial learning paradigm. Intracerebroventricular 5,7-dihydroxytryptamine (5,7-DHT) was used to produce a widespread depletion of brain 5-HT (without the peripheral effects associated with systemic treatment). It has been demonstrated that the 5,7-DHT neurotoxin administered to adult animals caused the distal loss of terminals, but had less effect on cell bodies in the raphe nu- clei [4, 6]. In contrast, when 5,7-DHT was administered intraventricularly to three-day-old rat pups, there was a long-lasting depletion of 5-HT and loss of both terminals and cell bodies [42]. All animals were pretreated with desipramine to protect noradrenergic neurons [5]. However, as 5-HT has widespread functions, it was possible that nonspecific effects of the lesion, such as motivational or motor effects, could con- found interpretation of the result [19, 23, 26].

The main goal of the present paper was to deter- mine whether neonatal 5-HT depletion would affect spatial learning and memory in the MWM and if so, whether motor, perceptual, and motivational dysfunction might occur in addition.

Materials and Methods

Animals

Rats were housed in a temperature-controlled room (20–22°C) under a 12 h light/dark cycle (light on at 7:00 p.m.) and 60% relative humidity, with access ad libitumto the granulated food (Labofeed H; WPiK, Kcynia, Poland) and tap water. All experiments were carried out between 8:00 a.m. and 4:00 p.m. The treatment of the rats was in accordance with ethical stan- dards of European and Polish regulations. All proce- dures were reviewed and approved by the Local Ethics Committee on Animal Studies.

Twenty two timed pregnant Sprague-Dawley rats were obtained from a licensed breeder (Jagiellonian University Collegium Medicum, Kraków, Poland) and were singly housed in clear plastic cages contain- ing wood chip bedding material. The age of newborn rats was determined by checking for births every day at 8:00 a.m. and 4:00 p.m. Three days after birth (the day of birth being postnatal day 0) the sex of pups was determined and males were submitted to further ex- perimentation as described below. Only one 5,7-DHT and one sham pup was taken from each litter.

Drugs

Drug or sham treatment was administered on postnatal day 3. 5,7-Dihydroxytryptamine creatinine sulfate (5,7- DHT) and desipramine hydrochloride (DMI) were pur- chased from Sigma-Aldrich, (St. Louis, USA). To pre- vent oxidation, the 5,7-DHT neurotoxin was dissolved in 0.1% saline solution of ascorbic acid.

Neonatal lesion of 5-HT system with 5,7-DHT

The detailed procedure was previously described by Jessa et al. [20]. Briefly, male pups (n = 22) were in- jected initially with DMI (20 mg/kg, ip) followed 60 min later by an intraventricular injection of 5,7-DHT. Neonates under ether anesthesia were placed on the flat, brightly illuminated surface. The sterile needle having a polyethylene sleeve up to 2 mm from the tip was positioned 1.5 mm anterior to lambda and 2 mm lateral to the sagittal plane. After the needle was lowered into two lateral ventricles, the 5,7-DHT solution was injected. The 5,7-DHT was administered at a dose of 70 µg of free base dissolved in

Effects of neonatal 5-HT depletion on spatial learning and memory

Agnieszka Piechal et al.

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the injector was left in place for 5 min to allow the neurotoxin to diffuse away from the site of the injection. Control sham rats (n = 18) received the DMI injection and vehiculum instead of 5,7-DHT. Dams were maintained with the litters until 28 days of age.

From postnatal day 28 to postnatal day 90 rats were housed two per cage. The 5,7-DHT-lesioned and control pups were weighted once a week. All experiments were carried out when animals reached the age of 3 months.

Water maze

The 5,7-DHT-lesioned and sham rats weighing 270–330 g at the beginning of the behavioral experiments were housed in a temperature-controlled room (20–22°C) under a 12 h light/dark cycle (light on at 7:00 p.m.) and 60% relative humidity. The MWM was used as described previously [30, 48]. The water tank was 180 cm in diameter, and the platform was 10 × 10 cm and submerged 2 cm beneath the water surface.

The inside of the tank was flat black, and the platform was camouflaged by being made of clear acrylic. The black visible platform was 10 × 10 cm and 2 cm above water level to make its location visible. Water temperature was maintained at 23°C. The surrounding environment of the maze contained visual cues such as windows, doors, cabinets, a clock and so on. For each trial, the rat was placed in the water facing the wall of the pool at one of three equally spaced starting points in a random order, excluding the quadrant with the platform. The performance of the rat was tracked automatically using a video tracking system (Chromo- track System; San Diego Instruments, San Diego, CA, USA).

Rats received six acquisition trials per day (limit 60 s per trial to find the platform) for 4 consecutive days. Upon finding the platform, rats were left there for 15 s. If they failed to locate the platform within 60 s, the trial was terminated, and they were placed on the platform for 15 s before being removed. For acquisition trials, the dependent measures were latency (rats’

actual swim time from a starting point to the platform), path length (rats’ actual swim distance from the starting point to the platform) and swim speed (path length divided by the latency in m/s).

form site crossings, path length and swim speed.

On day 6, a visible platform task was given to rats.

The visible platform procedure was similar to that of the hidden platform version described above. The difference was that the platform was 2 cm above water level to make its location visible. Also, the platform location and the start position were changed on every trial and no probe trial was given. The time limits and number of trials given per day were the same as for the hidden platform condition. The dependent measures were latency (rats’ actual swim time from a starting point to the platform), path length (rats’ actual swim distance from the starting point to the platform) and swim speed (path length divided by the latency in m/s).

Twenty four hours after the last behavioral session, all rats were decapitated and brains were quickly removed and dissected on an iced glass plate into three regions: the frontal cortex, hippocampus and striatum.

Tissues were kept at –70°C until use.

Neurochemistry

5-HT, 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed simultaneously using HPLC system with electrochemical detection (for details see Kostowski et al. [22]). Contents of monoamines were measured using a liquid chromatography with electrochemical detection HPLC system (Shimadzu, Japan) with a pro- grammable flow rate LC-9A pump equipped with a 20 µl injection loop (Rheodyne, CA, USA). Separa- tion of monoamines and their metabolites was carried out on a Nucleosil 7C-1B column (Macherey-Nagel, Germany) thermostated at 32°C in a Shimadzu CTO-6A column oven. Integration of the chroma- tograms was performed with a Shimadzu C-R4AX Chromatopac-computing integrator. Dihydroxyben- zylamine (DHBA) was used as an internal standard.

Minimum level of detection was 0.5 ng/ml injected onto the column. Levels of monoamines and their metabolites (ng/g of wet tissue) were then calculated.

Statistical analysis

The statistical analysis was performed using Statistica 7.0 software package (StatSoft, Inc., Tulsa, USA).

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Data are presented as the group means (± SEM).

Two-way analyses of variance (ANOVAs) for repeated measures with a split-plot design (between- within subjects) were used to assess the significance with a group as between-subject factor and block and trial as within subject factor. For neurochemical analysis, an one-way ANOVA was used. All post-hoc tests were performed using Newman-Keuls procedure. All testing hypotheses used a significance level of 0.05.

Results

Effects of neonatal 5-HT depletion on acquisition trials

The effects of neonatal 5-HT depletion on latency during acquisition are shown in Figure 1 (top panel).

An ANOVA with repeated measures on latency data revealed a significant group difference [F (1, 38) = 5.15, p < 0.05], trial difference [F (5, 190) = 9.06, p <

0.001], and a significant interaction between groups and consecutive days [F (3, 114) = 22.83, p < 0.001].

Post-hoc analyses showed that the mean escape latency for the 5-HT-lesioned group was significantly longer than the sham-operated control group on day 1 (p < 0.05). On days 2, 3 and 4 escape latency for the 5-HT-lesioned group was still longer but did not reach statistical significance.

The effects of neonatal 5-HT depletion on path length during acquisition are shown in Figure 1 (middle panel). No significant differences were observed on any behavioral readout.

The effects of neonatal 5-HT depletion on swim speed during acquisition are shown in Figure 1 (bottom panel). An ANOVA with repeated measures on swim speed data revealed a significant group difference [F (1, 38) = 17.67, p < 0.01], trial difference [F (5, 190) = 2.55, p < 0.05], and a significant interaction between groups and consecutive days [F (3, 114) = 5.05, p < 0.05]. The sham-operated control group was swimming faster than the 5-HT-lesioned group and this difference reached significance (p < 0.001) on days 2, 3 and 4.

Effects of neonatal 5-HT depletion on probe trial

Results from the probe trial are illustrated in Figure 2.

There were no significant differences on the number of platform site crossing [F (1, 38) = 0.34, p > 0.05]

(Fig. 2, top panel). A significant neonatal 5-HT depletion effect was observed for path length [F (1, 38) = 7.42, p < 0.01] (Fig. 2, middle panel) and swim speed [F (1, 38) = 8.45, p < 0.01] (Fig. 2, bottom panel).

Fig. 1.Effects of neonatal 5-HT depletion on latency to reach the submerged platform (top panel), path length taken to reach the submerged platform (middle panel) and swim speed (m/s) (bottom panel) over the 4 days of training (six acquisition trials/day). Note that the speed of swimming was significantly weakened by the neonatal exposure to the 5,7-DHT treatment; * p < 0.05, *** p < 0.001 compared to the sham-operated control animals. Data are expressed as the mean ± SEM

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Effects of neonatal 5-HT depletion on visible platform task

Results from the visible platform task are illustrated in Figure 3. A significant neonatal 5-HT depletion effect was observed for latency [F (1, 38) = 4.68, p <

0.05] (Fig. 3, top panel) and swim speed [F (1, 38) = 5.45, p < 0.05] (Fig. 3, bottom panel). There were no significant differences on path length [F (1, 38) = 0.21, p > 0.05] (Fig. 3, middle panel).

In the frontal cortex (Tab. 1), neonatal 5-HT depletion was associated with significant reductions in 5-HT [F (1, 38) = 246.91, p < 0.001], 5-HIAA [F (1, 38) = 209.77, p < 0.001] and the ratio of 5-HIAA/5-HT [F (1, 38) = 846.26, p < 0.001]. No significant changes were observed in the NA, DA, DOPAC, HVA and HVA/DA levels.

In the hippocampus (Tab. 1), neonatal 5-HT depletion was associated with significant reductions in 5-HT

Fig. 3.Effects of neonatal 5-HT depletion on latency to reach the visible platform (top panel), path length taken to reach the visible platform (middle panel) and swim speed (m/s) (bottom panel) during the visible platform task (four trials). Note that the speed of swimming was significantly weakened by the neonatal exposure to the 5,7-DHT treatment; * p < 0.05 compared to the sham-operated control animals. Data are expressed an the mean ± SEM

Fig. 2.Effects of neonatal 5-HT depletion on the number of platform site crossings (top panel), path length (middle panel) and swim speed (m/s) (bottom panel) during the probe trial. Note that the path length and speed of swimming were significantly weakened by the neonatal exposure to the 5,7-DHT treatment; ** p < 0.01 compared to the sham-operated control animals. Data are expressed as the mean

± SEM

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[F (1, 38) = 175.85, p < 0.001], 5-HIAA [F (1, 38) = 22.51, p < 0.001] and the ratio of 5-HIAA/5-HT [F (1, 38) = 30.49, p < 0.001]. No significant changes were observed in the NA, DA, DOPAC and HVA levels.

In the striatum (Tab. 1), neonatal 5-HT depletion was associated with significant reductions in 5-HT [F (1, 38)

= 272.21, p < 0.001], 5-HIAA [F (1, 38) = 482.76, p <

0.001] and the ratio of 5-HIAA/5-HT [F (1, 38) = 15.84, p < 0.001]. No significant changes were observed in the NA, DA, DOPAC, HVA and HVA/DA levels.

Discussion

The main finding of this study was that neonatal 5-HT depletion caused no spatial learning and memory impairment. The brain regions required to navigate the MWM include the striatum [47], prefrontal cortex [29], and especially the hippocampus [17, 30, 31].

These brain regions are also susceptible to the 5-HT- ergic neurotoxicity seen following 5,7-DHT treatment [42]. In the present study, we saw a severe and permanent reduction in hippocampal, prefrontal and striatal 5-HT levels, yet the depletion of 5-HT during neonatal stages did not affect the processing of spatial infor- mation in the adult rat. Thus, our results may suggest limited involvement of neonatal 5-HT brain depletion on spatial learning and memory processes. It is also possible that neonatal rats treated with neurotoxic doses of 5,7-DHT show differential learning and memory responses in the MWM depending on the

stage of brain development. In fact, Broening et al. [7]

demonstrated that 3,4-methylenedioxymethamphetamine (MDMA) exposure on days 11–20 (analogous to late human third trimester brain development) re- sulted in impairment of spatial learning and memory, whereas exposure on days 1–10 (analogous to early human third trimester brain development) had no effect on spatial learning and memory. This effect may be the result of an undeveloped DA-ergic system [38], since rat pups less than 28 days old with undeveloped DA-ergic systems do not exhibit MDMA-induced neurotoxicity, however, at 35 days the DA-ergic system is developed and the neurotoxicity occurs [3].

Although the mechanisms of MDMA-induced neurotoxicity are different from those of 5,7-DHT [37], the level of 5-HT-ergic depletion seen in our study is similar to that which can be produced with MDMA [33]. However, Vorhees et al. [46] reported that neonatal MDMA treatment during any one of four different five-day intervals from birth to postnatal day 20 caused impaired spatial learning and memory in the MWM when tested as adults. These results suggest that the effects of developmental MDMA are not eas- ily predicted based on treatment period alone and have different exposure-duration sensitivities.

There is substantial literature that reports intact learning and memory processes in animal studies using various pharmacological/neurotoxin methods of 5-HT depletion [1, 25, 32, 34, 40, 43, 45]. No deficits in spatial, egocentric, or novel object recognition learning were found in Pet-1^/mice with 80% fewer 5-HT-expressing neurons and 70–80% reductions in 5-HT levels that originate from early in development onward [36]. Also, the results of our experiment pres-

Tab. 1.Tissue levels (ng/g of wet tissue weight) of monoamines and their metabolites in different brain regions of the 5-HT-lesioned (n = 22) and sham-operated (n = 18) rats. Data are expressed as the mean ± SEM. 5-HT serotonin; 5-HIAA 5-hydroxyindoleacetic acid; NA nora- drenaline; DA dopamine; DOPAC 3,4-dihydroxyphenylacetic acid; HVA homovanillic acid. ** p < 0.001 vs. sham-operated controls, ND  not determined

Frontal cortex Hippocampus Striatum

5-HT-lesioned rats

Sham-operated rats

5-HT-lesioned rats

Sham-operated rats

5-HT-lesioned rats

Sham-operated rats

5-HT 258.56 ± 16.45** 2250.9 ± 151.8 265.54 ± 19.45** 2304.9 ± 142.6 233.54 ± 10.76** 2503.7 ± 168.2 5-HIAA 113.43 ± 8.43 ** 996.4 ± 15.1 98.45 ± 15.54** 806.9 ± 70.5 110.32 ± 9.34** 1207.9 ± 35.8 NA 298.8 ± 30.9 250.1 ± 28.6 242.1 ± 24.2 266.3 ± 30.1 364.31 ± 21.2 335.3 ± 20.6

DA 1123.2 ± 111.8 1250.9 ± 121.5 ND ND 12724.1 ± 481.12 11585.1 ± 506.3

DOPAC 198.5 ± 15.16 188.9 ± 17.9 ND ND 7956.3 ± 142.5 7457.9 ± 177.9

HVA 6162.2 ± 325.3 6302.7 ± 260.5 4380.6 ± 472.5 4195.8 ± 361.9 9019.6 ± 322.1 8831.9 ± 348.5

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DHT-provoked global serotonin depletion were able to acquire a place learning task in the MWM as well as control sham-lesioned animals.

With the near total destruction of the 5-HT system on day 3 of life, it may be that other neurotransmitter systems act to compensate for the permanent loss in 5-HT. Indeed, the 5-HT depletion was able to potenti- ate the place learning deficits associated with lesions of the septum [32] or the nucleus basalis magnocellularis [35]. These results emphasize the importance of 5-HT-ergic/cholinergic interactions in the brain and indicate that in the 5-HT-depleted rat brain, remaining neural systems are able to mediate spatial learning at normal levels of proficiency. Several other studies have reported that the learning and memory deficits that result from combined lesions to the 5-HT and cholinergic system are greater than the deficit following the single treatments [10, 25, 32, 39, 44]. It is likely that associated with enhanced spatial learning and memory acetylcholine release [15, 39] could serve as a compensatory mechanism to maintain the integrity of spatial processing in our neonatal 5,7- DHT-lesioned rats. This could be confirmed by meas- uring acetylcholine levels directly in our lesioned animals and may warrant future analysis. In addition, 5-HT-ergic lesion-induced upregulation of growth factors may contribute to such a compensatory mechanism [12, 18, 27, 41].

The most likely nonspecific effects that could con- found spatial learning and memory after neonatal 5-HT depletion are sensorimotor disturbances. In- deed, motor performance, as measured by the speed of swimming, was significantly weakened by the lesion, although some fluctuation of performance could be detected across training sessions. This may suggest that the increase in the latency over the period of the trials was not due to impaired spatial learning. In- stead, the spatial learning and memory deficits in the MWM were likely to be the result of swimming dis- turbance. As the swim speed was also affected during the visible platform task, it may be assumed that in the present experiment motor effects were sufficient to affect performance of the water maze task. When the same subjects were tested in an activity pattern monitor (our unpublished data), the lesion group showed aberrant patterns of behavior and decreased locomotion during the 30 min test session. Thus, it is

posure to the 5,7-DHT treatment.

In conclusion, we present evidence that destruction of 5-HT in early period of life does not influence spatial learning and memory. Limited involvement of chronic 5-HT depletion on learning and memory does not exclude the possibility that 5-HT has an important neuromodulatory role in these processes. In fact, a number of studies have demonstrated that 5-HT has a modulatory effect on hippocampal long-term potentiation, a cellular mechanism of synaptic plasticity be- lieved to underlie learning and memory [24]. Future studies will be needed to identify the nature of the compensatory processes that are able to allow normal proficiency of spatial learning in 5-HT depleted rats.

Acknowledgment:

The study was supported by Grant No. 3 PO5A 068 24 from the Ministry of Science and Higher Education, Warszawa, Poland.

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Received:May 23, 2011; in the revised form: November 3, 2011;

accepted:November 21, 2011.