Early postnatal protein malnutrition causes resistance ...

Nutritional Neuroscience, February/April 2007; 10(1/2): 23–29

Early postnatal protein malnutrition causes resistance to the anxiolytic effects of diazepam as assessed by the fear-potentiated startle test ˜ O2, & S. S. ALMEIDA1 A. L. FRANC ¸ OLIN-SILVA1, M. L. BRANDA 1

Laboratory of Nutrition and Behavior, Department of Psychology, FFCLRP, University of Saõ Paulo, Avenida dos Bandeirantes, 3900, 14040-901 Ribeiraõ Preto SP, Brazil, and 2Laboratory of Psychobiology, Department of Psychology, FFCLRP, University of Saõ Paulo, Avenida dos Bandeirantes, 3900, 14040-901 Ribeiraõ Preto SP Brazil (Received 24 October 2006; revised 20 November 2006; accepted 12 December 2006)

Abstract Given that protein malnutrition induces structural, neurochemical and functional changes in the CNS, the present study aimed to investigate the effects of different early periods of protein malnutrition on the behavior and reactivity to diazepam (DZ) in a model of anxiety: the fear-potentiated startle (FPS). Male Wistar rats (n ¼ 110) from well-nourished (16%-protein) or malnourished (6%-protein) litters were distributed in five different groups: W (well-nourished), M7 (malnourished for 7-days, since day 0), M14 (14-days), M21 (21-days) and M28 (28-days). The results obtained in FPS revealed that malnourished-animals acquired the startle response, irrespective of the time they were exposed to the diet. Besides, DZ reduced the startle amplitude in the noise-alone and light-noise trials. The data concerning the total freezing time showed that the expression of this response was affected by malnutrition and varied in accordance with the findings of previous studies in which malnutrition procedures was imposed for long periods (more than 50 days). Therefore, we suggest that early protein malnutrition: (a) did not produce deficits in the associative learning process of these animals in the FPS, and (b) decreased freezing time in the FPS and produce hyporeactivity to the effects of DZ in rats malnourished for 21 days or more, indicating alterations in the GABAergic neurotransmitter system.

Keywords: Early malnutrition, fear-potentiated startle, freezing, diazepam, startle reflex

Introduction Protein or protein-calorie malnutrition imposed early in life is a well-known environmental factor that produces alterations in brain. These alterations can affect morphological, neurochemical, neurophysiological and functional aspects of the developing brain (Morgane et al. 1978, 1992, 1993; Bedi 1987, 1991; Dobbing 1987; Tonkiss et al. 1993; Almeida et al. 1996a). However, the majority of the reported procedures in this area begin the malnutrition insult early in life (gestation and/or lactation periods) and prolong the exposition to the deficient diet for a long time (more than 50 days) before testing the animals in the behavioral paradigms (Almeida et al. 1996a). Regarding the functional aspects it has been demonstrated that malnourished-animals submitted

to experimental models of anxiety such light – dark transition (Brioni et al. 1989; Santucci et al. 1994), elevated plus-maze (EPM) (Almeida et al. 1994; 1996b) and elevated T-maze (Almeida et al. 1996c; Hernandes and Almeida 2003) showed behavioral alterations that have been interpreted as lower anxiety levels and/or higher impulsiveness. However, little is known about the effects of early protein malnutrition on experimental models of conditioned fear. The pattern of behavioral and cardiovascular change characteristic of an animal’s reaction to threatening or stressful stimuli is often referred to as “defense reaction” (Bandler and Carrive 1988). These defensive reactions are elicited by the appearance of predator and by the sudden appearance of innocuous objects. These responses are always near threshold

Correspondence: S. S. Almeida, Laboratory of Nutrition and Behavior, Department of Psychology, FFCLRP, University of Saõ Paulo, Avenida dos Bandeirantes, 3900, 14040-901, Ribeiraõ Preto SP Brazil. Tel: 55 16 36023663. Fax: 55 16 36335015. E-mail: [email protected] ISSN 1028-415X print/ISSN 1476-8305 online q 2007 Informa UK Ltd. DOI: 10.1080/10284150601168346

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A.L. Françolin-Silva et al.

so that the animal will take flight, freeze or threaten, whenever any novel stimulus event occurs. The animal presents a repertoire of responses (behavioral and neurovegetative) that characterize a fear reaction. Bolles (1970) named this repertoire of responses (characteristics of the species) species-specific defense reaction (SSDRs). The usual defense reaction presented by mice and rats is freezing, and it was defined by the absence of body movement together with the absence of the whisker and nose movements, characteristic of sniffing (Bolles and Collier 1976). A widely used model to investigate an animal’s reaction to threatening or stressful stimuli is the fearpotentiated startle (FPS). Startle is a fast twitch of facial and body muscles evoked by a sudden and intense tactile, visual or acoustic stimulus (Brown et al. 1951; Davis et al. 1993; Koch 1999). The startle pattern consists of eyelid-closure and contraction of facial, neck and skeletal muscles, as well an arrest of ongoing behaviors and an acceleration of the heart rate (Koch 1999). The response pattern is suggestive of a protective function of startle against injury from a predator or from a blow, and of the preparation of a flight/flight response (Koch 1999). Bremner (2004) demonstrated that the amplitude of the acoustic startle reflex in the rat can be augmented by presenting the eliciting auditory stimulus in the presence of a cue (e.g. a light) that has previously been paired with a shock, a phenomenon termed the “fear-potentiated startle effect”. In this test a central state of fear is considered to be the conditioned response (Brown et al. 1951; Davis et al. 1993). Conditioned-fear is operationally defined by elevated startle amplitude in the presence of a cue previously paired with a shock. The conditioned-stimulus does not elicit startle (Davis et al. 1993). A great variety of drugs that reduce anxiety in people decrease FPS. Drugs such as clonidine, diazepam (DZ), morphine, buspirone an alcohol, which differ considerably in their mechanism of action, all block-potentiated startle (Davis et al. 1979, 1993; Davis 1992; Hijzen et al. 1995; Josselyn et al. 1995; Patrick et al. 1996). Since that FPS response is altered by anxiolytic benzodiazepinic drugs (Patrick et al. 1996) and that early protein malnutrition changes the GABAergic neurotransmitter systems (Morgane et al. 1992; 1993; Almeida et al. 1996a), the main aim of the present study was to investigate if different early periods of protein malnutrition alters the response of animals to the DZ in the FPS test.

Material and methods Subjects Male Wistar rats from the animal colony of Ribeiraõ Preto Campus, University of Saõ Paulo were used. Each litter was culled to 6 male and 2 female pups

on the day of birth. The dams and the pups were housed in transparent plastic cages (40 £ 30 £ 20 cm) and assigned randomly to a 6% or a 16% protein ad libitum diet for different periods (0 –28 days). Male rats (n ¼ 110) from well-nourished (16%-protein) or malnourished litters (6%-protein) were distributed in five different groups: W (wellnourished), M7 (malnourished from birth to 7 days of age), M14 (malnourished from birth to 14 days of age), M21 (malnourished from birth to 21 days of age) and M28 (malnourished from birth to 28 days of age). After weaning (21 days), the animals started receiving a regular lab chow diet ad libitum until the end of the experiment, with the exception of one group that started receiving commercial diet after postnatal age 28. As the nutritional treatments were applied to the litters and not to individual animals, only one male pup per litter was chosen by draw and used for behavioral test. All animals were tested at 70-days old. The diets have been described elsewhere (Almeida et al. 1994). Briefly, the protein-deficient diet contained approximately 8% casein (6% protein), 5% salt mixture, 1% vitamin mixture, 8% corn oil, 0.2% choline and 77.8% corn starch (w/w). The regular protein diet contained approximately 20% casein (16% protein), 60.8% corn starch and the same percentage of the other constituents as the proteindeficient diet. The two diets were supplemented with L -methionine (2.0 g/kg protein) since casein is deficient in this amino acid. Only male rats were used in this study. The animals were maintained on a 12:12-h light/ dark cycle (lights on at 6:00 am) with room temperature kept at 23 –258C, and with free access to water and food throughout the experiment. The behavioral tests were conducted during the light period in the morning (8:00 – 12:00 am). The experiments were performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior (SBNeC), which are based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.

Apparatus and procedure Matching To record the amplitude of the startle response (ASR), two stabilimeter devices were used simultaneously. The rats were placed into a stabilimeter, which consisted of a wire-mesh cage (16.5 £ 5.1 £ 7.6 cm) suspended within a PVC frame (25 £ 12 £ 12 cm), which was firmly placed on the response platform by four thumbscrews. The stabilimeter and platform were located inside a ventilated polywood soundattenuating chamber (64 £ 60 £ 40 cm). The floor of the stabilimeter consisted of six 3.0-mm-diameter stainless steel bars, spaced 1.5 cm apart. The startle

Short-term malnutrition and fear-potentiated startle reflex reaction of the rats generated a pressure on the response platform and analog signals were amplified, digitized and analyzed by software (Startle Reflex, version 4.1, Med Associates Inc., VT, USA) provided by the manufacturer of the equipment. The presentation and sequencing of the acoustic and visual stimuli were also controlled by the same software and the appropriate interface (Med Associates Inc., VT, USA). A loudspeaker, located 10 cm behind the wiremesh cage, was used to deliver both the acoustic startle stimuli and a continuous background noise (55 dB SLP), and a white 6.0 W bulb located in the ceiling of the chamber delivered the visual stimuli. The startle stimulus was a 100 dB, 50 ms burst of white noise, having a rise-decay time of 5 ms and delivered through the same speakers as the background noise. The startle reaction was recorded within a time-window of 200 ms after the startle stimulus onset. Calibration procedures were conducted before the experiments to ensure equivalent sensitivities of the response of platforms. The behavior of animals were recorded by a video camera (Everfocus, USA) positioned behind the stabilimeter, allowing the discrimination of all possible behavior, with a signal being relayed to a monitor in another room via a closed circuit. A red light bulb (6.0 W) was located on top of the isolation chamber, to provide illumination for the camera. On the first 2 days, the animals were placed in the stabilimeter for 5 min for habituation, and afterwards a total of 30 startle stimuli with an intensity of 100 dB at a variable inter-stimulus interval of 30 s on average. The duration of each matching session was 20 min. For each experiment the animals were matched into two equivalent groups well nourished or malnourished and saline or DZ, based on their mean startle amplitude across the 30 noise bursts on the last matching before training began. Training The animals were conditioned to light (conditionedstimulus, CS) in a box (20 £ 20 £ 25 cm) with ceiling, side and back walls being constructed of stainless steel and the front door made of transparent Plexiglas. The grid floor of this chamber consisted of stainless-steel rods spaced 1.5 cm apart. The box was located within a ventilated, sound-attenuated chamber (55 £ 55 £ 57 cm). On each of the 2 consecutive days, animals were placed in the training cage, and 5 min later each rat received 10 CS– unconditioned-stimulus (US) pairings, using a 4 s light CS coterminating with a 1 s, 0.6 mA footshock (US) (Kim and Fanselow 1992; Phillips and Le Doux 1992; Silva et al. 2002, 2004). The shocks were delivered through the training cage floor by a constant current generator built with a scrambler (Albarsh Instruments, Brazil). The CS was a light presented through a bulb (6.0 W, 127 V) located in the ceiling

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of the chamber. Stimulus presentation was controlled by a microprocessor and an I/O board (Insight Equipment, Brazil). The inter-trial interval varied randomly between 60 and 180 s. Each animal was removed 5 min after the last shock and was returned to its home cage. The duration of the session was approximately 40 min. At the testing day the rats were injected intraperitoneally 25 min before being placed in the FPS box, with saline or 2 mg/kg DZ. Testing Testing sessions were conducted in the box where the FPS procedure was done. The behavior of animals was recorded by a video camera (Everfocus) positioned in front of the observation chambers, allowing the discrimination of all possible behaviors, with the signal being relayed to a monitor in another room via a closed circuit. A red light from a bulb (6.0 W) located on the back panel of the chamber was turned on during the session to provide minimal illumination of the box. The measure used to assess fear was the time spent in a freezing response during the test period. Freezing was operationally defined as the total absence of movement of the body and vibrissae, accompanied by atleast two of the following responses: arched back, retraction of the ears, piloerection or exophthalmia. The freezing behavior was scored during testing and also subsequently from videotapes by an observer. The rats were placed in testing cages, and 5 min later presented with 60 startle stimuli (noise bursts) at a 30 s inter-stimulus interval. The intensity of the startle stimulus used was 100 dB. Half of startle stimuli were presented in the absence of the CS to provide a baseline (noise-alone trials), and the other half were presented in the presence of the CS (lightnoise trials). In the light-noise trials, the startle stimulus was presented in the last second of a 4 s presentation of light, similar to training sessions. Startle response amplitudes collected from this experiment were stored on computer hard disk, and transferred to tables in a spreadsheet program (Excel; Microsoft Corp.) for the off-line analysis. We also measured body weight from birth until the end of the experiment. Only data from the day of experiment were used. Statistical analysis Body weight data are reported as means ^ SEM and were analyzed by one-way analysis of variance (ANOVA). Behavioral data also are reported as means ^ SEM. The total freezing time was analyzed by two-way (diet X treatment) ANOVA and startle response amplitude for condition (noise-alone and light-noise) was analyzed by three-way (diet X treatment X condition),

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with diet and treatment as between-group and condition as within-group factors. When appropriate, post hoc comparisons were made using the Newman– Keuls test. The level of significance was set at 0.05.

Results Body weight On day 70, the animals of malnourished groups weighed less than well-nourished animals as indicated by a significant effect of diet treatment [F (4.105) ¼ 48.20; p , 0.05]. Post hoc analysis showed a significant higher weight for W as compared with all other M groups, according to Newman–Keuls test, p , 0.05 (Table I).

Behavioral measures Figure 1 illustrates the startle amplitude in the noisealone and light-noise conditions after treatment with saline or DZ. The three-way ANOVA showed a significant effect of treatment [F (1,100) ¼ 5.08; p , 0.05] with DZ decreasing the startle amplitude in both conditions (light-noise and noise-alone). ANOVA also showed a significant effect of condition [F (1,100) ¼ 48.94; p , 0.05] with the startle response intensified, in all groups, in light-noise condition as compared to noise-alone condition. Finally, no significant effects were found on the diet factor or any of the factors interaction. Figure 2 illustrates the freezing time response. A two-way ANOVA showed a significant effect of diet [F (4,100) ¼ 2.58; p , 0.05] with higher freezing duration on W as compared with M7, M14, M21 and M28 groups. Post hoc analysis showed that freezing times of M21 and M28 groups were significantly lower as compared with freezing time of W group. ANOVA also showed a significant effect of treatment [F (1,100) ¼ 29.76; p , 0.05] with DZ decreasing freezing duration. Finally, there was a significant diet X treatment interaction [F (4,100) ¼ 3.51; p , 0.05] in total freezing time. Post hoc analysis showed that DZ decreased freezing time on W and M7 groups but did not produce effect on M14, M21 and M28 groups, i.e. Table I. Body weight of rats at the age 70 days when the behavioral tests were carried out. Data are reported as means ^ SEM for W (n ¼ 22), M7 (n ¼ 22), M14 (n ¼ 22), M21 (n ¼ 24) and M28 (n ¼ 20). *Compared with well nourished group (W), according to Newman–Keuls test ( p , 0.05). Groups Well-nourished Malnourished7* Malnourished14* Malnourished21* Malnourished28*

Weight 434.25 ^ 13.2 388.97 ^ 11.0 359.97 ^ 6.9 287.60 ^ 6.5 287.42 ^ 6.7

as the exposition time to malnutrition increase also increase the resistance to the effects of DZ. Discussion The results presented here show postnatal protein malnutrition, even when imposed for short periods in early life, significantly reduced the body weight of the animals, as previously reported by our group (Santucci et al. 1994; Rocinholi et al. 1997; Fukuda et al. 2002; Françolin-Silva and Almeida 2004) and others (Finger and Green 1983; Bedi 1994; Peeling and Smart 1994; Rocha and Mello 1994). Our results in the ASR indicate that the association was made with success. The amplitudes were higher in the light-noise trial, than in the noise-alone trials, showing that learning occurred in all of the nutritional conditions. So it can be suggested that early malnutrition did not affect the rats’ ability to learn this specific kind of response. In different experimental models that involve associative learning the literature shows that previously malnourished-animals present lower latencies of escape and avoidance in passive-avoidance procedures (De Oliveira and Almeida 1985; Almeida and De Oliveira 1994). It has therefore been suggested that behaviors related to individual survival are preserved (the startle reflex was found in every species of mammals already studied—Koch 1999), even when the animal has been malnourished in early life. This pattern of response can be interpreted as a mechanism of self protection against injury from a predator or from a blow, and of the preparation of a flight/flight response (Koch 1999). On the other hand, malnourished-animals present deficits in other learning tasks. Early postnatal protein malnutrition affects learning (acquisition) and memory (retention) in the distal but not in the proximal cue version of the Morris water maze and changes learning and memory in spaced but not in condensed trials in the same test (Fukuda et al. 2002; Valadares and Almeida 2005). The present data show that the magnitude of the startle response to light-CS was higher than to noise alone presentations in the testing sessions in wellnourished animals. This finding demonstrates that the anxiety-like state of these animals, and not just the aversiveness of the loud-noise, contributes to the fear potentiated startle in rats previously submitted to the association of light and foot shock. As expected, FPS and freezing responses were inhibited by DZ. Especially on the freezing response it was demonstrated those longer periods of exposition to protein malnutrition early in life (14, 21 and 28 days) causes resistance to the effects of DZ as compared with shorter malnutrition periods (7 days) or even with W condition. It is possible that this effect may be attributed to a deficit in performance rather than to a learning deficit. In fact, these animals show

Short-term malnutrition and fear-potentiated startle

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Figure 1. Startle amplitude response on noise-alone and light-noise conditions. Bars represent the mean of ASR and the vertical lines the SEM. W (n ¼ 22), M7 (n ¼ 22), M14 (n ¼ 22), M21 (n ¼ 24) and M28 (n ¼ 20). *Compared with noise-alone condition, in the same nutritional group, according to Newman–Keuls test ( p , 0.05). Sal, saline and DZ, DZ.

Figure 2. Effects of DZ on time of freezing. Bars represent the mean of time of freezing and the vertical lines the SEM. W (n ¼ 22), M7 (n ¼ 22), M14 (n ¼ 22), M21 (n ¼ 24) and M28 (n ¼ 20). *Compared with well-nourished group (W), in the saline (Sal) condition. #Compared with the saline groups in the same nutritional condition, according to Newman–Keuls test ( p , 0,05).

hyperactivity when exposed to a hypoproteic diet for 21 days or more (Almeida et al.1993, 1994, 1998; Moreira et al. 1997). The resistance to the anxiolytic effects of the DZ caused by early protein malnutrition (14, 21 and 28 days) may be due to the reported neural and/or neurochemical alterations in limbic structures such as hippocampus and amygdala (Del Angel-Meza et. al. 2002; Mokler et. al. 2003; Millan 2003; Steiger et. al. 2003; Bremner 2004; Granados-Rojas et. al. 2004; Lister et. al. 2005). In particular, neurochemical changes in the GABAergic hippocampal system may underlie the resistance to the anxiolytic effects of DZ observed in malnourished-animals tested in FPS. The lower freezing time demonstrated by rats malnourished by 21 and 28 days could also be interpreted as the inability of these animals in expressing behavioral inhibition, probably due to their impulsiveness (Tonkiss et al. 1990). Impulsiveness and freezing are mutually exclusive events, which means that if one occurs, the other do not. In this case if the impulsiveness is higher in malnourished-animals, a decrease in freezing response, indeed, would be expected.

Interestingly enough, these rats malnourished by 21 or 28 days show normal baseline acoustic startle responding is spite of decreased inability of performing freezing behavior. This finding supports earlier evidence from this laboratory showing that FPS and freezing are dissociated (Silva et al. 2004; Borelli et al. 2005). The present data also indicate that early postnatal protein malnutrition causes resistance to the anxiolytic effects of DZ as assessed by the FPS test in spite of the reduction in freezing behavior probably caused by the hyperactivity of these animals. Thus, caution needs to be taken in the assessment of fear states in undernourished-animals particularly in tests which the motor reactivity of the animals may be a confounding factor. In summary, animals undernourished early in life and submitted to mild stressful conditions show fear associated with a reduction in the freezing response to light-CS cues. This latter reaction has been attributed to a performance deficit since they are fearful, as assessed by the enhanced startle response displayed by the same animals. Besides, the FPS is resistant to the anxiolytic action of the benzodiazepine DZ. These findings support the contention that early protein

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malnutrition, even imposed for short periods, alters the reactivity of the animals to stressful conditions and to the anxiolytic effects of DZ. The longer the exposure to the deficient diet, the lower the response to the anxiolytic compounds and/or higher the impulsiveness of the animals.

Acknowledgements This work was supported by research grants from FAPESP-Brazil (02/05674-5, 02/03705-0) and CNPq-Brazil (470415/2003-7 and 411029/2003-7). ALFS and was the recipient of a research scholarship from FAPESP-Brazil (00/06330-2). SSA was a recipient of a research scholarship from CNPqBrazil (351507/1996-5). The authors thank Dalmo C. P. Nicola for technical assistance.

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