NGF expression in the developing rat brain: effects of maternal separation

Developmental Brain Research 123 (2000) 129–134 www.elsevier.com / locate / bres

Research report

NGF expression in the developing rat brain: effects of maternal separation Francesca Cirulli a

a,b ,

*, Enrico Alleva a , Alessia Antonelli c , Luigi Aloe c

Section of Behavioural Pathophysiology, Laboratorio di Fisiopatologia di Organo e di Sistema, Istituto Superiore di Sanita` , Viale Regina Elena 299, I-00161 Rome, Italy b Neurosciences Program, Stanford University School of Medicine, Stanford, CA 94305, USA c Institute of Neurobiology, Consiglio Nazionale delle Ricerche, Viale Marx 15, I-00156 Rome, Italy Accepted 8 August 2000

Abstract A number of studies have shown that mothering style in rodents can produce neuroendocrine, neurochemical and behavioural changes in the adult, although the basic mechanisms initiating this cascade of events still need to be investigated. Long term changes in neuronal function might be due to alterations in the expression of neurotrophins which have been shown to promote neuronal survival, differentiation and function during development, such as Nerve Growth Factor (NGF). NGF is essential for proper development of sympathetic and neural crest-derived sensory neurons of the peripheral nervous system as well as of central cholinergic neurons. In previous studies, using a maternal separation paradigm, we have shown that NGF expression is increased in the dentate gyrus and the hilus of the hippocampus as a result of brief (45 min) maternal separations. In the present study neonatal rats were separated for longer periods of time (up to 3 h) and at different ages during development (9 and 16 days postnatally). Results indicate that the effects of maternal separation on NGF expression are stronger with longer separations and are not restricted to the hippocampal region but can be seen also in other brain areas. Overall these results indicate that external factors, such as the presence / absence of the mother, can modify neurotrophic factor’s availability in the brain, thus indicating NGF as a potential player in environmentally-mediated brain plasticity during development.  2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotrophic factors: expression and regulation Keywords: Nerve growth factor; Gene expression; Development; Maternal separation; Central nervous system; Rat

1. Introduction A number of developmental studies conducted in rodents have clearly indicated that the quality, quantity, and timing of infant stimulation can have long-lasting effects on both behaviour and physiology. Brief (3–15 min) daily manipulations of newborn rats during the first few weeks of neonatal life result in adult subjects which show reduced endocrine responses to stress as well as reduced neurodegeneration and more limited cognitive deficits in old age. Opposite effects can be seen with longer periods of maternal separation (3–6 h) (see [14,18]). *Corresponding author. Tel.: 139-06-4990-2480; fax: 139-06-4957821. E-mail address: [email protected] (F. Cirulli).

Although the long-term effects of early manipulations have been studied quite extensively, little is known of the short-term mechanisms which are activated by these events and which may be responsible for determining individual differences in vulnerability to stress and disease. Separation of the mother from its offspring in rodents initiates a complex response involving both physiological and behavioural changes [11,12]. Most notably, these modifications include a decrease in the synthesis of ornithine decarboxylase as well as changes in hormones secretion [5,12]. Interestingly, maternal deprivation results in an increased expression of the immediate early genes c-fos and the nerve growth factor (NGF)-inducible gene NGFI-B in both the paraventricular nucleus of the hypothalamus and in the cerebral cortex [23]. Neurotrophic factors, such as NGF, play a fundamental role during brain development

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02844-4

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affecting proliferation, survival and neurochemical differentiation of selected neuronal populations both in the peripheral and central nervous system [16,19]. In addition, NGF may also play a role in activity-dependent synaptic plasticity thus contributing to the shaping of neural connections during brain development [3,25]. NGF expression has been localised mainly to the hippocampus and neocortex which represent target areas for basal forebrain cholinergic neurons [9,13,17]. Since almost all granule cells in the rat dentate gyrus are generated postnatally and synaptogenesis and myelination occur in the hippocampal formation well into the fourth postnatal week, changes in the expression of this neurotrophin during critical developmental periods could modify cell number and exert long term effects on neuronal function in this structure [8]. We have hypothesised that some of the long-term differences in brain functioning and in behavioural patterns characterising subjects handled during infancy might be mediated through changes in the expression of neurotrophins such as NGF. In a previous study we have shown that in 3-day-old rats NGF expression is increased in the dentate gyrus and the hilus of the hippocampus as a result of a single brief (45 min) maternal separation [7]. Aim of the present study was to assess the effects of separating neonatal rats from the mother on brain NGF expression using a longer separation period (up to 3 h) and two different developmental ages (9 and 16 days postnatally) when significant neuroanatomical and neurophysiological changes are still taking place [8].

to litter effects. Six litters were used (3 litters on postnatal day (PND) 9 and 3 litters on PND 16). Only three pups were used in each litter; (a) one subject was sacrificed immediately (time 0); (b) one subject was removed and placed in an incubator set at 328C for 1 h; and (c) one subject was removed and placed in an incubator set at 328C for 3 h. Neither food nor water were available during the deprivation period. At the end of the separation period, subjects were immediately sacrificed, their brains dissected, fresh-frozen in iso-pentane and stored at 2808C. All procedures involving animals were performed according to the European Communities Council Directive of November 24th 1986 (86 / 609 / EEC) as implemented in Italy by the Legislative Decree 116 / 1992.

2.3. In situ hybridization Coronal brain sections (15 mm thick) were cut on a cryostat at 2158C. Before hybridization, the sections were fixed in 4% paraformaldehyde (Merck / BDH, Germany). For detection of NGF mRNA an anti-sense (45)-mer oligonucleotide complementary to a region of the NGF mRNA was used. The sequence of the oligonucleotide was as follows: 59-TCGATGCCCCGGCACCCACTCTCAACAGGATTGGAGGCTCGGCAC-39. The oligonucleotide probes were labelled at the 39 end with alpha35 S-dATP (DuPont, Italia) using terminal deoxynucleotidyl transferase (Boehringer Mannheim). In situ hybridization was performed as previously described [7].

2. Materials and methods

2.4. In situ hybridization data analysis 2.1. Animals The subjects used in these experiments were the offspring of male and female Sprague Dawley rats bred in the laboratory. The breeder adult rats were supplied by Charles River Laboratories (Calco, Italy). Upon arrival in the laboratory, animals were housed separately in 40325320 cm Plexiglas boxes, with sawdust as bedding and a metal top, in an air-conditioned room under standard laboratory conditions (temperature 21618C, relative humidity 60610%, lights on from 0930 h to 2130 h). Two weeks later, breeding pairs were formed. The males were removed 15 days after mating and, starting on day 20 post-mating, the females were inspected daily at 0900 for delivery. All litters were culled to 8 pups, 4 males and 4 females, the day following birth (day of birth5day 0) to ensure a balanced sex ratio [6]. Only male pups were used since we have previously shown no sex differences in NGF expression following maternal separation [7].

2.2. Maternal separation procedure A split-litter design was used to avoid confoundings due

For quantitation of NGF mRNA expression in brain sections, positive cells were counted in the dentate gyrus, frontal cortex and the paraventricular nucleus of the hypothalamus. Cell counts were carried out using a Zeiss Axiophot (Zeiss, Germany) microscope and the results analysed by an automatic image analysis system (IAS 2000, vers. 4.0, Delta Sistemi, Rome, Italy). Quantitation of grain clusters was done under dark field illumination at 403 magnification. An average of 6 sections from level A 26 onward [22] were chosen for quantitation of granulebearing cells in the regions selected for each subject (n53 subjects in each final group). Over 110 cells were screened per region and in each section. Three independent hybridizations were performed. Data from sections hybridized with sense probes were used as background and the resulting values subtracted from the original measurements to obtain the final values. All labelled cells showing a grain density at least three times higher than background were considered as positive cells. Data are represented as percentage of positive cells over the total number of cells analysed in each region. Statistical differences between mean percentages of positive cells in the different ex-

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perimental groups were assessed by parametric t-tests for independent samples.

3. Results Overall, NGF expression was affected by maternal separation at all ages considered (Figs. 1–3). A greater number of NGF mRNA positive neurons was found as a result of time following separation in the dentate gyrus of 9- and 16-day-old rats (Fig. 1). Specifically, a higher number of positively labelled cells was found following 1 h of maternal separation in PND 9 rats, t(4)53.98, P,0.01

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and following 3 h of separation in PND 16 pups, t(4)5 3.67, P,0.02 (Fig. 3). Interestingly, changes in NGF expression as a result of maternal separation were also found in the paraventricular nucleus of the hypothalamus. Specifically, significant increases in the percentage of cells positive for NGF mRNA were found following both 1 and 3 h of separation in PND 9 subjects [t(4)510.25, P,0.01; t(4)536.02, P,0.01 respectively], while at the later age a significant increase in NGF expression was seen only following 3 h of separation, t(4)53.38, P,0.02 (Figs. 2 and 3). Changes in NGF expression as a result of maternal separation characterised also the frontal cortical regions (Fig. 3). Nonetheless, these changes were less dramatic

Fig. 1. Changes in NGF expression following maternal separation in the dentate gyrus of neonatal rats. (A) Control slice. In situ hybridization for NGF mRNA in PND 9 subjects at time 0 (403). (B) In situ hybridization for NGF mRNA in PND 9 subjects separated from the mother and kept in an incubator for 1 h (203). (C) NGF expression in the dentate gyrus following 3 h of maternal separation on PND 9 (203). (D) NGF expression following 3 h of maternal separation in PND 16 pups (203).

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Fig. 3. Results of the data quantitation, each data point represents the mean1S.E.M. of the percentage of positive cells over total cells counted in each brain area examined in the different experimental groups (n53 in each final group). Sections from the separated (1 h vs. 3 h) or nonseparated (time 0) subjects were hybridized to an NGF antisense riboprobe. Overall, NGF expression increased with the length of the separation, an effect more pronounced in the hippocampal and hypothalamic areas. Significant intragroup comparisons are marked. * P,0.05; ** P,0.01.

Fig. 2. NGF expression in the paraventricular nucleus of the hypothalamus following maternal separation in PND 9 rat pups. (A) Basal (time 0) NGF expression (203). (B) NGF expression following 1 h of separation (403). (C) Labelled hypothalamic cells in subjects separated for 3 h (403).

compared to the other areas previously described and did not reach statistical significance. No main signs of cell death were found in the brain tissues inspected.

4. Discussion Overall, results from this experiment indicate that, during development, NGF expression can be increased in a number of brain regions and in a time dependent manner, by means of a simple manipulation, i.e. following a brief maternal separation of rat pups. In a previous study we have shown increased NGF expression in the dentate gyrus ad the hilus of the hippocampus of 3-day-old rat pups following 45 min of

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maternal separation [7]. Compared to the previous study, we have extended the length of the separation (3 h) and tested pups at older ages (9 and 16 days postnatally). At both ages tested we found high levels of NGF in the hippocampus, a region where this neurotrophin is known to be highly expressed, as well as in the hypothalamus [2,13]. Other areas of expression include the frontal cortex. Overall, greater increases in NGF expression were seen with longer separation periods, highest levels characterising older subjects. This can be explained considering that the effects of maternal separation on NGF expression are superimposed over the normal developmental programme of cellular differentiation and maturation [13]. Indeed, following 3 h of maternal separation, the increase in NGF expression in the dentate gyrus was greater in PND 16, compared to PND 9 pups. While frontal cortical areas appeared less affected by the manipulations, significant changes in NGF expression were detected in the paraventricular nucleus of the hypothalamus. NGF and its receptors have been previously shown to be present in the hypothalamus, suggesting that this neurotrophin might be involved in neuroendocrine function. Indeed, previous studies have shown changes in NGF expression in this structure as a result of a social-type stressful events in adult subjects [1,24]. As for the mechanism regulating NGF expression, a number of studies suggest that, in contrast to the periphery, in the central nervous system neurotrophins are synthesised in an activity-dependent manner and that they are released upon depolarization of CNS neurons [26]. Regulation of NGF expression by physiological activity has also been reported by Spillantini et al. [24] who have shown increased levels of hypothalamic NGF mRNA following intraspecific aggression in mice, a highly arousing situation. Although the direct mechanism responsible for increasing NGF expression during the maternal separation procedure is still unclear, it is unlikely that glucocorticoid hormones, such as corticosterone, may play a role since significant elevations in this hormone can be seen only after longer separation periods [15]. It has been speculated that the effects of early manipulations on brain and behaviour are not due to the separation procedure per se, but rather to specific changes in maternal behaviour patterns, or lack of, resulting from the manipulation [11]. Mother-infant relationships are dyadic and reciprocal in nature: the mother regulates the infant’s physiology in a highly specific fashion. If the mother is removed, a number of physiological responses occur, a reduction in growth hormone secretion and in ornithine decarboxylase activity, and an increase in corticosterone secretion [5,11,12,15]. Previous studies have indicated that physiological stimuli, such as light, can regulate the production of BDNF and trkB in the visual system [4]. At the younger age tested (PND 9) rat pups have their eyelids still closed, although they are already fully mobile and can move away from the nest area. Our data indicate that an

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increase in NGF expression represents yet another physiological response to maternal separation, although the specific maternal behavior (or lack of) involved in this response needs to be investigated. Changes in NGF expression in the postnatal rat brain as a result of maternal separation could have both short- and long-term consequences [21]. We don’t know whether, following a brief separation, NGF levels could be normalized by maternal presence. Also, the expression of this neurotrophin could be still elevated in previously separated adult subjects [20]. One aspect that will need to be addressed is how long NGF expression stays elevated, after it has been induced. Another important question is whether such changes do affect brain plasticity and how. This issue has not been specifically addressed in this experiment but is currently under study. It has been argued that early handling is one of the strongest models for environmentally-mediated brain plasticity whereby the development of those circuits regulating the stress response is matched with specific environmental demands [10]. Because of the fundamental role played by NGF in brain development, changes in the expression of this neurotrophin following external stimulation suggest that it might be one of the mediators of such plasticity.

Acknowledgements This work was supported by ISS (grant 1997–2000), ‘‘Role of NGF in stress / coping and in pain regulation’’. Supported by ASI, grant ASI-ARS-98-45. We thank Dr. Alessandra Micera and Dr. Flavia Chiarotti for their help in data analysis and Ms. Carla Tascone for formatting the manuscript.

References [1] E. Alleva, S. Petruzzi, F. Cirulli, L. Aloe, NGF regulatory role in stress and coping of rodents and humans, Pharmacol. Biochem. Behav. 54 (1996) 65–72. [2] C. Ayer-LeLievre, L. Olson, T. Ebendal, A. Seiger, H. Persson, Expression of the b-Nerve Growth factor gene in hippocampal neurons, Science 240 (1988) 1339–1341. [3] A. Cellerino, L. Maffei, The actions of neurotrophins in the development and plasticity of the visual cortex, Progr. Neurobiol. 49 (1996) 53–71. [4] E. Castren, F. Zafra, H. Thoenen, D. Lindholm, Light regulates expression of brain-derived neurotrophic factor mRNA in rat visual cortex, Proc. Natl. Acad. Sci. USA 89 (1992) 9444–9448. [5] F. Cirulli, D. Santucci, G. Laviola, E. Alleva, S. Levine, Behavioral and hormonal responses to stress in the newborn mouse: Effects of maternal deprivation and chlordiazepoxide, Dev. Psychobiol. 27 (1994) 301–316. [6] F. Cirulli, W. Adriani, G. Laviola, Sexual segregation in infant mice: Behavioural and neuroendocrine responses to d-amphetamine administration, Psychopharmacology 134 (1997) 140–152. [7] F. Cirulli, A. Micera, E. Alleva, L. Aloe, Early maternal separation

134

[8]

[9] [10] [11] [12]

[13]

[14] [15]

[16]

[17] [18]

F. Cirulli et al. / Developmental Brain Research 123 (2000) 129 – 134 increases NGF expression in the developing rat hippocampus, Pharmacol. Biochem. Behav. 59 (1998) 853–858. B. Crain, C. Cotman, D. Taylor, G. Lynch, A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat, Brain Res. 63 (1973) 195–204. A. Cuello, Effects of trophic factors on the CNS cholinergic phenotype, Progr. Brain Res. 109 (1996) 347–358. D. Francis, M.J. Meaney, Maternal care and the development of stress responses, Curr. Op. Neurobiol. 9 (1999) 128–134. M.A. Hofer, Relationships as regulators: A psychobiologic perspective on bereavement, Psychosom. Med. 46 (1984) 183–197. C.M. Kuhn, S.R. Butler, S.M. Schanberg, Selective depression of serum growth hormone during maternal deprivation in rat pups, Science 201 (1978) 1034–1036. T.H. Large, S.C. Bodary, D.O. Clegg, G. Weskamp, U. Otten, L.F. Reichardt, Nerve growth factor gene expression in the developing rat brain, Science 234 (1986) 352–355. S. Levine, Infantile experience and resistance to physiological stress, Science 126 (1957) 405–406. S. Levine, D.M. Huchton, S.G. Wiener, P. Rosenfeld, Time course of the effect of maternal deprivation on the hypothalamic-pituitaryadrenal axis in the infant rat, Dev. Psychobiol. 24 (1991) 547–558. R. Levi-Montalcini, B. Booker, Destruction of sympathetic ganglia in mammals by an antiserum to the nerve-growth promoting factor, Proc. Natl. Acad. Sci. USA 42 (1960) 373–384. G.R. Lewin, Y.-A. Barde, Physiology of neurotrophins, A. Rev. Neurosci. 19 (1996) 289–317. M.J. Meaney, D.H. Aitken, S. Bhatnagar, C. Van Berkel, R.M.

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

Sapolsky, Postnatal handling attenuates neuroendocrine, anatomical, and cognitive impairments related to the aged hippocampus, Science 238 (1988) 766–768. W.C. Mobley, J.L. Rutkowsky, G.I. Tennekoon, K. Buchanan, M.V. Johnston, Choline acetyltransferase activity in striatum in neonatal rats increased by nerve growth factor, Science 229 (1985) 284–287. A.K. Mohammed, B. Winblad, T. Ebendal, L. Larkfors, Environmental influence on behaviour and nerve growth factor in the brain, Brain Res. 528 (1990) 62–72. U. Otten, J.B. Baumann, J. Girard, Stimulation of the pituitaryadrenocortical axis by nerve growth factor, Nature 282 (1979) 413–414. N.M. Sherwood, P.S. Timiras (Eds.), A Stereotaxic Atlas of the Developing Rat Brain, University of California Press, Berkeley, 1970. M.A. Smith, S.Y. Kim, H.J. van Oers, S. Levine, Maternal deprivation and stress induce immediate early genes in the infant rat brain, Endocrinology 138 (1997) 4622–4628. M.G. Spillantini, L. Aloe, E. Alleva, R. De Simone, M. Goedert, R. Levi-Montalcini, Nerve growth factor mRNA and protein increase in a mouse model of aggression, Proc. Natl. Acad. Sci. USA 86 (1989) 8555–8559. H. Thoenen, Neurotrophins and neuronal plasticity, Science 270 (1995) 593–598. F. Zafra, B. Hengerer, J. Leibrock, H. Thoenen, D. Lindholm, Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors, EMBO J. 9 (1990) 3545–3550.