Prenatal Stress Alters Spine Density and Dendritic Length of Nucleus Accumbens and Hippocampus Neurons in Rat Offspring RUBELIA ISAURA MARTINEZ-TELLEZ, �� �� ELIZABETH HERNANDEZ-TORRES,�� CITLALLI GAMBOA, AND GONZALO FLORES* Laboratorio de Neuropsiquiatr��a, �� Instituto de Fisiolog��a, �� Universidad Autonoma �� de Puebla, 14 Sur 6301, CP 72570, Puebla, Mexico�� KEY WORDS prenatal stress restrained movement Golgi-Cox stain schizophre- nia dendritic morphology spine density ABSTRACT Prenatal stress alters neuronal morphology of mesocorticolimbic struc- tures such as frontal cortex and hippocampus in the adult offspring. We investigated here the effects of prenatal stress on the spine density and the dendrite morphology of hippocampal pyramidal neurons and medium spiny cells from nucleus accumbens in prepubertal and adult male offsprings. Sprague-Dawley pregnant dams were stressed by restraining movement daily for 2 hours from gestational day 11 until delivery. Con- trol mothers remained free in their home cage without water and food during the stressful event. Male offsprings from immobilized and control rats were left to grow until postnatal day (PD) 35 for the prepubertal group, and until PD 65 for the adult group. Spontaneous locomotor activity was assessed and then brains were removed to study the dendritic morphology by the Golgi-Cox stain method followed by Sholl analy- sis. Prenatally stressed animals demonstrated increased locomotion and alterations in spine density in the hippocampus and nucleus accumbens at both ages. However, pre- pubertal males showed an increase in spine density in the CA1 hippocampus with a decrease in CA3 hippocampus, whereas the adult group showed a decrease in the spine density in both of the regions studied. These results suggest that prenatal stress carried out during the middle of pregnancy affect the spine density and basal dendrites of py- ramidal neurons of hippocampus, as well as the dendritic morphology of nucleus accum- bens which may reflect important changes in the mesocorticolimbic dopaminergic trans- mission and behaviors associated with the development of psychiatric diseases such as schizophrenia. Synapse 63:794���804, 2009. V2009 V C Wiley-Liss, Inc. INTRODUCTION Stressful experiences during pregnancy have been implicated in the development of several human dis- orders, which become evident in prepubertal as well as in adult stages (Fumagalli et al., 2007 Knackstedt et al., 2005 Kofman, 2002 Weinstock, 2001, 2008). Results suggest that the younger the individual, the more harmful the stress at later times. These impair- ments include neuroendocrine abnormalities, the hypothalamic-pituitary-adrenal (HPA) axis (Bowman et al., 2004 Richardson et al., 2006 Van den Hove et al., 2006 Weinstock, 2005), metabolic disorders such as low birth weight and/or higher incidence of diabetes (D���mello and Liu, 2006 Seckl and Meaney, 2006) emotional alterations, behavioral and cognitive problems involving hyperactivity, attention deficit in children (Weinstock, 2005), and increased vulnerabil- ity to develop psychiatric disorders such as depression and schizophrenia (Berger et al., 2002 Koeniga et al., 2005 Newport et al., 2002 Van den Hove et al., 2005). Nevertheless, it has been observed that these disorders may not appear if the adequate postnatal Contract grant sponsor: VIEP-BUAP Contract grant number: FLAG-SAL08- G (to G.F.) Contract grant sponsor: PROMEP Contract grant number: BUAP- CA-120 (to G.F.). *Correspondence to: Gonzalo Flores, Laboratorio de Neuropsiquiatr��a, �� Insti- tuto de Fisiolog��a, �� Universidad Autonoma �� de Puebla, 14 Sur 6301, CP 72570 Puebla, Mexico. �� E-mail: gflores@siu.buap.mx Received 28 September 2008 Accepted 23 January 2009 DOI 10.1002/syn.20664 Published online in Wiley InterScience (www.interscience.wiley.com). V V C 2009 WILEY-LISS, INC. SYNAPSE 63:794���804 (2009)

stimulation is given (Barros et al., 2006 Vallee �� et al., 1997). Recently, prenatal stress (PS) research has been focused on procedures applied mainly to pregnant rats due to their resemblance to early embryonic stages of the human brain (Weinstock, 2001) and to the number of animals which can be included to explore the neuronal mechanisms involved in stress- ful events. Findings reveal that both mild and strong PS methods are potentially harmful for the offspring (Fumagalli et al., 2007 Richardson et al., 2006). Since neuronal connections are vulnerable to stress hor- mones during brain development and this may affect plasticity, synaptic formation, neurotransmitter release, dopaminergic and glutamatergic transmis- sion, receptor expression, calcium and ion channels activity, corticostriatal and corticolimbic pathways, and dendritic branching in the offspring (Barros et al., 2006 Berger et al., 2002 Cai et al., 2007 Murmu et al., 2006 Ulupinar et al., 2006 Van den Hove et al., 2006 Weinstock, 2008 Wu et al., 2007). These changes could remain in the offspring and induce neurobiological alterations implicated in the development of schizophrenia and other psychiatric disorders. The information about the effects of PS on neuronal morphology is limited (for a recent review see Wein- stock, 2008), in contrast to postnatal stress studies (for review see Radley and Morrison, 2005). However, in prenatal (Michelsen et al., 2007 Murmu et al., 2006) as well as in postnatal stress (Brown et al., 2005 Cook and Wellman, 2004 Radley et al., 2006a,b), morpho- logical changes on apical dendritic arbor are reported mainly in the prefrontal cortex and hippocampus. Pre- natal studies have shown reduced cell proliferation in the hippocampus, the dentate gyrus (Kofman, 2002), and NAcc (Kawamura et al. 2006 McClure et al., 2004). More recently, alterations have been reported mainly in the apical dendritic arbor of CA3 neurons of hippocampus and pyramidal neurons from PFC (Michelsen et al., 2007). PS studies in basal dendrites of pyramidal neurons from mesocorticolimbic structures as well as in spine density are quite recent (Hosseini-Sharifabad and Hadinedoushan, 2007 Michelsen et al., 2007 Murmu et al., 2006). Findings suggest that PS may induce changes on these dendritic structures according to the age they are observed and to the brain regions studied. Basal and apical dendrites exhibit different input domains, excitability, physical properties, and time development, which may modulate synaptic transmission (for review see Spruston, 2008). Golgi- Cox method has been the most used stain procedure to visualize these processes including spines (Gibb and Kolb, 1998), and in this context, we have pro- posed here to study the effect of PS in the basal den- dritic morphology and the spine density of neurons from the hippocampus and nucleus accumbens in pre- pubertal and adult offspring. Pregnant rats were stressed by restraining move- ment during 2 hours from gestational day 11 until delivery (Patin et al., 2004). Only male pups were included in this study and they remained with the mothers until weaning at postnatal day 21 (PD 21) and were assessed until postnatal day 35 (PD 35) for the prepubertal group or until postnatal day 65 (PD 65) for the postpubertal or adult group. Control ani- mals were born from not immobilized mothers, and were evaluated at the same age. Golgi-Cox staining procedure and Sholl analysis were used to measure branching, dendritic length, and spine density to examine changes in neuronal morphology (Gibb and Kolb, 1998). Additionally, locomotor activity in prepu- bertal and adult animals was assessed as a behav- ioral test. Results indicate significant alterations in dendritic length and the spine density of basal den- drites in pyramidal neurons of hippocampus, as well as important changes in dendritic length and spine density in medium spiny neurons of nucleus accum- bens. Our findings suggest that PS may modify den- dritic length and spine density of basal dendrites in neurons of mesocorticolimbic structures. MATERIALS AND METHODS Animals Fourteen nulliparous female Sprague-Dawley rats weighing 230���250 g (PD 100) were obtained from our facilities at the University of Puebla (BUAP). Rats were housed in a temperature (20���238C), humidity (40���50%), and controlled environment on a 12���12- hour light���dark cycle with free access to food and water. Vaginal smears were collected to determine the stage of estrous cycle and the day of conception. Sub- sequently, each female rat was housed with one male Sprague-Dawley rat on proestro day. The male rats were obtained from the same bioterium at the Univer- sity of Puebla. The day the spermatozoa were found in the smear was designated as day 1 of pregnancy. After birth, the offsprings were housed under the same conditions. All experimental procedures were Abbreviations CNS central nervous system C35 control group of postnatal day 35 C65 control group of postnatal day 65 E35 experimental group of postnatal day 35 E65 experimental group of postnatal day 65 HPA hypothalamic-pituitary-adrenal MPFC medial prefrontal cortex NAcc nucleus accumbens NS nonstressed group S stressed group PD postnatal days PS prenatal stress 795 EFFECTS OF PS ON NEURONAL MORPHOLOGY Synapse