471-479 pascual.qxd

Acta Neurobiol Exp 2007, 67: 471–479 Effects of neonatal maternal deprivation and postweaning environmental complexity on dendritic morphology of prefrontal pyramidal neurons in the rat Rodrigo Pascual1 and S. Pilar Zamora-León2 1Laboratory of Neuroscience, School of Kinesiology, Faculty of Basic Sciences and Mathematics, Catholic University of Valparaiso, Avenida Brasil 2950, Valparaíso, Chile; 2Laboratory of Developmental Neurobiology, Institute of Basic Science, Catholic University of Maule Abstract. It has been reported that periodic maternal separation in rats leads to a variety of endure behavioral, neurochemical and microstructural sequelae associated with the pathophysiology of anxiety disorders. Since it has been proposed that these changes might be permanent, we examined whether environmental complexity aid to recover the structural dendritic impairment induced by neonatal maternal deprivation in the medial prefrontal cortex of the rat. In addition, the anxiety-like behavior was assessed in the elevated plus- maze. Repeated maternal separation between postnatal days 6–21 (3 hours daily) significantly reduced the dendritic material in layer II/III pyramidal neurons and induced anxiety-like behaviors in the elevated plus maze.
Furthermore, environmental stimulation (twice a day, 1 h each) during 12 consecutive days (postnatal days 23–35) failed to recover the neuronal and behavioral disorders induced by neonatal maternal separation. The results demonstrated that (i) neonatal maternal separation severely altered pyramidal dendritic outgrowth in close association with anxiety-like behavior assessed in the elevated plus maze, and (ii) postweaning environmental complexity was unable to recover neither the prefrontocortical neuronal impairment nor the novelty-induced anxiety-like behavior triggered by early maternal deprivation.
Correspondence should be addressed to R. Pascual, Key words: dendritic impairment, environmental complexity, maternal separation, medial prefrontal cortex, elevated-plus maze 472 R. Pascual and S.P. Zamora-León tion may compensate neuronal impairments induced by neonatal MS. Thus, the objective of the present Clinical studies have suggested that disruption of the study was to determine (i) whether MS alters dendritic mother-infant socioemotional bond during the first length and branching of mPFC pyramidal cells, which year of life constitutes a severe early life stressful are involved in the regulation of complex socio-emo- experience that may contribute to the pathophysiology tional behaviors, and (ii) if postweaning EC amelio- of some psychiatric disorders, such as anxiety sub- rates the MS-induced neuronal impairments. In addi- stance abuse disorders and antisocial behavior (Foley tion, we evaluated the effect of both MS and EC on the et al. 2004, Lipman et al. 2001, MacMillan et al. 2001). anxiety-like behavior in the elevated plus-maze, the In addition, several studies performed with laboratory most widely screening assay employed for anxiolytic animals have clearly demonstrated that repeated mater- and anxiogenic agents in rodents.
nal separation (3–6 h) during the first 2–3 weeks of life have long-term consequences on endocrine, behav- ioral, and brain development later in life. For example, maternal separation (MS) in rats showed stress hyper- Animals and experimental design reactivity, anxious behavior in the elevated plus-maze test (EPM), anhedonia, increased ethanol consump- Adult female Sprague-Dawley rats were mated with tion, and hyper-reactivity of the hypothalamic-pitu- colony breeder male rats. One week before birth, dams itary-adrenal (HPA) axis in response to stress (Caldji et were individually housed in standard laboratory al. 2000, Francis and Meaney 1999, Hofer 1996, Huot Plexiglas cages (40 × 30 × 18 cm), with sawdust as et al. 2001, Wigger and Neumann 1999). Most of these bedding in an air-conditioned room under standard behavioral and endocrinal dysfunctions persist laboratory conditions: 12 h light-dark cycle (lights on throughout adulthood (Daniels et al. 2004). In addtion, at 7:00 AM), 22 ± 2°C, and free access to pellets and it has been shown that these long-term effects induced water. The date of birth was designated as postnatal by MS appear to depend upon changes in the structure day 0 (P0). One day after delivery, litters (10–14 pups) and function of the medial prefrontal cortex (mPFC) were cross-fostered and culled to 10 pups each neurons, which are involved in the regulation of the (5 males and 5 females) and housed together with stress response (Spencer et al. 2005, Williams et al. a mother in standard rat cages (50 × 30 × 20 cm). Half 2006). In fact, mPFC pyramidal neurons of MS rats of the litters were randomly assigned to the maternal showed changes in dendritic branching, spine density, separation group (MS, n=20) and the other half to the and monoaminergic fiber innervation (Bock et al. mother-reared control group (MR, n=20). MS pups 2005, Helmeke et al. 2001, Ovtscharoff and Braun were separated every day from their dams for 3 h 2001, Poeggel et al. 2003), indicating that early (1:00–4:00 PM) from P6 until P21. During that time adverse socioemotional experiences, such as maternal MS pups were kept individually in small aluminium deprivation, interfere with the development of neu- foil nests (12 cm diameter, 15 cm height) filled with ronal and synaptic composition of the mPFC.
clean bedding and placed on a 36°C heating blanket.
On the other hand, several reports in laboratory ani- There was no visual, tactile, olfactory or auditory mals indicated that environmental complexity (EC) communication between the pups during the depriva- enhanced the individual’s brain and behavioral devel- tion period. After isolation, the pups were placed back opment. For instance, rats exposed to EC showed an in their home cage. MR pups were left undisturbed, increase in brain weight, cortical depth (Rosenzweig except for cage cleaning 2 times a weak. At P21, all and Bennett 1996), dendritic outgrowth (Green et al. animals were weaned and housed in groups of 4 rats 1983), synaptogenesis (Comery et al. 1996) and neuro- per cage under standard laboratory conditions. At P23, genesis (Olson et al. 2006). Additionally, it has been half of the MS (n=10) and MR (n=10) animals were demonstrated that environmental stimulation during randomly selected (MS-EC and MR-EC groups) and the postweaning period reverses the effects of MS on transferred to an enriched environment (EC), twice both HPA axis and behavioral responses to stress a day for 1 h each during 12 consecutive days (Francis et al. 2002). However, there are no studies (P23–P35). The EC consist of a large Plexiglas/alu- examining whether a similar environmental manipula- minum cage (100 × 100 × 70 cm) with a variety of

Neonatal maternal deprivation 473 Fig. 1. Experimental design. (P1–P35) postnatal days; (MR) maternally-reared animals; (MS) maternally-separated ani- mals; (MR-EC) maternally-reared animals submitted to environmental complexity; (MS-EC) maternally-separated animals submitted to environmental complexity; (EPM) ele- vated-plus maze.
objects, such as tunnels, shelves, running wheels, lad- ders, and different kinds of manipulable objects (e.g., Fig. 2. Representative Golgi-impregnated pyramidal neuron jars, glass balls, wooden objects) that where changed of the rat’s mPFC. Scale bar is 45 mm.
twice a week in order to avoid habituation. At P36, all animals were behaviorally evaluated and, at the fol- Neuronal evaluations lowing day, sacrificed under deep pentobarbital anes- thesia (50 mg/kg, i.p.). The experimental design is In order to study the impact of periodic MS on shown in Fig. 1.
mPFC pyramidal dendritic development, the brains All experimental protocols followed guidelines were stained with the Golgi-Cox-Sholl procedure given at “Principles of laboratory animal care” (NIH (MR, n=10; MS, n=10; MR-EC, n=10; MS-EC, n=10; publication No. 86-23, revised 1996) and were Sholl 1953; Fig. 2), technique that stains 1–5% of ran- approved by the Institutional Animal Care and Use domly distributed neurons (Pasternak and Woolsey Committee at Universidad Católica del Maule.
1975). After 60 days of slow impregnation, brains were dehydrated in ethanol-acetone and ethanol-ether solu- Elevated plus maze tions (50%/50%), embedded in celoidin, and hardened in chloroform vapors. Coronal sections (120 µm thick- At P36, all animals were evaluated in the elevated ness) were cut using a sledge microtome and mounted plus maze (EPM). The EPM was made of black with DPX (Fluka). Plexiglas and consisted of 2 open arms (50 × 15 cm) In order to qualify for the morphometrical evalua- and 2 closed arms (50 × 15 × 20 cm). The apparatus tions, pyramidal cells should fulfill the following crite- was mounted on a fixed base, 50 cm above the floor. ria: (i) have a well-defined pyramidal shape, (ii) show Each rat was placed in the center of the EPM facing an an adequate staining of the soma and dendrites, open arm, and was allowed to freely explore the EPM (iii) have no extensive processes overlapping neigh- for 5 minutes. The number of entries and the time boring neurons, and (iv) be located in a cortical strip spent in the open arms was recorded. Measures were between 100 and 350 mm under the pial surface carried out under dim light condition between (2.2 mm and 3.2 mm anterior to bregma; Paxinos and 8:00–11:00 PM by 2 observers, who were “blind” to Watson 1998). The first 10–12 cells per brain section the rearing condition of the pups. The EPM was meeting the above criteria were traced with the aid of cleaned between each pup with 5% ethanol to remove a camera lucida (Olympus, model BH-DA-LB, 400× odor cues. This test has been validated in rodents to magnification). Two dendritic variables were quanti- evaluate anxiety related behaviors (Andreatini and fied: (i) the mean basilar dendritic length/neuron by Bacellar 2000).
means of an electronic map reader (Pascual et al.

474 R. Pascual and S.P. Zamora-León Fig. 4. Effect of maternal separation and environmental complexity on basal dendritic length. (MR), (MS) maternal- ly-reared and maternally-separated animals, respectively; (MR-EC), (MS-EC) maternally-reared and maternally-sepa- rated animals submitted to environmental complexity, respectively. *P<0.05, **P<0.01 (means ± SD).
Fig. 3. Effect of maternal separation and environmental complexity on (A) time spent (s) and (B) number of entries into the open arms of the elevated-plus maze. (MR), (MS) maternally-reared and maternally-separated animals, respec- tively; (MR-EC), (MS-EC) maternally-reared and maternal- ly-separated animals submitted to environmental complexi- ty, respectively. **P<0.01 (means ± SD).
Fig. 5. Effect of maternal separation and environmental 1996), and (ii) the mean basilar dendritic ramifica- complexity on basal dendritic branching. (MR), (MS) mater- tion/order, determined according to the method of nally-reared and maternally-separated animals, respectively; Coleman and Riesen (1968). This method states that (MR-EC), (MS-EC) maternally-reared and maternally-sepa- dendrites coming out directly from the cell body are rated, animals submitted to environmental complexity, considered as first order, direct branches from first respectively. *P<0.05, **P<0.01.
order dendrites are considered as second order, and so on. A total of 391 cells were sampled from mPFC (MR: time and did not enter the open arms as many times as 87; MS: 98; MR-EC: 94; MS-EC: 112). All cells were the MR control rats (Fig. 3A and B, respectively; drawn and analyzed by the same person in a blind man- P<0.01). Unexpectedly, these behavioral alterations ner to maximize reliability.
appeared to be permanent, since MS environmentally stimulated animals (MS-EC group) did not differ from Statistical analysis MS non-stimulated rats (Fig. 3A and B). On the other hand, MS rats displayed a decrease in dendritic length Unpaired two-tailed t-test statistical analyses were compared to the MR control group ( 28%; P<0.05; performed. Results were expressed as means ± SD and Fig. 4). Again, the EC condition was unable to amelio- differences were considered significant when P<0.05.
rate the dendritic impairment induced by MS ( 44%; P<0.01; Fig. 4). Surprisingly, MS did not affect the number of dendritic branches per neuron (Fig. 5). Only control rat pyramidal cells submitted to EC condition Rats exposed to maternal separation (MS group) did (MR-EC group) showed a significant increase in den- not explore the open arms of the EPM as much as the dritic branching (dendritic orders 4–7; P<0.05, control rats (MR group). In addition, MS rats spent less P<0.01; Fig. 5). Those results indicate that post-

Neonatal maternal deprivation 475 vation on mPFC pyramidal cells. Bock and coauthors (2005) examined the effects of brief and repeated maternal deprivation periods (P1–P3, P5–P7, or P14–P16) on dendritic length development in the rat’s mPFC. These authors showed, opposite to our results, that repeated maternal deprivation during the ontoge- netic stage P5–P7 and P14–P16 induced a significant increase in dendritic length of layer II/III pyramidal neurons. In that report, MS was induced in alternated and shorter periods (1 h); in our study, the pups were submitted to MS during continued and prolonged peri- ods (3 h). This is a critical methodological difference, since it has been demonstrated that short periods of MS actually enhance maternal care and leads to an increased exploratory behavior, less defecation, and a reduced taste neofobia on handled pups (reviewed in Sánchez et al. 2001) and a to a more effective hypo- thalamic-pituitary-adrenal (HPA) axis regulation (Liu et al. 2000). In addition, short alternating periods of MS are similar to normal wild-type maternal behavior, since dams often leave the nest 15–30 min to forage (Jans and Woodside 1990). Therefore, short periods of MS could enhance dendritic outgrowth (or avoid its Fig. 6. Representative camera lucida drawings of prefronto- damage), as reported in the study of Bock and others cortical layer II/III pyramidal cells. (MR), (MS) maternally- (2005). On the contrary, longer periods of MS alter reared and maternally-separated animals, respectively; (MR- maternal care quality and impair HPA negative feed- EC), (MS-EC) maternally-reared and maternally-separated back (Berger et al. 2000, Brake et al. 2004). These animals submitted to environmental complexity, respective- findings suggest that postnatal manipulations per- ly. Scale bar is 50 µm.
formed at different periods can induce opposite effects on neuronal development.
weaning environmental complexity appears to be Even though the mechanisms involved in neuronal unable to offset the neurobehavioral alterations alterations induced by maternal deprivation are not induced by early maternal separation. Representative well known, we speculate that the endocrine response camera lucida drawings of mPFC pyramidal cells are induced by stress, and/or changes in neurotrophin shown in Fig. 6. expression, could be involved. One of the most well At weaning, measurements of body weight did not characterized biological features of maternal depriva- show significant differences between the experimental tion in laboratory animals (McCormick et al. 1998) and groups (MR: 40.6 ± 4.2; MS: 43.7 ± 3.8; n.s.) human infants (Buss et al. 2003, Gunnar et al. 2001) is the hyper-reactivity of the HPA axis in response to socio-emotional stressful experiences. As a conse- quence, glucocorticoids (GCs) levels are extremely We demonstrated that repeated maternal separation elevated (Kalinichev et al. 2002), exerting a broad during the early preweaning period (P6–P21) induced range of deleterious effects on developing GCs recep- a significant dendritic impairment in mPFC pyramidal tor expressing neurons, i.e., prefrontal (Meaney and neurons and a significant reduction in the rat’s explorato- Aitken 1985) or hippocampal pyramidal cells (Meaney ry behavior in the EPM, alterations that were not attenu- et al. 1988). It has been demonstrated that chronic ated by postweaning environmental stimulation. social stress or direct administration of GCs produce To our knowledge, there is only one report (Bock et dendritic atrophy in prefrontal (Wellman 2001) and al. 2005) analyzing the impact of early maternal depri- hippocampal (McKittrick et al. 2000) neurons.
476 R. Pascual and S.P. Zamora-León Alternatively, it has been demonstrated that the adult rats (P90), when the critical dendritic develop- expression of brain ornithine descarboxylase (ODC), the mental period is currently over (Petit et al. 1988). first and rate-limiting enzyme in the synthesis of It is possible that EC, in combination with antide- endogenous polyamines (Kuhn et al. 1978), is dramati- pressant drugs, may be effective in promoting dendrit- cally decreased in MR rats. Since ODC is involved in ic outgrowth. This hypothesis is supported by at least neural growth and differentiation (Shimizu et al. 1965, 2 evidences. First, paroxetine or reboxetine, adminis- Tabor and Tabor 1984), the reduced ODC activity tered during P21–P28, reversed most of the emotional observed in MS rats could induce dendritic outgrowth abnormalities detected in MS rats (Huot et al. 2001, abnormalities in specific neuronal groups (Soulet and Ladd et al. 1999). Second, BDNF expression, the Rivest 2003). On the other hand, since MS significantly major neurotrophin involved in dendritic outgrowth decreases growth hormone (GH) secretion (Kuhn et al. and plasticity (McAllister 1999), is increased in 1978), and GH can regulate neuronal differentiation response to EC (Spires et al. 2004) and antidepressants (Turnley 2005), it is possible that the dendritic out- drugs (such as desipramine or phenelzine) (Dias et al.
growth impairment observed in the current study could 2003). Accordingly, it is possible that the administra- be induced by low GH levels in the brain. Finally, since tion of antidepressants drugs combined with EC could dendritic outgrowth is regulated by the action of brain- attenuate the dendritic impairment induced by neonatal derived neurotrophic factor (BDNF; McAllister 1999), MS. Further preclinical studies will be performed in and MS animals express lower levels of BDNF on both order to prove these hypotheses.
prefrontal and hippocampal neurons (Roceri et al. 2002, The EPM test is widely used to assess anxiety-like 2004; but see Greisen et al. 2005), we cannot discard behaviors and is based on unconditioned responses to that the downregulation of BDNF expression could a potentially dangerous environment, i.e., the avoid- induce neuronal atrophy in MS rats. Further preclinical ance of open and novel spaces. We observed, consis- studies, at the molecular and cellular levels, are required tent with previous reports, that MS rats entered the in order to prove these pathophysiological relationships. EPM open arms less times compared to MR controls Environmental complexity (EC) was not effective (Fig. 3; Daniels et al. 2004, Boccia and Pedersen 2001, recovering both dendritic impairment and exploratory McIntosh et al. 1999, Romeo et al. 2003). The fact that behavior in the EPM. Since EC is a condition of inan- the anxiety-like behavior continued even after 30-days imate and social over-stimulation, it is possible that the of resocialization or EC, suggests that early neonatal social challenge and the novel environment could act MS produced endured behavioral sequelae. Opposite as a stressful condition in vulnerable animals, such as to our results, Hellemans and coauthors (2004) showed MS pups. As a consequence, the “therapeutic” effects that EC significantly ameliorated the anxiety-like of the EC on dendritic and behavioral parameters behavior induced by postweaning social isolation. In described in other studies (Green et al. 1983, that report, social deprivation was imposed later in Rosenzweig and Bennett 1996) could not be effective development (postweaning period) when the environ- in previously deprived animals. This suggestion is con- mental hostile experiences may be less harmful. In our sistent with the fact that EC experienced by control rats study, maternal deprivation was imposed during (MR) significantly promoted mPFC dendritic develop- preweaning, a more vulnerable period. These opposite ment (see Figs 4 and 5). However, the dramatic neu- results suggest how important and critical is the time ronal plasticity observed in mPFC neurons of MR ani- when the deprivation period begins.
mals contrasted with previous data reported by Kolb and colleagues (2003), study that failed to demonstrate any significant change in mPFC dendritic outgrowth of rats submitted to EC. The most critical methodological The present data demonstrated that neonatal mater- difference between both studies was the age of the ani- nal deprivation impaired postnatal mPFC neuronal mals at the time of the EC stimulation. We submitted development and caused long-term anxiety disorders.
the rats to EC during the early postweaning period In addition, postweaning environmental enrichment (P23–P35), when mPFC neurons are highly plastic and was unable to ameliorate any of the neurobehavioral modifiable (Wedzony et al. 2005); in contrast, Kolb variables studied, suggesting that MS induces perma- and others (2003) performed the EC manipulation in nent neurobehavioral sequelae.
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Received 19 January 2007, accepted 4 July 2007

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Bad axe, michigan

Bad Axe, Michigan Wednesday, December 28, 2011 The regular meeting of the Huron County Board of Commissioners was held on Wednesday, December 28, 2011, commencing at 10:15 a.m. in the Board of Commissioners room, Third Floor, Huron County Building, Bad Axe, Michigan. PRAYER AND PLEDGE: The meeting was called to order by Chairman Wruble with The Lord's Prayer and Pledge to the Flag of the United States of America. ROLL CALL: Commissioners present: Chairman Ron Wruble, Steve Vaughan, John Nugent, Clark Elftman, John Horny, John Bodis and Dave Peruski. AGENDA: Commissioner Elftman moves Consent Resolution #31 to the New Business agenda. Motion by Vaughan, seconded by Bodis to approve the agenda as amended. Motion carried. APPROVAL OF MINUTES: Motion by Elftman, seconded by Bodis to approve the Committee of the Whole minutes of December 13, 2011 and the minutes of the regular meeting of December 13, 2011 and Committee of the Whole minutes of December 20, 2011. Motion carried. COMMUNICATIONS: Chairman Wruble turns the following communications over to proper committee.

Ug symposium program-2012

6th Annual North Park University Undergraduate Research Symposium Tuesday, April 17, 2012 North Park University Chicago, Illinois Dr. Rachel Schmale Session 1 John-Tyler Carlson Session 2 Closing Remarks 5:20–5:25 pm Dr. Matthew Schau Following the symposium: Discussion and dinner (served at 5:45 pm) for presenters and advisors in Olssson Lounge, Seminary Building.