Introduction 50 1990 1995 1995 2004 1978 1998 1995 1998 1998 2002 1998 2004 2005 1962 1994 2002 1994 2003 2001 Material and methods Animals The offspring of 4 male and 8 female C57BL/6J mice (8 weeks old) obtained from Taconic Europe (Taconic Farms Inc., DK) were used in this study. The animals were acclimatized to the animal facility for 1 week. Multiparous females were used, since there is a higher rate of offspring survival. The animals were housed under a 12:12 h light/dark cycle (lights on at 6 a.m.) with constant temperature (21 ± 2°C) and humidity (52 ± 2%) in Macrolon type III cages with environmental enrichment in the form of wood splints bedding (aspen 4HV), wood shavings, 1 piece of Aspen Corner 15, 1 standard mouse house made of recycled cardboard, 1 aspen chewing stick size medium, and pads of nesting material (all obtained from Brogaarden, DK). Food (Altromin pills NR 1324) and tap water were available ad libitum. Two females were placed in a male’s cage for a period of 1 week to ensure conception, followed by separation of the two females to their own cages. Maternal separation N The 24-h deprivation was carried out on PND 9 starting at 8 a.m. The pups remained in the home cage but were placed in a separate room with the same temperature, humidity and lighting conditions as the home stable. The cage was placed on a heating pad, which had a constant temperature of 31°C. No food or water was available during the separation. The dam was placed in a cage with similar facilities as the home cage in the home stable. The pups were checked every 3 h, using a red light during the night. Body weights were recorded before and after separation. Immediately after 24 h the dams were returned to the home cage and reunited with the pups. Test for anxiety and fear related behaviour 1934 1955 1985 Open field test (OFT) The open field consisted of a circular wooden platform (diameter 90 cm) surrounded by a 43 cm high wall with a camera mounted directly above. A central circle of 31 cm diameter was defined in the behaviour analysis software. Three 60-W light bulbs illuminated the arena. On the day of testing each animal was transported in a cardboard box to the centre of the open field and behaviour was recorded for 10 min. After the trial the maze was cleaned with a solution of acetic acid and soap water and faecal boli were counted. The following parameters were calculated; total distance moved (cm), time spent in central circle and time spent in peripheral zone (expressed as % of session duration). Elevated plus maze The plus maze was elevated 50 cm above the ground and consisted of two opposing open arms (21 cm × 8 cm) connected by a central square (8 cm × 8 cm) to two opposing enclosed arms of the same size with 32 cm high walls. A video camera placed above the maze recorded the animals’ behaviour. On the day of testing, the animal was placed in the centre of the maze and behaviour was recorded for 10 min. Between trials the maze was cleaned as described for the OFT. The following parameters were calculated; total duration (s) in open and closed arms, the number of entries into the open and closed arms and the total distance moved (cm). From these parameters the ratio of entries into the open arms to the total number of arm entries, and the ratio of time spent in the open arms to time spent in both open and closed arms was calculated. Test for spatial memory; Barnes maze The Barnes maze (Barnes 1975) consisted of a circular, white-coated platform 90 cm in diameter and elevated 50 cm over the ground. Sixteen 5 cm wide holes were evenly distributed around the perimeter, 2.5 cm from the edge. A pair of rails was placed under two opposing holes to hold the hidden escape box. The escape box was a dark plastic storage box with a 5 cm diameter hole in the lid. A dark cylindrical cardboard tube (7.5 cm × 7 cm high) with a lid was used as the transport and start chamber. Three 60-W bulbs illuminating the maze and high irregular rock and techno music played from a computer in a random manner provided the aversive stimuli. As with the previous test, a digital video camera mounted above the maze recorded animal’s behaviours. Shaping For 2 days before testing commenced, the animals were trained to enter the hidden escape box. Using the transport cylinder, the mouse was placed near the edge of the target hole with the hidden escape box underneath. Two cardboard walls blocked entry to adjacent holes. Only dim lightning and no noise was used during this phase of the experiment. The animal was allowed 5 min to enter the escape box and if this failed, it was placed manually inside. When the animal entered the box it was quickly carried to the home cage. Acquisition trials Six consecutive trials were given, one per day. The animal was placed in the transport cylinder, oriented in a random direction, in the centre of the maze. The aversive stimuli were turned on and the cylinder removed. Recording in the behaviour-observation software began immediately after the experimenter had left the room. The trial ended after 5 min or when the animal entered the hidden escape box. If the animal failed to enter the box or re-entered the maze after recording was stopped, the aversive stimuli were turned back on and the animal was allowed 5 min more to enter the escape box. If that also failed, the animal was manually placed in the box. After completion of each trial, the box was placed in the home cage. Reversal trials Three days after acquisition trials, the escape box was placed underneath the hole opposite to the hole that had been the target during acquisition training. Reversal training was conducted for seven consecutive days as described above. Parameters Each of the 16 escape holes in the Barnes maze was defined as a separate zone-of-interest in the behaviour analysis software. The following parameters were analysed: latency to target [time from start of the trial to first entry into the target hole zone (s)], total distance moved (cm) and error frequency (number of visits to other holes than the target hole). During reversal training two additional parameters were analysed: the mean number of visits to the old target hole over the seven trials (the hole where the escape box was located during acquisition training) and the mean number of visits to the two holes adjacent to the old target hole. 1995 1979 1995 2000 2002 2004 1 Fig. 1 Random Serial Spatial  Fixation and embedding 2 Sectioning The whole hemisphere was cut horizontally with a Leica (model SM 2400) microtome with a microtome setting of 40 μm thickness. All sections were mounted on silicone-coated glass (Frost +). After a minimum of 24 h in a 40°C heating cupboard, the sections were sampled systematic uniformly randomly (SURS). The first section was randomly selected from a random number table, hereafter every 6th section was sampled systematically for staining and counting. This provided a total of 8–10 sections containing the hippocampus per specimen. To be able to account for block advance (BA) the block height was measured for every 100th section. The BA determines the hitting probability of the particles within the block (see later for calculation). Furthermore, the paraffin shrinkage effect was calculated and amounted to about 70%. The sections were stained with a modified Vogt’s Cresyl violet acetate (Armed forces). Optical design equipment x y z Definitions and divisions of the hippocampus the dentate gyrus the hilus of the dentate gyrus regio inferior region superior subiculum 1956 2 1991 Fig. 2 A schematic drawing of the hippocampus with the five subregions identified in this study. Dg, the granule cell layer of the dentate gyrus; h/CA4, hilus of the dentate gyrus; ri, regio inferior; CA3/2, rs, region superior; CA1, s, subiculum  Stereological equations N N \documentclass[12pt]{minimal}
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$$ N = \frac{1} {\text{ssf}} \times \frac{1} {\text{asf}} \times \frac{1} {\text{hsf}} \times \sum {Q^{ - } } \times 2 $$\end{document} Q − x y \documentclass[12pt]{minimal}
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$$ \text{asf} = \frac{a({\text{frame}})\,\upmu{\text{m}}^{2} } xy\,\upmu{\text{m}}^{2} $$\end{document} \documentclass[12pt]{minimal}
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$$ {\text{hsf}}: = \frac{h} {{{{\mathop t \limits^ -}_{{q^{ - } } } }}}\quad {\text{where}}\  {\mathop t\limits^ - }_{{q^{ - } }} = \frac{{{\sum\limits_i {{\left( {t_{i} q^{ - }_{i} } \right)}} }}} {{{\sum\limits_i {q^{ - }_{i} } }}} $$\end{document} q − 1988 2001 Estimation of total volume—the Cavalieri estimator 1987 N V 3 Error predictions 1987 1999 1 1999 Table 1 Example of how the CE is calculated for neuron number and volume respectively Section Q j − Q i  Q i Q i Q i Q i  Q i Section P j − P i P i P i P i P i  P i 1 11 121 616 231 1 2 4 22 8 2 56 3,136 1,176 1,064 2 11 121 44 33 3 21 441 399 378 3 4 16 12 12 4 19 361 342 57 4 3 9 9 3 5 18 324 54 198 5 3 9 3 9 6 3 9 33 48 6 1 1 3 3 7 11 121 176 165 7 3 9 9 6 8 16 256 240 352 8 3 9 6 15 9 15 225 330 90 9 2 4 10 8 10 22 484 132 – 10 5 25 20 – 11 6 36 – – 11 4 16 – – Sum 198 5,514 3,498 2,583 Sum 41 223 138 97 \documentclass[12pt]{minimal}
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\begin{document}$$ {\text{Noise}} = {\sum {Q^{ - } = 198} } $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \begin{aligned}{} {\text{Noise}} &=  0.0724 \times \frac{b} {{{\sqrt a }}} \times {\sqrt {n \times {\sum P }} } \\ &=  0.0724 \times 10 \times {\sqrt {11 \times 41} } \\ &= 15.4 \\ \end{aligned} $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \begin{aligned}{} {\text{Var}}_{{({\text{surs}})}} &= {\left( {\frac{{{\sqrt {3{\left( {A - {\text{Noise}}} \right)} - 4B + C} }}} {{240}}} \right)} \\ &= {\left( {\frac{{{\sqrt {3{\left( {5,514 - 198} \right)} - 4 \times 3,498 + 2,583} }}} {{240}}} \right)} \\ &= 18.9 \\ \end{aligned} $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \begin{aligned}{} {\text{Var}}({\sum a }) = & {\left( {\frac{{{\sqrt {3{\left( {A - {\text{Noise}}} \right)} - 4B + C} }}} {{240}}} \right)} \\ = & {\left( {\frac{{{\sqrt {3{\left( {223 - 154} \right)} - 4 \times 138 + 97} }}} {{240}}} \right)} \\ = & 0.70 \\ \end{aligned} $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \begin{aligned}{} {\text{CE}}_{{{\left( {Q^{ - } } \right)}}} = & \frac{{{\sqrt {{\text{Noise}} + {\text{Var}}_{{{\text{surs}}}} } }}} {{{\sum {Q_{i} } }}} \\ = & \frac{{{\sqrt {198 + 18.91} }}} {{198}} \\ = & 0.07 \\ \end{aligned} $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \begin{aligned}{} {\text{CE}}{\left( {{\text{Vol}}} \right)} = & \frac{{{\sqrt {{\text{Noise}} + {\text{Var}}_{{{\text{surs}}}} } }}} {{{\sum {Q_{i} } }}} \\ = & \frac{{{\sqrt {15.4 + 0.70} }}} {{41}} \\ = & 0.098 \\ \end{aligned} $$\end{document} 1987 1988 1990 Q − Statistical analyses t U P Results Only significant behavioural results are depicted and statistically elaborated. Body weights P t P t Open field test P t Elevated plus maze P P t 3 Fig. 3 N N P P t  F (9,171)  P F (9,11) P F (9,11)  P F (9,171) P There was no significant difference in any of the other parameters analysed. Barnes maze Acquisition trials F (4.3,77.4)  P F (5,90)  P F (3.9,70.1) P Reversal trials 4 5 6 F (3.1;55.95)  P F (1.85;33.41)  P F (3.43,61.68) P F (1, 18) P 6 5 P Fig. 4 Left Trial progress Trial x Group P Right P U P N N  Fig. 5 P P U  Trial x Group N N  Fig. 6 N N Trial progress P N N  4 t (17.5) P t (17.5) P 1950 1995 1997 Search strategies 7 (2)  P (2)  P Fig. 7 N N  Neuron number 2 Table 2 Overview of the stereological sampling used in this study  Subiculum CA4 CA1 CA3 DG MS (maternal separated) 2 770–1,160 710–1,100 259 259 259 Z 20 20 20 20 20 X Y 175 80 100 100 170 Q − 208 ± 36.5 140 ± 23.8 254 ± 15.7 193 ± 10.3 205 ± 15.4 tq − 39.5 ± 0.2 40.0 ± 0.30 39.0 ± 0.3 38.9 ± 0.30 39.3 ± 0.30 Height sampling fraction, hsf 1.97 ± 0.01 2.00 ± 0.01 1.95 ± 0.02 1.95 ± 0.01 1.96 ± 0.02 Area sampling fraction, asf 57.4 ± 11.5 16.8 ± 4.06 38.7 ± 0.00 30.7 ± 0.01 111 ± 0.00 Section sampling fraction (k), ssf 6 6 6 6 6 Guard zone, (μm) 5.5–7 5.5–7 5.5–7 5.5–7 5.5–7 SFR (Control) 2 1,222 ± 61.6 1,388 ± 34.7 197 ± 0.1 240 ± 6.2 197 ± 0.1 Z 20 20 20 20 20 X Y 200 95 100 100 170 Q − 245 ± 14.7 190 ± 14.4 215 ± 13.6 188 ± 11.8 201 ± 7.5 tq − 39.0 ± 0.60 39.1 ± 0.5 38.5 ± 0.6 38.2 ± 0.4 38.6 ± 0.4 Height sampling fraction, hsf 1.95 ± 0.03 1.95 ± 0.02 1.93 ± 0.03 1.91 ± 0.02 1.93 ± 0.02 Area sampling fraction, asf 33.3 ± 1.62 6.46 ± 0.29 50.9 ± 0.03 42.0 ± 1.28 147 ± 0.07 Section sampling fraction (k), ssf 6 6 6 6 6 Guard zone, (μm) 5.5–7 5.5–7 5.5–7 5.5–7 5.5–7 3 P t 8 3 Table 3 Total estimated neuron number in five subregions of the hippocampus N Sex Subiculum CA4 CA1 CA3 DG Total Subject 3 CE 3 CE 3 CE  3 CE  3 CE 3 CE  Female 331 0.08 42.9 0.13 292 0.06 190 0.07 672 0.07 1.529 0.09  Female 264 0.10 34.8 0.13 243 0.06 189 0.07 576 0.07 1.307 0.09  Female 371 0.08 18.6 0.18 303 0.06 226 0.06 474 0.08 1.393 0.09  Female 107 0.08 21.2 0.08 142 0.08 112 0.09 334 0.10 716 0.09  Female 148 0.07 23.1 0.07 213 0.07 144 0.08 354 0.10 882 0.08  Female 132 0.07 20.6 0.08 262 0.06 174 0.07 665 0.07 1.254 0.07  Female 138 0.07 20.2 0.07 155 0.08 163 0.08 552 0.07 1.028 0.07  Female 189 0.12 67.9 0.13 202 0.07 134 0.08 460 0.08 1.053 0.10  Female 144 0.05 23.1 0.07 230 0.07 171 0.07 597 0.07 1.166 0.07  Female 197 0.05 20.9 0.07 229 0.07 203 0.07 484 0.09 1.135 0.07  Male 237 0.10 67.2 0.12 257 0.06 191 0.07 487 0.09 1.239 0.09  Male 242 0.10 37.4 0.13 224 0.06 186 0.07 788 0.06 1.488 0.09 Mean  208 0.08 33.2 0.11 229 0.07 174 0.07 538 0.08 1.182 0.08  N  0.50  0.36  0.28  0.21  0.26  0.25   Female 186 0.07 26.3 0.07 285 0.07 175 0.08 804 0.07 1.476 0.07  Female 176 0.07 30.2 0.09 240 0.07 191 0.08 658 0.08 1.296 0.08  Female 169 0.07 31.5 0.07 273 0.07 205 0.07 655 0.08 1.333 0.07  Female 194 0.05 25.3 0.09 182 0.08 186 0.07 617 0.06 1.204 0.08  Female 162 0.08 34.1 0.09 278 0.07 199 0.08 725 0.07 1.397 0.05  Male 157 0.08 20.3 0.09 206 0.08 115 0.09 613 0.09 1.112 0.07  Male 221 0.06 28.0 0.07 285 0.07 191 0.07 644 0.09 1.368 0.07 Mean  181 0.07 27.9 0.08 250 0.07 180 0.08 674 0.08 1.312 0.08 CV  0.07  0.12  0.17  0.06  0.11  0.08  t  P  P  P  P  P  P  P Fig. 8 N N P t filled circle open circle Sub CA 4 DG  Volume t 4 Table 4 Total estimated volume in five subregions of the hippocampus N Sex Subiculum CA4 CA1 CA3 DG Total Subject 2 CE 2 CE 2 CE 2 CE 2 CE 2 CE  Female 4.33 0.05 0.69 0.08 1.46 0.05 1.52 0.05 1.80 0.03 9.79 0.05  Female 3.38 0.05 0.69 0.06 1.14 0.06 1.39 0.05 1.23 0.04 7.84 0.05  Female 4.25 0.05 0.59 0.07 1.54 0.05 1.72 0.04 1.18 0.04 9.29 0.05  Female 1.66 0.12 0.60 0.07 0.52 0.09 1.00 0.06 1.06 0.04 4.84 0.08  Female 2.40 0.09 0.86 0.06 0.79 0.08 1.26 0.05 1.29 0.04 6.59 0.07  Female 1.73 0.12 0.65 0.07 1.07 0.06 1.39 0.05 1.33 0.03 6.17 0.07  Female 2.26 0.10 0.69 0.06 0.68 0.09 1.42 0.05 1.22 0.04 6.26 0.07  Female 2.29 0.07 1.04 0.08 1.00 0.06 1.26 0.05 1.12 0.06 6.71 0.06  Female 2.15 0.07 0.79 0.06 0.91 0.07 1.45 0.05 1.13 0.03 6.43 0.06  Female 3.00 0.06 0.74 0.06 0.98 0.07 1.47 0.05 1.13 0.06 7.32 0.06  Male 2.98 0.06 1.01 0.06 1.07 0.06 1.39 0.05 1.15 0.05 7.00 0.06  Male 3.31 0.05 0.91 0.05 1.22 0.06 1.63 0.04 2.18 0.03 9.25 0.05 Mean  2.81 0.08 0.77 0.07 1.03 0.07 1.41 0.05 1.32 0.04 7.34 0.06  N  0.32  0.20  0.29  0.13  0.25  0.20   Female 2.97 0.08 0.78 0.07 1.26 0.06 1.69 0.04 1.56 0.03 8.25 0.06  Female 2.29 0.09 0.91 0.07 1.01 0.06 1.22 0.05 1.45 0.03 6.88 0.07  Female 2.51 0.09 0.87 0.06 1.16 0.06 1.88 0.04 1.38 0.04 7.82 0.06  Female 2.55 0.09 0.52 0.09 0.85 0.07 1.30 0.05 0.73 0.04 6.05 0.07  Female 2.10 0.11 0.86 0.07 0.98 0.07 1.48 0.05 1.30 0.04 6.73 0.07  Male 2.32 0.10 0.52 0.10 1.03 0.07 1.19 0.05 1.73 0.05 6.78 0.08  Male 2.83 0.08 0.70 0.07 1.17 0.05 1.46 0.05 1.58 0.04 7.72 0.06  Mean  2.51 0.09 0.75 0.08 1.07 0.05 1.46 0.05 1.39 0.04 7.18 0.07 CV  0.12  0.19  0.13  0.17  0.23  0.11  t  P  P  P  P  P  P  Discussion 1993 1993 1994 1999 2001 2004 1999 1999 1999 2004 2004 2004 2002 2002 1999 1988 1988 1995 1979 2002 1979 1995 1965 1975 1982 1996 1998 1997 1997 1998 1998 2001 1999 2000 2000 2004 2004 2005 1997 1998 1999 2000 2000 2005 We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain. We found that a single 24 h maternal separation on PND 9 could elicit a reduced stress response in the elevated plus maze and induce perseveration behaviour in the Barnes maze. Further, a 20% reduction in total neuron numbers was found in the dentate gyrus of the hippocampus. Ethics The experiment was carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the Danish legislation regulating animal experiments (Animal care and housing BEK nr 687 from 25/07/2003). The Danish Animal Experiments Inspectorate approved the protocols (Journal No. 2003/561–781).