Neuroanatomical changes in the adult rat brain after neonatal lesion of the medial prefrontal cortex

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Experimental Neurology 209 (2008) 199 – 212 www.elsevier.com/locate/yexnr

Neuroanatomical changes in the adult rat brain after neonatal lesion of the medial prefrontal cortex Steffen Klein a,⁎, Michael Koch a , Kerstin Schwabe b a

Brain Research Institute, Department of Neuropharmacology, University of Bremen, PO Box 33 04 40, 28334 Bremen, Germany b Department of Neurosurgery, Medical University, MHH, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Received 21 March 2007; revised 12 August 2007; accepted 17 September 2007 Available online 29 October 2007

Abstract After neonatal lesions of the medial prefrontal cortex (mPFC) the lesion cavity are considerably replaced by neuronal tissue (“filled-in-tissue”). This is accompanied by sparing of some behavioral functions related to the mPFC, while others are deteriorated. We here investigated the neuroanatomical integration of the filled-in-tissue. Bilateral neonatal (postnatal day 7) excitotoxic lesions were induced by microinjection of ibotenate into the mPFC. Immunohistochemical staining for the neuronal marker NeuN and the glia cell marker GFAP in adult rats confirmed that the filled-in-tissue consists of neuronal tissue with disturbed lamination but without reactive gliosis. The afferents to the mPFC were marginally altered as shown by almost normal distribution of retrogradely labeled cells in regions projecting to the mPFC after injection of the retrograde tract tracer fluorogold into the filled-in-tissue. Although virtually no transport was found after injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the filled-in-tissue, injections of fluorogold into the nucleus accumbens and mediodorsal thalamus revealed that neurons of the filled-in-tissue project at least to the nucleus accumbens. Additionally, after neonatal mPFC lesions the density of parvalbumin-immunoreactive, presumably GABAergic interneurons, was increased in the amygdala and hippocampus and myelin sheaths were reduced in several cortical and subcortical regions as shown by goldchloride staining. Together, these findings indicate restored anatomical connectivities of the filled-in-tissue that might be responsible for the sparing of behavioral function in some mPFC related tasks, but may also explain disturbed function as a consequence of faulty information transfer between subcortical regions and the filled-in-tissue of the mPFC. © 2007 Elsevier Inc. All rights reserved. Keywords: Neurodevelopment; Altered connectivity; Inhibitory neurons; Tract tracing; Parvalbumin; Myelin

Introduction The lesion cavity after neonatal lesion of the rat medial prefrontal cortex (mPFC) inflicted around postnatal day (PND) 10 has been shown to be filled in to a considerable extent (e.g., De Brabander et al., 1991a,b; Kolb et al., 1996a,b; Kolb and Whishaw, 1981; Schwabe et al., 2004), which is in the following termed “filled-in-tissue” to contrast the “scar” found after adult lesion (Kolb et al., 1998a,b). This anatomical recovery of the mPFC lesion is accompanied by sparing of a variety of mPFC-related functions that have been shown to be compromised by adult lesions of the mPFC, like working memory (e.g., Kolb and Gibb, 1991, 1993; Kolb and Whishaw, 1981). Notably, the functional sparing after neonatal lesion ⁎ Corresponding author. Fax: +49 421 218 4932. E-mail address: [email protected] (S. Klein). 0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2007.09.015

correlates with the extent of the filled-in-tissue and can be reversed by second, adult lesions of this tissue, indicating that the functional sparing is not merely a consequence of compensation by other brain regions, but that the filled-in-tissue itself is of functional importance (Dallison and Kolb, 2003). However, function of other behavioral domains, e.g., social behavior, perseveration, and food hoarding, have been shown to be compromised after neonatal mPFC lesions (De Brabander et al., 1991a; Kolb et al., 1996a; Kolb and Whishaw, 1981; Schneider and Koch, 2005a,b; Schwabe et al., 2004). In line with this, neonatally lesioned animals show various neuroanatomical and physiological alterations, e.g., the development of an abnormally thin cortex (Kolb et al., 1983, 1996b; Kolb and Gibb, 1990, 1991; Kolb and Whishaw, 1981, 1989) and abnormalities in the projection pattern of several subcortical brain regions (Kolb et al., 1992, 1994). Additionally, neonatally lesioned animals show increased responsiveness to dopamine

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receptor agonists and stress. This has been related to compromised function of the nucleus accumbens (NAC), a main projection site of the intact mPFC (Bennay et al., 2004; Flores et al., 1996). The process of refilling after neonatal lesions has been investigated in several studies (e.g., Kolb et al., 1998a,b), but to date, to the best of our knowledge, no one comprehensively investigated the integration of the filled-in-tissue itself into neuronal circuits by examining its anatomical connectivity. The expression of the calcium binding protein parvalbumin is important for neuronal network function, since parvalbumin expressing interneurons synapse on the proximal dendrites or initial axon segment of pyramidal projection neurons (Lewis, 1995, 2000; Lewis et al., 2005; Woo and Lu, 2006). Interestingly, the expression of this protein starts around the time of our lesions (Solbach and Celio, 1991). Another process that might be influenced by neonatal lesion is myelination, which is important for speed and accuracy of neurotransmission. The developmental peak of myelination in rodents occurs around day 14, (Rice and Barone, 2000), i.e., shortly after infliction of the mPFC lesions in the present study. Additionally, a preliminary qualitative study in our laboratory already pointed to a myelination deficit after neonatal mPFC lesion (Schneider and Koch, 2005a). We first used immunohistochemical double labeling for NeuN and GFAP to confirm that the filled-in-tissue consists of neurons without reactive gliosis similar as found by De Brabander et al. (1991a). Subsequently, the anterograde and retrograde anatomical connections were evaluated after injection of the anterograde anatomic tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde tracer fluorogold (F-gold), respectively, into the filled-in-tissue after neonatal mPFC lesions and corresponding intact mPFC of control rats. Additionally, we assessed the relative density of parvalbumin-immunoreactive interneurons and myelination in various brain regions. Materials and methods Animals All rats used in this study were offspring of Wistar rats from Harlan–Winkelmann (Borchen, Germany). The litters were culled to eight male pups directly after birth. In case of less than eight male pups, females were used to fill up the litters. The day of birth was designated as postnatal day 0 (PND 0). The pups were weaned on PND 21 and then housed in groups of four to six in Makrolon cages (type IV) under controlled environmental conditions (ambient temperature 22–24 °C, 12 h light–dark cycle, lights on at 7 a.m.). The rats had free access to tap water and were initially fed ad libitum. Since the male rats were used for noninvasive behavioral experiments, their food was restricted to 12 g of standard laboratory chow after reaching a body weight of 180 g, thereby keeping the rat's body weight on approximately 85% of the free-feeding weight throughout their testing period. The females were fed ad libitum throughout the whole study. All experiments were done in accordance with the European Communities Council Directive of November 24th 1986 (86/ 609/EEC) and were approved by the local ethical committee.

All efforts were made to minimize the number of animals and their suffering. Neonatal lesion On PND, 7 rats were randomly assigned to an ibotenate lesion (males: n = 10; females: n = 8), sham lesion (males: n = 4), or naive control group (males: n = 5; females: n = 4). All rats, except for the naive controls, were anesthetized by hypothermia (placed on ice for 15–20 min) and positioned in a stereotaxic frame adapted for neonatal surgery (TSE, Bad Homburg, Germany). The tooth bar was adjusted so that bregma and lambda were at the same height. The skull was penetrated with a microliter syringe (SGE, Deutschland GmbH, Darmstadt), which was lowered into the mPFC using the following coordinates relative to bregma: anterior-posterior (AP) +2.7; lateral (L) ± 0.3 mm, and ventral (V) 3.2 mm). Ibotenic acid (2 μg; Sigma Deisenhofen, Germany) in 0.3 μl phosphate buffered saline (PBS; pH 7.4) for the lesion group or the equal volume of PBS for the sham group was infused over 3 min bilaterally into the mPFC (0.1 μl/min). After completion of the injections, the cannulae were left in place for additional 5 min in order to prevent spreading of the neurotoxin along the needle track. These coordinates and procedures have been shown to induce selective lesions within the mPFC in previous studies (Schwabe et al., 2004, 2006). The skin was closed with medical tissue adhesive. The pups were allowed to recover on an electric warming pad before being returned to their mothers, where they remained until weaning. Tracer application Adult male rats (i.e., after PND 90) were injected with the neuroanatomical tracers F-gold and PHA-L into the mPFC in a second stereotaxic surgery. Rats were anesthetized with chloralhydrate (360 mg/kg, i.p.; Sigma-Aldrich, Steinheim, Germany) and positioned in a stereotaxic frame, with the tooth bar set at − 3.3 mm. Glass electrodes were pulled on an electrode puller, the tips shortened down to tip diameter of 10 to 15 μm and filled with F-gold or PHA-L. Thereafter, electrodes were lowered through drilled holes into the mPFC at the following coordinates; AP +2.7 mm; L ± 0.5 mm; V 4.5 mm. Both tracers were applied iontophoretically (5 μA positive current, 7 s dutycycle), F-gold for 15 min and PHA-L for 10 min. In preliminary work, these parameters have been shown to produce injection sites of similar size in the mPFC of lesioned and control rats. The female rats were injected with F-gold into either the NAC (AP +1.2 mm; L + 1.3 mm; V − 6.8 mm) or the mediodorsal thalamic nucleus (MDT; AP − 1.8 mm; L − 0.5 mm; V − 6.2 mm; lesioned n = 4; control n = 2 in each group, respectively). Histological procedures Ten days after the tracer application, all animals were deeply anesthetized with chloralhydrate (720 mg/kg, i.p.) and transcardially perfused with phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M

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phosphate buffer (pH 7.4). The brains were removed from the skulls and cryoprotected in 30% sucrose for at least 48 h. Each brain was cut in six series of 40 μm coronal sections. To determine placement and extent of the lesions, one series of sections was Nissl-stained with thionine (series 1). The second series was mounted on slides and coverslipped with mounting medium (DAKO, Glostrup, Denmark) for fluorescence-micro-

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scopical evaluation of the transport of F-gold (series 2). The third, fourth, and fifth series were immunohistochemically processed, either for fluorescent double-labeling of NeuN and GFAP (series 3), PHA-L (series 4), or Parv (series 5) using peroxidase-labeling. For histological evaluation of the relative density of myelinated fibers, the sixth series was mounted on slides and stained with goldchloride. Care was taken to use

Fig. 1. The medial prefrontal cortex of a control (A) and a neonatally lesioned rat (B). A1 and B1 show Nissl-stained sections of a 9-day-old control rat and of a rat 2 days after ibotenate injection, respectively. A/B2,3 show Nissl-stained sections of an adult control and a neonatally lesioned rat with the area marked in A/B2 shown in higher magnification in A/B3. Arrows point to occasional cell clusters within the condensed cellular layers in panel B3. The insert in panel B3 shows one of the cell clusters in higher magnification. A schematical drawing of a coronal section of the medial prefrontal cortex is shown in panel C with dotted lines indicating the borders of subregions (according to the atlas of Paxinos and Watson, 1998). Scale bars: A/B1/2: 1000 μm; A/B3: 100 μm. Cg1—cingulate cortex 1; PL—prelimbic cortex; IL—infralimbic cortex.

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sections of all treatment groups within one passage of each staining thus reducing the error of different background staining between groups. Nissl-staining Sections were mounted on slides with chromalaun-gelatine. After drying, the sections were rehydrated in graded ethanol concentrations and stained in aqueous thionine solution for 90 s. Thereafter, the sections were dehydrated in graded ethanol concentrations, cleared in xylene, and coverslipped with Entellan® (Merck, Darmstadt, Germany). Immunohistochemical processing For immunohistochemical staining, free-floating sections were preincubated in blocking buffer containing normal goat serum (10%; Linaris, Wertheim-Bettingen, Germany) and 0.3% Triton-X 100 in PBS for 60 min. The blocking buffer of the sections processed for Parv or PHA-L additionally contained 0.01% H2O2. Thereafter, the sections were incubated for 48 h at 7 °C in blocking buffer that additionally contained the IgG-antibody, specifically series 3 with monoclonal mouse α-NeuN (1:1000; Chemicon Int., Temecula, Canada) and rabbit α-GFAP (1:5000; DAKO, Glostrup, Denmark), series 4 with rabbit α-PHA-L (1:5000; Vector, Wertheim, Germany), and series 5 with monoclonal mouse α-Parv (1:5000; Sigma, Deisenhofen, Germany). After washing three times in PBS, sections were placed in PBS containing 0.2% bovine serum albumine (PBSA, pH 7.4) for 60 min. Thereafter, the sections were directly placed in the adequate secondary antibody solution (all Sigma Deisenhofen, Germany; 1:2000 in PBSA) for 24 h, specifically, series 3 in biotin-labeled IgG goat α-rabbit and Cy2 labeled IgG goat α-mouse, series 4 and 5 in biotin labeled IgG goat α-rabbit or goat α-mouse. Thereafter sections were again washed three times in PBS and then placed in PBSA for 1 h. Series 3 was then incubated in PBSA containing Cy3 labeled streptavidin (1:500), and series 4 and 5 were incubated in PBSA containing biotin and peroxidase-labeled streptavidin (1:1000; Linaris, Wertheim, Germany) for 6 h and subsequently washed three times in PBS. The sections stained for NeuN and GFAP were mounted on slides and coverslipped with fluorescence-mounting medium (DAKO, Glostrup, Denmark). The sections used for peroxidase labeling (series 4 and 5) were further incubated in a solution containing 0.05% 3,3diaminobenzidine tetrahydrochloride, 0.6% nickel sulfate, and 0.01% H2O2 for 3 min. Thereafter, these sections were dehydrated in graded ethanol, cleared in xylene, and finally coverslipped with Entellan® (Merck, Darmstadt, Germany). Same procedures leaving out the primary antibodies were used as negative controls to ensure specificity of immunohistochemical staining. Goldchloride staining For goldchloride staining, the mounted sections were placed in a 0.2% goldchloride solution (tetrachloroaurate-trihydrate

(AuCl4), Carl Roth GmbH and Co. Karlsruhe, Germany) in 0.02 M PBS (pH 7) for about 2 h until the CA1-3 region of the dorsal hippocampus (HIPP) appeared as purple-brown structure. Sections were then rinsed with distilled water for 2 h. Thereafter, they were fixed in a 2.5% sodiumthiosulfate solution (Merck, Darmstadt, Germany). Finally, the sections were dehydrated in graded ethanol, cleared in xylene, and coverslipped with Entellan®. Analysis of histological staining All histological analyses were done using a Zeiss Axiophot microscope (Göttingen, Germany) equipped with the digital camera RT Slider Spot and the image analysis software MetaMorph 4.6 (both from Visitron Systems GmbH, Puchheim, Germany). To determine the regions of interest, we used the atlas of Paxinos and Watson (1998). Nissl-stained sections were used to determine location and extent of the neonatal lesions. The distribution of neurons and glia cells was evaluated using the fluorescent lamp of the microscope and appropriate filters. Only animals with exact placement of the lesions in the mPFC were used for subsequent analysis. To detect F-gold labeled cells, we used the fluorescent lamp and appropriate filters while PHA-L labeling of fibers was analyzed by light microscopy. The transport of F-gold or PHAL was only evaluated in case of exact injection into the mPFC of control rats or into the filled-in-tissue of neonatally lesioned rats. First, the transport of F-gold and PHA-L to brain regions known to be interconnected with the mPFC (see for example (Conde et al., 1990, 1995; Heidbreder and Groenewegen, 2003; Vertes, 2004) was confirmed in control rats. Thereafter, we assessed whether the tracers were transported to these regions after injection into the filled-in-tissue of neonatal lesioned animals. Finally, the whole brains were scanned in order to find transport into regions normally not labeled after injection into the mPFC. The transport of F-gold and PHA-L in lesioned and

Fig. 2. Schematic drawings of coronal sections of the medial prefrontal cortex (Paxinos and Watson, 1998) indicating the injection sites of the neuroanatomical tracers PHA-L and F-gold in relation to the neonatal medial prefrontal cortex lesions. The dashed line indicates the smallest and the continuous line the largest area with cytoarchitectonic abnormalities after neonatal lesion. The black area marks the smallest and the dotted area the biggest injection site of the tracers.

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Fig. 3. Typical distribution of fluorescence labeled cells after injection of F-gold into the medial prefrontal cortex of a control (A) and a neonatal lesioned rat (B). Schematic drawings are based on transverse sections (distance in mm to bregma indicated) from the atlas of Paxinos and Watson (1998). The hatched oval indicates the injection site of F-gold and the black area in panel B the cavitation in the neonatal lesioned animal. The distribution of labeled cells in different brain regions after neonatal lesion and in control rats is shown as dots.

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control rats was qualitatively compared. After the injection of F-gold into the NAC and MDT, the amount and distribution of labeled cells in the mPFC of control rats or neonatally lesioned rats were likewise qualitatively compared. All Parv-immunoreactive cells were bilaterally counted in the stratum pyramidale of the CA1 and CA3 region of the dorsal HIPP, the CA1 region of the ventral HIPP as well as in the basolateral amygdala (BLA) on three subsequent sections (distance between sections 240 μm) and the sums of these measures were used for statistical evaluation. The first section used for the BLA was −2.2 mm from bregma, for the dorsal HIPP − 2.7 mm from bregma, and for the ventral HIPP − 4.8 mm from bregma (according to the atlas of Paxinos and Watson, 1998). These sections were chosen on the basis of their importance for the regulation of behavior and because they can be easily and reproducibly picked up while sectioning the brain. The number of Parv-immunoreactive neurons within the different brain regions of the left and right hemispheres was determined per section for the entire area by counting all Parv-immunoreactive neurons emerging into focus through the 40 μm sections at 200 times magnification. Since the area of the different regions was expected to differ at least to some extent between groups, the boundaries of the areas used for cell counting were measured by using the digital camera and the image analysis system mentioned before. We then calculated the number of Parv-immunoreactive neurons per area (in mm2) of the different regions (i.e., a measure of cell density). Relative density of myelinated fibers was determined on three subsequent sections in the NAC, the BLA, the central amygdaloid nucleus (CeN), the MDT, stratum radiatum (SRad), and stratum oriens (SO) of the dorsal and ventral HIPP and in the primary motor cortex. The first sections used for this evaluation within the NAC and the primary motor cortex were +1.6 from bregma. For the BLA and the dorsal and the ventral HIPP, we used sections adjacent to the ones used for evaluation of Parv-immunoreactive cells. The first section used to measure the degree of myelination within the CeN was − 2.3 mm from bregma and the first one used for the MDT was − 1.9 mm from bregma. First, a gray-scale image was taken at 100 times magnification using the digital camera and the MetaMorph software. If the brain region could not be visualized within one frame, additional images were taken. The image was first calibrated to transfer pixel units into micrometer units. Stained fibers were then efficiently enhanced from background staining by using the gradient operation “Sobel filter”. This filter improves the signal/noise ratio by calculating the difference of adjacent pixels values in both horizontal and vertical direction. Subsequently, the enhanced edges were automatically selected by an “Auto Threshold” operation. This threshold operation analyzes the histogram fitting a Gaussian to the background peak in the histogram and setting the threshold to 2 times the standard deviation from the Gaussian. Making use of position and width of the image's background peak, this analysis is independent of camera exposure time and brightness of illumination. A binary copy of the thresholded image was

made, which has a value of 1 only at pixels that accomplish the thresholded area but is 0 anywhere else. This binary image was then added to the original gray-scale image in order to visually control the detection of fibers enhanced by the “Auto Threshold” operation of the software. After defining a region of interest, the percentage of thresholded area within this region and the area in μm2 was measured in order to calculate the relative density of fibers within this region. Statistical analysis Differences in Parv-staining and myelinated fibers between groups were analyzed by Student t-test. All tests were performed two-sided and p b 0.05 was considered significant. Table 1 Transport of F-gold into different ipsilateral brain regions after injection into the mPFC after neonatally induced lesions or in controls Region

Control (n = 4)

Lesion (n = 3)

Cortex and forebrain Cingulate cortex Orbital cortex Agranular insular cortex Perirhinal cortex Piriform cortex Entorhinal cortex Claustrum Septal nucleus Endopiriform nucleus Horizontal limb of the diagonal band Bed nuclei of stria terminalis Ventral pallidum

100% 50% 75% 25% 50% 25% 75% 25% 50% 50% 50% 50%

100% 0% 100% 33% 100% 0% 100% 33% 33% 100% 100% 100%

Limbic system Basolateral amygdala Basomedial amygdala Lateral amygdala Hippocampus Amygdalohippocampal transition zone

100% 100% 25% 100% 75%

100% 67% 0% 100% 0%

75% 50%

100% 100%

Thalamus Anteromedial, centromedial and intermediodorsal nucleus Interanteromedial thalamic nucleus Mediodorsal thalamic nucleus Paracentral thalamic nucleus Paraventricular thalamic nucleus Ventrolateral thalamic nucleus Ventromedial thalamic nucleus Nucleus reuniens Rhomboid thalamic nucleus Zona incerta

25% 75% 100% 25% 75% 25% 25% 75% 75% 75%

0% 33% 100% 67% 67% 0% 33% 100% 33% 67%

Hypothalamus Ventral tegmental area Substantia nigra pars compacta Lateral hypothalamic area

100% 25% 75%

33% 0% 100%

Midbrain Nucleus ruber Occulomotoric nucleus

Indicated is the percentage of the rats used for evaluation (controls: n = 4; lesions: n = 3).

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Results Neonatal lesions Assessment of the mPFC 2 days after injection of ibotenate showed an almost complete loss of neurons at the injection site, which was restricted to the infralimbic, prelimbic, and the ventral part of the anterior cingulate cortices (Figs. 1A/B1). In adult rats lesioned on PND, 7 Nissl-stained sections showed atrophy and retraction of tissue thus causing a slight cavity at the site of neonatal ibotenate injection (Fig. 1B2). Only animals with placement of the cavity at the infralimbicprelimbic cortices were used for further evaluation (males, n = 7; females, n = 8). Similar to previous studies (e.g., Schwabe et al., 2004; Schneider and Koch, 2005a), this cavity was surrounded by neuronal tissue with cytoarchitectonic abnormalities, such as condensed cell layers and occasional cell clusters, which affected most parts of the infralimbic–prelimbic cortices and the ventral part of the anterior cingulate cortex (Fig. 1B2,3). These alterations were generally found within the anterior–posterior coordinates approximately from 3.7 to 2.2 from bregma, according to the atlas of Paxinos and Watson (1998) and correlated with the extent of neonatal lesions observed in neonatally lesioned rats which were killed 2 days after introduction of the lesion (Figs. 1A/B1). Other areas of the frontal cortex adjacent to the anterior cingulate and infralimbic cortices were spared from the lesion. Similar to previous studies (De Brabander et al., 1991a), qualitative evaluation of the sections processed for immunohistochemical double-labeling of NeuN and GFAP (lesioned: n = 5, sham and control: n = 5) showed that after neonatal ex-

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citotoxic lesion neurons were scattered throughout the filled-intissue, but with condensed cellular layers and some occasional cell clusters, whereas distribution of glia cells was unaffected with the exception of a small rim of gliosis surrounding the filled-in-tissue (data not shown). Sham-lesioned rats showed no morphological changes or detectable degeneration at the injection site of the mPFC (Fig. 1A1–3). Therefore, for the subsequent analysis, the data from the two control groups were combined for comparison to neonatally lesioned rats, as done in previous studies (Bennay et al., 2004; Schwabe et al., 2006). Afferent connections Only animals with exact placement and comparable size of filled-in-tissue as well as F-gold injection located within tissue of cytoarchitectonic abnormalities were used for analysis of retrograde connectivity (Fig. 2). These rigid criteria reduced the number of animals used for further evaluation to 4 controls and 3 lesioned rats. Overall, the afferent connections of the mPFC were similar to those described in detail before (Conde et al., 1990, 1995; Heidbreder and Groenewegen, 2003), but with reduced amount of labeled cells in neonatally lesioned animals (Fig. 3; Table 1). Transport of F-gold into cortical and subcortical areas was generally similar in both groups except for the orbital cortex, where no labeling was found after neonatal mPFC lesion, and in some subcortical forebrain regions (the ventral pallidum, the horizontal limb of the diagonal band, and the bed nucleus of stria terminalis) where cell labeling was found in a higher percentage of lesioned rats (for a detailed description see Table 1).

Fig. 4. Microphotographs of coronal sections of a control (A) and a neonatally lesioned rat (B). A/B1 show the injection site in the prefrontal cortex. A/B2 show a Nisslstained section with the striatum and the nucleus accumbens, A/B3,4 show details of the areas marked in A/B2 with arrows on the adjacent section processed for PHA-L immunoreactivity at higher magnification. While in control rats numerous fibers were labeled in the medial striatum (A3) and the nucleus accumbens (A4), only few weakly stained fibers were found in the medial striatum (B3) and in the nucleus accumbens (B4) of neonatal lesioned rats (arrows). Scale bar: A/B12: 1000 μm; A/B3,4: 100 μm.

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Efferent connections Only animals with exact placement and comparable size of the lesion as well as injection sites and diffusion halo of PHA-L within the boundaries of cytoarchitectonic abnormalities in the mPFC were used for analysis of anterograde tracing (controls: n = 5, lesioned rats: n = 4). After injection of PHA-L into the mPFC of controls, we found stained fibers, both surrounding the injection site, as well as in most of the previously described projection areas of the mPFC (Vertes, 2004), i.e., in the neigh-

boring prefrontal areas, the medial striatum, the NAC, the claustrum, the bed nucleus of stria terminalis, the amygdaloid complex, and several thalamic and hypothalamic nuclei. In contrast, except for weakly stained fibers around the injection site, and some solitary fibers in the medial striatum and the NAC, no fibers were labeled in projection areas of the mPFC of neonatally lesioned rats (see Fig. 4 as an example of fiber labeling after injection of PHA-L into the intact mPFC of a control rat in comparison to the same brain region after injection into previously lesioned mPFC).

Fig. 5. Microphotographs showing the injection site of F-gold in the nucleus accumbens (NAC; A/B1,2) or the mediodorsal thalamus (MDT; C/D1,2), as well as the transport of F-gold into the medial prefrontal cortex (mPFC; A–D3,4) in control rats (A/C1–4) and after neonatal lesion (B/D1–4). A–D1,3 show Nissl-stained sections and A–D2,4 the adjacent sections with F-gold labeled cells. Scale bars: A–D1,3: 1000 μm; A–D2: 500 μm; A–D4: 250 μm.

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However, in light of the functional sparing as well as the functional deficit after adult lesion of the filled-in-tissue, a complete loss of anterograde connectivity seemed unlikely to us. Thus, we attempted to verify this finding by evaluating retrograde transport of F-gold to the mPFC after injection into two regions that were heavily labeled after injection of PHA-L into the mPFC of controls, i.e., the NAC and the MDT. After injection of F-gold into the NAC of lesioned animals and controls, a similar pattern of labeled cells was found within the mPFC of both groups, indicating that at least some of the efferent projections are preserved after neonatal lesion of the mPFC (Figs. 5A/B). However, after injection of F-gold into the MDT, no cell labeling was found within the filled-in-tissue, whereas numerous cells were labeled within the mPFC after injection into the MDT of control rats (Figs. 5C/D).

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Parvalbumin For analysis of Parv-immunoreactive cells, we used six lesioned and six control rats. Statistical analysis revealed that after neonatal mPFC lesions the number of Parv-immunoreactive cells per mm2 was significantly increased in the BLA ( p = 0.003), as well as in the dorsal ( p = 0.003) and in the ventral HIPP compared to controls ( p = 0.001; Fig. 6). Notably, the area of these different regions did not differ between groups (all pvalues N 0.19). Goldchloride For analysis of myelination, we used nine controls and seven lesioned rats. After digital enhancement of the contrast in

Fig. 6. Microphotographs of coronal sections showing parvalbumin immunoreactivity in the ventral hippocampus of a control (A) and a neonatally lesioned rat (B). A/B2 show the area marked in A/B1 at higher magnification. Scale bar: A/B1: 1000 μm; A/B2: 100 μm. Density of parvalbumin immunoreactive cells (C; cells/mm2) in the basolateral amygdala (BLA) and the dorsal and ventral hippocampus (HIPP). Data are means + S.E.M. of lesioned (n = 6, black bars) and control rats (n = 6, white bars). Significant differences between groups are indicated as asterisks (⁎p b 0.05).

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Fig. 7. Microphotographs of coronal sections showing the goldchloride-stained ventral hippocampus of a control (A) and a neonatally lesioned rat (B). A/B2 show the area marked in A/B1 at higher magnification. Scale bar: A/B1: 1000 μm; A/B2: 100 μm. Relative density of goldchloride staining in percent for several brain regions (C). Data are means ± S.E.M. of lesioned (n = 7, black bars) and control rats (n = 9, white bars). Significant differences between groups are indicated as asterisks (⁎p b 0.05). Abbreviations: NAC—nucleus accumbens; BLA—basolateral amygdala; CeN—central amygdaloid nucleus; MDT—mediodorsal thalamic nucleus; vHIPP So/Sr—ventral hippocampus stratum oriens/stratum radiatum; dHIPP So/Sr—dorsal hippocampus stratum oriens/stratum radiatum; M1—primary motor cortex.

goldchloride-stained sections, myelinated fibers were reliably detected by the software. Compared to controls, the relative density of goldchloride-stained fibers was reduced in most examined brain regions of neonatally lesioned rats, i.e., the NAC, the BLA, and CeN of the amygdaloid complex, different regions of the dorsal and ventral HIPP (SO and SRad) and the primary motor cortex (all p-values b 0.03; Fig. 7). In the MDT, the difference between groups failed to reach significance ( p = 0.051). Discussion Similar to previous reports (e.g., De Brabander et al., 1991a) after neonatal injection of ibotenate into the mPFC, the lesion

cavity was filled in to a considerable extent with neuronal tissue that is, however, reduced in volume and that shows cytoarchitectonic abnormalities. We assume that this is largely due to neurogenesis and not merely a consequence of redistribution of the surrounding cortical tissue, since (1) using BrdU labeling, a cellular marker for mitosis (Kolb et al., 1998b) showed various BrdU labeled NeuN-immunoreactive cells in the filled-in-tissue of the mPFC after lesions between postnatal days 7–10, (2) the mPFC is a late developing region of the neocortex and is still under construction at the time of the lesion although neurogenesis itself is regarded to be completed at that time (Fuster, 2002; Van Eden and Uylings, 1985; Van Eden et al., 1990), (3) the mPFC is in very close proximity to the subventricular zone, which is the origin of newly generated neurons for the olfactory

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bulb, a zone of ongoing neurogenesis through lifetime of rodents (Ng et al., 2005). On its way to the olfactory bulb, these newly generated neurons pass directly by the mPFC (Lois and Alvarez-Buylla, 1994) and some of the migrating neurons on their destination to the olfactory bulb may come to lie in the lesioned mPFC area as suggested by Kolb et al. (1998b). Notably, the subventricular zone reacts to injuries of mPFC and other cortical areas like the sensorimotor cortex with increased proliferation (Kolb et al., 1998b; Szele and Chesselet, 1996). However, since we did no double labeling with BrdU in the present study, we cannot rule out that some of the neurons originate from the tissue surrounding the lesion side, or – even if we here and previously showed that 2 days after injection of ibotenate virtually no neurons are left around the injection site (unpublished observations) – were spared by the neonatal lesion. Therefore, the filled-in-tissue may be of mixed origin from newly generated neurons as well as from neurons originating from the surrounding neuronal tissue. Using retrograde tracing, we here showed that the filled-intissue of the mPFC receives neuronal projections from most of its normal input regions. At the time of the lesion (PND 7), the mPFC is almost completely differentiated and most of the afferent connections from other brain regions have already been established (Van Eden et al., 1990; Van Eden and Uylings, 1985). Since the lesion cavity has been shown to be filled in within days following neonatal mPFC lesions (Kolb et al., 1998b), these efferent axons may simply maintain their normal physiological connections. The few differences in labeling within some forebrain regions may be related to differences in the exact placements and size of the injection sites rather than to differences in retrograde connectivity, especially considering that only few animals with exactly matching lesions and injection sites within the filled-in-tissue could be used for analysis, which, unfortunately, does not allow for a quantitative analysis. However, at least to some extent the maintenance of connections might also be the result of axonal sprouting of projection neurons that normally innervate the areas surrounding the filled-in-tissue. Additionally, we cannot rule out the possibility, that F-gold was picked up by fibers of passage, leading to some false positive results. However, since we found retrogradely labeled cells only in brain areas known to be anterogradely connected with the mPFC (Conde et al., 1990, 1995), we are confident, that most of the labeling reflects retrograde connectivity of the filled-in-tissue. In contrast, evaluation of PHA-L transport suggests a complete loss of efferent connections of the filled-in-tissue since no substantial transport of PHA-L to any brain region could be observed in lesioned animals. However, since (1) a previous study had already shown that at least the connections to the striatum and the parietal cortex recover after mechanical neonatal lesion of the mPFC (Kolb et al., 1998b), and (2) adult rats show behavioral deficits when the filled-in-tissue after neonatal lesions is lesioned again in adult rats (Dallison and Kolb, 2003), we attempted to verify our finding by using a different anatomical tracer. For this purpose, we injected the retrograde tracer F-gold into the NAC and the MDT, i.e., two main projection areas of the mPFC that are heavily labeled after injection of

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PHA-L in control animals. While injection of F-gold into the NAC resulted in robust cell labeling in all parts of the filled-intissue, which was comparable to the distribution of labeled cells within the mPFC after injection into the NAC of control rats, no cell labeling was found after injection of F-gold into the MDT of rats with neonatal mPFC lesions. This indicates that efferent connections of the filled-in-tissue obviously depend on the projection site. Possibly, this is simply a question of distance, since in rats the distance from the mPFC to the MDT is more than twice the distance to the NAC (Paxinos and Watson, 1998). Additionally, the connections from the mPFC to the MDT have been reported to develop until PND 14 (Van Eden, 1986; Van Eden and Uylings, 1985), i.e., a time when the reconstruction of the lesioned tissue is still on its way (Kolb et al., 1998b). Therefore one may surmise that once the neurons have found their position within the regenerating brain region, the appropriate growth factors that would guide the axons to the MDT are not available anymore. Unfortunately, we are not aware of any study that examined the development of connections from the mPFC to the NAC. In light of the anatomical projection to the NAC, it is interesting to note that several previous studies indicated compromised NAC function after neonatal mPFC lesions. The mPFC influences dopaminergic neurotransmission in the NAC by presynaptic contacts on dopaminergic fibers arising from the ventral tegmental area (Carr and Sesack, 1996; Carr and Sesack, 2000) and neonatal mPFC-lesioned rats show increased sensitivity to the stimulating effects of dopamine agonists (Flores et al., 1996). Moreover, electrophysiological studies revealed that neonatal mPFC lesions change the firing pattern of NAC core and shell neurons together with increased sensitivity to a dopamine receptor agonist (Bennay et al., 2004). Taken together, these findings implicate disturbed function of the anatomical projection of the mPFC to the NAC after neonatal lesion, as indicated by failure of PHA-L transport from this region, too. However, whether these anatomic connections are of functional significance cannot be answered with the methods used in this study. We also cannot rule out the possibility that some neurons survived the ibotenic acid lesion and simply maintained their anatomical connectivity. Analysis of histological staining revealed that after neonatal lesion the number of Parv-immunoreactive cells was increased in the BLA, as well as in the dorsal and ventral HIPP. Parv is a molecular marker for a subset of GABAergic interneurons, specifically chandelier and wide-basket cells, which either synapse on the soma and proximal dendrites (basket neurons) or on the initial axon segment of excitatory neurons (Hardwick et al., 2005). Accordingly, both subtypes are thought to have stronger impact on pyramidal excitability and network function than other local GABAergic circuit neurons that synapse more distally along the dendritic tree (Blum and Mann, 2002; Lewis, 2000; Lewis et al., 2005). The calcium binding protein Parv regulates calciumdependent aspects of neuronal function by effectively buffering intracellular calcium transients, thus shortening the duration between action potentials (Caillard et al., 2000; Chard et al., 1993). Parv-containing GABA interneurons are therefore

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known for their pattern of firing at an extremely high frequency and in bursts (Cauli et al., 1997; Kawaguchi and Kubota, 1993). Since the increased density of Parv-immunoreactive neurons found in the present study was not attributed to shrinkage of brain tissue, the most likely explanation is an increase in its cellular expression and consequent detectability in neurons, but not the emergence of a new set of neurons. In this context, it is of special interest that increased bursting activity in GABA interneurons has been shown to induce an increase in the level of calcium-binding proteins (Chard et al., 1993). Therefore, we assume that the increased density of Parv immunoreactive neurons may be a neuroprotective response against excitotoxicity from prolonged activity in subcortical brain regions related to compromised function after neonatal mPFC lesion, especially in regions where principal neurons of the mPFC have been shown to exert inhibition like within the amygdala (Milad and Quirk, 2002; Rosenkranz and Grace, 2001). So increased Parv expression might reflect the brain's attempt to reinstall homeostasis after loosing parts of its inhibitory control. Interestingly, an increase in Parv immunoreactivity within different brain regions has also been reported after prolonged amphetamine and cocaine treatment (Liu et al., 2005; Mohila and Onn, 2005) and after prenatal (intrauterine) cocaine exposure (Wang et al., 1996). Evaluation of goldchloride-staining revealed that the relative density of myelin was reduced in all examined brain areas, notably even in the primary motor cortex, which has no direct anatomical connections with the mPFC. This is an extension of a previous finding of our group, where a lesion-induced loss of myelin staining has been qualitatively evaluated in selected brain regions (Schneider and Koch, 2005a). In the central nervous system, myelin sheaths are built by oligodendrocytes, which develop shortly after the beginning of neurogenesis. The differentiation of these glia cells follows axiogenesis and is controlled by complex actions of various trophic factors between the oligodendrocytes and surrounding neurons, which has been demonstrated to be vulnerable to perturbation at any time of this process (Bartzokis, 2002; Davis et al., 2003; Hof et al., 2002; Rice and Barone, 2000). Notably, the developmental peak of myelination occurs in the second postnatal week, i.e., within a few days after infliction of lesions in the present study (Davis et al., 2003; Rice and Barone, 2000). Although excitotoxins, such as the glutamate receptor agonist ibotenate, are “axon-sparing”, i.e., do not damage neuronal fibers at the injection site, it has been shown that overactivation of glutamate receptors damages oligodendroglia cells (Davis et al., 2003). In this context, ibotenate injection has been shown to reduce white matter in neonatal mice, possibly by microglia activation and astrocytic death (Dommergues et al., 2003; Rogido et al., 2003; Tahraoui et al., 2001). Interestingly, this excitotoxic effect has only been shown until PND 10. Since the mPFC lesions in the present study were inflicted on PND 7, we surmise that the acute excitatory effect of ibotenate may have disturbed myelination processes in brain areas receiving dense glutamatergic innervations from the mPFC and related subcortical network. However, since myelination is reduced in all examined brain regions, including the primary motor cortex, it is also possible that the reduced myelination is caused by

compensatory rewiring of corticolimbic circuits after neonatally induced lesions and is not due to a general loss of input from the mPFC. It also remains unclear whether some of the decreased myelination is due to fiber degeneration after neonatal lesion. Together, we found that although the lesion cavity is filled-in to a vast extent after neonatal mPFC lesions and the afferents to the filled-in tissue are largely spared, the efferent connections to subcortical brain regions are partially lost. This is accompanied by an increase in Parv-immunoreactive neurons in the BLA and in the HIPP indicating enhanced neuronal activity in this class of neurons, which points to hyperactivity in these brain regions after neonatal mPFC lesions. The deficit in myelination also suggests disturbed nerve impulse conduction speed and accuracy, probably leading to disturbed network function. Interestingly, in a recent study (Harich S.; personal communication) we showed that after neonatal lesions of the entorhinal cortex Parv-immunoreactivity was reduced in the BLA and in the HIPP, while myelination was decreased similar to the findings of the present study. These findings might have possible impact on stem cell research, since it would be of great interest to elucidate the differences between cortical areas that are able to restore lost tissue and function and those that are not. Notably, in contrast to the mPFC, the entorhinal cortex is not refilled after neonatal lesions, which may be the reason for the differences in Parv expression between these two studies. On the other hand, in contrast to the entorhinal cortex, the mPFC is classically thought to be involved in inhibitory control, so the different outcome of neonatal lesions of these two brain areas on the expression of Parv may also be due to their defined and different role in inhibitory control. Since reduced myelination and parvalbumin-expressing interneurons are discussed as risk factors of certain psychiatric disorders (e.g., Lewis et al., 2005; Torrey et al., 2005; Zhang and Reynolds, 2002), these anatomical findings should encourage further studies of network function after neonatal lesions of different brain regions. Acknowledgments This project was supported by the DFG (SFB517; TP A11) and by a grant of the University of Bremen. We thank Maja Brand for technical assistance and Dr. Wolfgang Feneberg from Visitron Systems for helping with the description of softwarefeatures. References Bartzokis, G., 2002. Schizophrenia: breakdown in the well-regulated lifelong process of brain development and maturation. Neuropsychopharmacology 27, 672–683. Bennay, M., Gernert, M., Schwabe, K., Enkel, T., Koch, M., 2004. Neonatal medial prefrontal cortex lesion enhances the sensitivity of the mesoaccumbal dopamine system. Eur. J. Neurosci. 19, 3277–3290. Blum, P.B., Mann, J.J., 2002. The GABAergic system in schizophrenia. Int. J. Neuropsychopharmacol. 5, 159–179. Caillard, O., Moreno, H., Schwaller, B., Llano, I., Celio, M.R., Marty, A., 2000. Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity. Proc. Natl. Acad. Sci. U. S. A. 97, 13372–13377.

S. Klein et al. / Experimental Neurology 209 (2008) 199–212 Carr, D.B., Sesack, S.R., 1996. Hippocampal afferents to the rat prefrontal cortex: synaptic targets and relation to dopamine terminals. J. Comp. Neurol. 369, 1–15. Carr, D.B., Sesack, S.R., 2000. Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J. Neurosci. 20, 3864–3873. Cauli, B., Audinat, E., Lambolez, B., Angulo, M.C., Ropert, N., Tsuzuki, K., Hestrin, S., Rossier, J., 1997. Molecular and physiological diversity of cortical nonpyramidal cells. J. Neurosci. 17, 3894–3906. Chard, P.S., Bleakman, D., Christakos, S., Fullmer, C.S., Miller, R.J., 1993. Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J. Physiol. 472, 341–357. Conde, F., Audinat, E., Maire-Lepoivre, E., Crepel, F., 1990. Afferent connections of the medial frontal cortex of the rat. A study using retrograde transport of fluorescent dyes. I. Thalamic afferents. Brain Res. Bull. 24, 341–354. Conde, F., Maire-Lepoivre, E., Audinat, E., Crepel, F., 1995. Afferent connections of the medial frontal cortex of the rat. II. Cortical and subcortical afferents. J. Comp. Neurol. 352, 567–593. Dallison, A., Kolb, B., 2003. Recovery from infant medial frontal cortical lesions in rats is reversed by cortical lesions in adulthood. Behav. Brain Res. 146, 57–63. Davis, K.L., Stewart, D.G., Friedman, J.I., Buchsbaum, M., Harvey, P.D., Hof, P.R., Buxbaum, J., Haroutunian, V., 2003. White matter changes in schizophrenia: evidence for myelin-related dysfunction. Arch. Gen. Psychiatry 60, 443–456. De Brabander, J.M., de Bruin, J.P., Van Eden, C.G., 1991a. Comparison of the effects of neonatal and adult medial prefrontal cortex lesions on food hoarding and spatial delayed alternation. Behav. Brain Res. 42, 67–75. De Brabander, J.M., Van Eden, C.G., de Bruin, J.P., 1991b. Neuroanatomical correlates of sparing of function after neonatal medial prefrontal cortex lesions in rats. Brain Res. 568, 24–34. Dommergues, M.A., Plaisant, F., Verney, C., Gressens, P., 2003. Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection. Neuroscience 121, 619–628. Flores, G., Wood, G.K., Liang, J.J., Quirion, R., Srivastava, L.K., 1996. Enhanced amphetamine sensitivity and increased expression of dopamine D2 receptors in postpubertal rats after neonatal excitotoxic lesions of the medial prefrontal cortex. J. Neurosci. 16, 7366–7375. Fuster, J.M., 2002. Frontal lobe and cognitive development. J. Neurocytol. 31, 373–385. Hardwick, C., French, S.J., Southam, E., Totterdell, S., 2005. A comparison of possible markers for chandelier cartridges in rat medial prefrontal cortex and hippocampus. Brain Res. 1031, 238–244. Heidbreder, C.A., Groenewegen, H.J., 2003. The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci. Biobehav. Rev. 27, 555–579. Hof, P.R., Haroutunian, V., Copland, C., Davis, K.L., Buxbaum, J.D., 2002. Molecular and cellular evidence for an oligodendrocyte abnormality in schizophrenia. Neurochem. Res. 27, 1193–1200. Kawaguchi, Y., Kubota, Y., 1993. Correlation of physiological subgroupings of nonpyramidal cells with parvalbumin- and calbindinD28k-immunoreactive neurons in layer V of rat frontal cortex. J. Neurophysiol. 70, 387–396. Kolb, B., Gibb, R., 1990. Anatomical correlates of behavioural change after neonatal prefrontal lesions in rats. Prog. Brain Res. 85, 241–255. Kolb, B., Gibb, R., 1991. Sparing of function after neonatal frontal lesions correlates with increased cortical dendritic branching: a possible mechanism for the Kennard effect. Behav. Brain Res. 43, 51–56. Kolb, B., Gibb, R., 1993. Possible anatomical basis of recovery of function after neonatal frontal lesions in rats. Behav. Neurosci. 107, 799–811. Kolb, B., Whishaw, I.Q., 1981. Neonatal frontal lesions in the rat: sparing of learned but not species-typical behavior in the presence of reduced brain weight and cortical thickness. J. Comp. Physiol. Psychol. 95, 863–879. Kolb, B., Whishaw, I.Q., 1989. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog. Neurobiol. 32, 235–276. Kolb, B., Sutherland, R.J., Whishaw, I.Q., 1983. Abnormalities in cortical and subcortical morphology after neonatal neocortical lesions in rats. Exp. Neurol. 79, 223–244. Kolb, B., Gibb, R., van der, K.D., 1992. Cortical and striatal structure and

211

connectivity are altered by neonatal hemidecortication in rats. J. Comp. Neurol. 322, 311–324. Kolb, B., Gibb, R., Van der Kooy, D., 1994. Neonatal frontal cortical lesions in rats alter cortical structure and connectivity. Brain Res. 645, 85–97. Kolb, B., Ladowski, R., Gibb, R., Gorny, G., 1996a. Does dendritic growth underlie recovery from neonatal occipital lesions in rats. Behav. Brain Res. 77, 125–133. Kolb, B., Petrie, B., Cioe, J., 1996b. Recovery from early cortical damage in rats, VII. Comparison of the behavioural and anatomical effects of medial prefrontal lesions at different ages of neural maturation. Behav. Brain Res. 79, 1–14. Kolb, B., Cioe, J., Muirhead, D., 1998a. Cerebral morphology and functional sparing after prenatal frontal cortex lesions in rats. Behav. Brain Res. 91, 143–155. Kolb, B., Gibb, R., Gorny, G., Whishaw, I.Q., 1998b. Possible regeneration of rat medial frontal cortex following neonatal frontal lesions. Behav. Brain Res. 91, 127–141. Lewis, D.A., 1995. Neural circuitry of the prefrontal cortex in schizophrenia. Arch. Gen. Psychiatry 52, 269–273. Lewis, D.A., 2000. GABAergic local circuit neurons and prefrontal cortical dysfunction in schizophrenia. Brain Res. 31, 270–276. Lewis, D.A., Hashimoto, T., Volk, D.W., 2005. Cortical inhibitory neurons and schizophrenia. Nat. Rev. Neurosci. 6, 312–324. Liu, J.J., Mohila, C.A., Gong, Y., Govindarajan, N., Onn, S.P., 2005. Chronic nicotine exposure during adolescence differentially influences calciumbinding proteins in rat anterior cingulate cortex. Eur. J. Neurosci. 22, 2462–2474. Lois, C., Alvarez-Buylla, A., 1994. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148. Milad, M.R., Quirk, G.J., 2002. Neurons in medial prefrontal cortex signal memory for fear extinction. Nature 420, 70–74. Mohila, C.A., Onn, S.P., 2005. Increases in the density of parvalbuminimmunoreactive neurons in anterior cingulate cortex of amphetaminewithdrawn rats: evidence for corticotropin-releasing factor in sustained elevation. Cereb. Cortex 15, 262–274. Ng, K.L., Li, J.D., Cheng, M.Y., Leslie, F.M., Lee, A.G., Zhou, Q.Y., 2005. Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science 308, 1923–1927. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego. Rice, D., Barone, S.J., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108, 511–533. Rogido, M., Husson, I., Bonnier, C., Lallemand, M.C., Merienne, C., Gregory, G.A., Sola, A., Gressens, P., 2003. Fructose-1,6-biphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain. Brain Res. Dev. Brain Res. 140, 287–297. Rosenkranz, J.A., Grace, A.A., 2001. Dopamine attenuates prefrontal cortical suppression of sensory inputs to the basolateral amygdala of rats. J. Neurosci. 21, 4090–4103. Schneider, M., Koch, M., 2005a. Behavioral and morphological alterations following neonatal excitotoxic lesions of the medial prefrontal cortex in rats. Exp. Neurol. 195, 185–198. Schneider, M., Koch, M., 2005b. Deficient social and play behavior in juvenile and adult rats after neonatal cortical lesion: effects of chronic pubertal cannabinoid treatment. Neuropsychopharmacology 30, 944–957. Schwabe, K., Enkel, T., Klein, S., Schutte, M., Koch, M., 2004. Effects of neonatal lesions of the medial prefrontal cortex on adult rat behaviour. Behav. Brain Res. 153, 21–34. Schwabe, K., Klein, S., Koch, M., 2006. Behavioural effects of neonatal lesions of the medial prefrontal cortex and subchronic pubertal treatment with phencyclidine of adult rats. Behav. Brain Res. 168, 150–160. Solbach, S., Celio, M.R., 1991. Ontogeny of the calcium binding protein parvalbumin in the rat nervous system. Anat. Embryol. 184, 103–124. Szele, F.G., Chesselet, M.F., 1996. Cortical lesions induce an increase in cell number and PSA-NCAM expression in the subventricular zone of adult rats. J. Comp. Neurol. 368, 439–454.

212

S. Klein et al. / Experimental Neurology 209 (2008) 199–212

Tahraoui, S.L., Marret, S., Bodenant, C., Leroux, P., Dommergues, M.A., Evrard, P., Gressens, P., 2001. Central role of microglia in neonatal excitotoxic lesions of the murine periventricular white matter. Brain Pathol. 11, 56–71. Torrey, E.F., Barci, B.M., Webster, M.J., Bartko, J.J., Meador-Woodruff, J.H., Knable, M.B., 2005. Neurochemical markers for schizophrenia, bipolar disorder, and major depression in postmortem brains. Biol. Psychiatry 57, 252–260. Van Eden, C.G., 1986. Development of connections between the mediodorsal nucleus of the thalamus and the prefrontal cortex in the rat. J. Comp. Neurol. 244, 349–359. Van Eden, C.G., Uylings, H.B.M., 1985. Cytoarchitectonic development of the prefrontal cortex in the rat. J. Comp. Neurol. 241, 253–267. Van Eden, C.G., Kros, J.M., Uylings, H.B., 1990. The development of

the rat prefrontal cortex. Its size and development of connections with thalamus, spinal cord and other cortical areas. Prog. Brain Res. 85, 169–183. Vertes, R.P., 2004. Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse 51, 32–58. Wang, X.H., Jenkins, A.O., Choi, L., Murphy, E.H., 1996. Altered neuronal distribution of parvalbumin in anterior cingulate cortex of rabbits exposed in utero to cocaine. Exp. Brain Res. 112, 359–371. Woo, N.H., Lu, B., 2006. Regulation of cortical interneurons by neurotrophins: from development to cognitive disorders. Neuroscientist 12, 43–56. Zhang, Z.J., Reynolds, G.P., 2002. A selective decrease in the relative density of parvalbumin-immunoreactive neurons in the hippocampus in schizophrenia. Schizophr. Res. 55, 1–10.