Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons

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IMPAIRED SYNAPTIC PLASTICITY IN THE PREFRONTAL CORTEX OF MICE WITH DEVELOPMENTALLY DECREASED NUMBER OF INTERNEURONS

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X. KONSTANTOUDAKI, a,c K. CHALKIADAKI, a,c S. TIVODAR, b,c D. KARAGOGEOS b,c AND K. SIDIROPOULOU a,c*

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a Department of Biology, University of Crete, Voutes University Campus, Vassilika Vouton, GR 70013 Heraklion, Greece

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b Medical School, University of Crete, Voutes University Campus, Vassilika Vouton, GR 71003 Heraklion, Greece

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changes in dendritic morphology observed in non-treated Rac1 cKO mice. Therefore, our data show that disruption in GABAergic inhibition alters glutamatergic function in the adult PFC, an effect that could be reversed by enhancement of GABAergic function during an early postnatal period. Ó 2016 Published by Elsevier Ltd. on behalf of IBRO.

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, Vassilika Vouton, GR 70013 Heraklion, Greece

Key words: synaptic plasticity, NMDA, MGE-derived interneurons, Rac1, dendritic spines, diazepam. 16

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Abstract—Interneurons are inhibitory neurons, which protect neural tissue from excessive excitation. They are interconnected with glutamatergic pyramidal neurons in the cerebral cortex and regulate their function. Particularly in the prefrontal cortex (PFC), interneurons have been strongly implicated in regulating pathological states which display deficits in the PFC. The aim of this study is to investigate the adaptations in the adult glutamatergic system, when defects in interneuron development do not allow adequate numbers of interneurons to reach the cerebral cortex. To this end, we used a mouse model that displays
50% fewer cortical interneurons due to the Rac1 protein loss from Nkx2.1/Cre expressing cells (Rac1 conditional knockout (cKO) mice), to examine how the developmental loss of interneurons may affect basal synaptic transmission, synaptic plasticity and neuronal morphology in the adult PFC. Despite the decrease in the number of interneurons, basal synaptic transmission, as examined by recording field excitatory postsynaptic potentials (fEPSPs) from layer II networks, is not altered in the PFC of Rac1 cKO mice. However, there is decreased paired-pulse ratio (PPR) and decreased long-term potentiation (LTP), in response to tetanic stimulation, in the layer II PFC synapses of Rac1 cKO mice. Furthermore, expression of N-methyl-D-aspartate (NMDA) subunits is decreased and dendritic morphology is altered, changes that could underlie the decrease in LTP in the Rac1 cKO mice. Finally, we find that treating Rac1 cKO mice with diazepam in early postnatal life can reverse

*Correspondence to: K. Sidiropoulou, Department of Biology, University of Crete, Voutes University Campus, GR 70013 Heraklion, Greece. E-mail address: [email protected] (K. Sidiropoulou). Abbreviations: aCSF, artificial cerebrospinal fluid; cKO, conditional knockout; EGTA, ethylene glycol tetraacetic acid; fEPSPs, field excitatory postsynaptic potentials; HEPES, 4-(2-hydroxyethyl)-1-piper azineethanesulfonic acid; LTP, long-term potentiation; MGE, medial ganglionic eminence; NMDA, N-methyl-D-aspartate; PBS, phosphatebuffered saline; PD, postnatal day; PFC, prefrontal cortex; PPR, paired-pulse ratio; PV, protein parvalbumin; SST, somatostatin; YFP, yellow fluorescent protein. http://dx.doi.org/10.1016/j.neuroscience.2016.02.048 0306-4522/Ó 2016 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Physiological studies (Haider and McCormick, 2009; Yizhar et al., 2011) and studies applying neurophysiological simulations using neuronal models (Konstantoudaki et al., 2014; Murray et al., 2014) have shown that excitation-inhibition balance is critical for maintaining proper functioning of the cerebral cortex. Excitation is provided by glutamate release from pyramidal neurons, while inhibition is provided by GABA release from several types of interneurons, which can be classified according to their protein expression, electrophysiological profile, and morphology. Two main types of interneurons include: (a) those that express the calcium binding protein parvalbumin (PV), have fast-spiking profile and target the soma and axon of the pyramidal neurons, and (b) those that express the neuropeptide somatostatin (SST), have a regular-spiking electrophysiological profile and primarily target the apical dendrites of the pyramidal neurons (Markram et al., 2004; Yizhar et al., 2011). Both cortical PV+ and SST+ interneurons originate from the medial ganglionic eminence (MGE) of the embryonic mouse brain (Wonders and Anderson, 2006). Many neuropsychiatric disorders, such as epilepsy, anxiety, schizophrenia and autism exhibit an imbalance between excitatory and inhibitory mechanisms, in several brain regions including the prefrontal cortex (PFC) (Lewis et al., 2003; Yizhar et al., 2011; Marı´ n, 2012). Specifically, reduction in interneuronal markers, such as GAD65/67 and PV, or GABA system adaptations have been correlated with several mental diseases, for example, schizophrenia (Lewis et al., 2003; Lodge et al., 2009; Hyde et al., 2011), autism (Fatemi et al., 2008a,b; Blatt and Fatemi, 2011), depression (Markram et al., 2004; Kalueff and Nutt, 2007; Yizhar et al., 2011; Mo¨hler, 2012) and epilepsy (Powell, 2013).

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Although a reduction in GABAergic markers has been observed in several neuropsychiatric illnesses, it is not known whether these changes are causative or an adaptation of other primary modifications. The enhanced knowledge with regard to the transcription factors and intracellular mediators regulating various aspects of interneuron development has resulted in the generation of transgenic mouse lines with fewer cortical interneurons due to impaired proliferation and/or migration (Lewis et al., 2003; Cobos et al., 2005; Butt et al., 2008; Kerjan et al., 2009; Yizhar et al., 2011; Marı´ n, 2012; Neves et al., 2012; Finlay and Uchiyama, 2015). These mice can be used to determine whether developmental defects in interneuron function would underlie network-wide changes in the brain. In this study, we utilize a transgenic mouse line that is missing the Rac1 gene from Nkx2.1/Cre-expressing cells (Rac1fl/fl/Nkx2.1+/Cre) of the MGE (referred to as the Rac1 (conditional knockout) cKO mouse hereafter; (Vidaki et al., 2012)). The Nkx1.2 transcription factor controls the generation of distinct interneuron subtypes originating from the MGE (Marin et al., 2000; Anderson et al., 2001). We have shown previously that the Rac1 cKO mice contain about 50% fewer MGE-derived GABAergic interneurons in the postnatal barrel cortex. This decrease results mainly from a longer G1 phase of MGE interneuron progenitors, leading to a delay in cell cycle exit, due to the Rac1 protein loss specifically from these cells (Vidaki et al., 2012). The interneurons found in the postnatal cortex have normal morphology, however the 50% that do not migrate remain aggregated in the ventral telencephalon and exhibit defective growth cones. Many mice die after 3 weeks of age (see the Experimental procedures section). Using the Rac1 cKO mice, our goal is to determine how the developmental decrease in the number of interneurons affects the glutamatergic transmission properties of PFC neurons in adult mice. We demonstrate that PFC neurons of Rac1 cKO mice exhibit: (a) decreased paired-pulse facilitation at 20-Hz frequency, (b) decreased long-term potentiation (LTP), (c) reduced NR2A and NR2B subunits of the N-methylD-aspartate (NMDA) receptors, and (d) reduced number of mushroom-type spines. In addition, we find that Rac1cKO mice treated with diazepam, a GABA-A receptor agonist, during the early postnatal period, do not develop the defect in dendritic morphology, suggesting that enhancement of GABA-A receptor function during that period reverses the detrimental effects of decreased number of interneurons in these mice.

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EXPERIMENTAL PROCEDURES

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Animals and housing

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Adult male mice, 60–80 days of age, were used for all experiments. Mice were housed in groups (3–4 per cage) and provided with standard mouse chow and water ad libitum, under a 12-h light/dark cycle (light on at 7: 00 am) with controlled temperature (21 ± 1 °C). The following genotypes were used for analysis:

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Rac1(fl/fl);Nkx2.1(+/Cre) (referred to as Rac1 cKO mice) and Rac1(+/fl);Nkx2.1(+/Cre), referred to as heterozygous mice. The heterozygous mice were used as the control group to Rac1 cKO. Animals carrying a floxed allele of Rac1 (Rac1fl/fl;Nkx2.1+/Cre) were previously described (Vidaki et al., 2012). Specifically, animals carrying a floxed allele of Rac1 (Rac1fl/fl) (the fourth and fifth exons of the Rac1 gene are flanked with loxP sites, (Walmsley et al., 2003) were crossed with Nkx2.1Tg(Cre) mice (Nkx2.1 transgenic Cre, (Fogarty et al., 2007)), in order to generate the Rac1fl/fl;Nkx2.1Tg(Cre) genotype. The ROSA26fl-STOP-fl-YFP allele was also inserted as an independent marker, to allow visualization of the MGE-derived interneurons in which the Rac1 protein is deleted and Rac1 heterozygous MGE-derived interneurons, via yellow fluorescent protein (YFP) expression (Srinivas et al., 2001). Mice used in these experiments were taken from crossing Rac1fl/fl;Nkx2.1+/+ with Rac1+/fl;Nkx2.1+/Cre genotypes. At least 80% of Rac1 heterozygous and Rac1 cKO animals came from the same litters. Fifty percent of the Rac1fl/fl;Nkx2.1+/cre (Rac1 cKO) die within 3 weeks after birth. Our experiments were conducted only in mice that survived at least until postnatal day (PD) 60. All procedures were performed according to the European Union ethical standards and the IMBB and University of Crete ethical rules.

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Electrophysiology

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Electrophysiological experiments were performed using the in vitro slice preparation. Mice were decapitated under halothane anesthesia. The brain was removed immediately and placed in ice cold, oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 3.5 KCl, 26 NaHCO3, 1 MgCl2 and 10 glucose (pH = 7.4, 315 mOsm/l). The brain was blocked and glued onto the stage of a vibratome (Leica, VT1000S, Leica Biosystems GmbH, Wetzlar, Germany). 400- lm-thick brain slices containing the PFC were taken and were transferred to a submerged chamber, which was continuously superfused with oxygenated (95% O2/5% CO2) aCSF containing (in mM): 125 NaCl, 3.5 KCl, 26 NaHCO3, 2 CaCl2, 1 MgCl2 and 10 glucose (pH = 7.4, 315 mOsm/l) at room temperature. The slices were allowed to equilibrate for at least one hour in this chamber before experiments began. Slices were then transferred to a submerged recording chamber, which was continuously superfused with oxygenated (95% O2/5% CO2) aCSF (same constitution as the one used for maintenance of brain slices) at room temperature. Extracellular recording electrodes filled with NaCl (2 M) were placed in layers II/III of PFC. Platinum/ iridium metal microelectrodes (Harvard apparatus UK, Cambridge, UK) were placed on layer II of the PFC, about 300 lm away from the recording electrode, and were used to evoke field excitatory postsynaptic potentials (fEPSPs). Responses were amplified using a Dagan BVC-700A amplifier (Dagan Corporation, Minneapolis, MN, USA), digitized using the ITC-18

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board (Instrutech, Inc) on a PC using custom-made procedures in IgorPro (Wavemetrics, Inc, Lake Oswego, OR, USA). The electrical stimulus consisted of a single square waveform of 100-lsec duration given at intensities of 0.05–0.3 mA generated by a stimulator equipped with a stimulus isolation unit (World Precision Instruments, Inc).

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Data acquisition and analysis

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Data were acquired and analyzed using custom-written procedures in IgorPro software (Wavemetrics, Inc). The fEPSP amplitude was measured from the minimum value of the synaptic response (4–5 ms following stimulation) compared to the baseline value prior to stimulation. Both parameters were monitored in realtime in every experiment. A stimulus-response curve was then determined using stimulation intensities between 0.05 and 0.3 mA. For each different intensity level, two traces were acquired and averaged. Baseline stimulation parameters were selected to evoke a response of 1 mV. The paired-pulse protocol consisted of two pulses at baseline intensity separated by 100, 50 and/or 20 ms. For the LTP experiments, baseline responses were acquired for at least 20 min, then three 1 s tetanic stimuli (100 Hz) with an inter-stimulus interval of 20 s were applied and finally responses were acquired for at least 50 min post-tetanus every 1-min. Synaptic responses were normalized to the average 10 min pre-tetanic fEPSP.

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Golgi–Cox staining

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Brains were removed and placed in Golgi–Cox solution (5% Potassium Dichromate, 5% Mercuric Chloride (sublimate), and 5% Solution of Potassium Chromate), which had been prepared at least 5 days earlier. Brains remained in Golgi–Cox solution for 10 days at room temperature, then placed in 30% sucrose solution and subsequently sliced (150- lm-thick slices) in a vibratome (Leica VT1000S, Leica Biosystems GmbH, Wetzlar, Germany). The slices were placed onto gelatin-coated microscope slides, covered with parafilm, and maintained in a humidity chamber for about 30–40 h. The parafilm was then removed, and the slides were incubated first in ammonium hydroxide for 15 min in a dark room and then in Kodak Fix solution for 15 min followed by washes with dH2O. The brain slices were then dehydrated with increasing concentrations of ethanol, incubated in xylene for 5 min and coverslipped with permount. The slides were kept for at least a month before imaging under the 60 lens of a Nikon Eclipse E800 microscope (Nikon Instruments Europe BV, Amsterdam, Netherlands). Initially, the PFC neurons to be analyzed were picked under 20 magnification. We analyzed 2–5 neurons per animal of both hemispheres, and measured the number of secondary dendritic segments, the length of apical dendrites, the number of spines on them and the dendritic diameter. The spines were analyzed separately according to the spine type: thin, stubby and mushroom spines (Horner and Arbuthnott, 1991).

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Western blot

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Mice were decapitated following cervical dislocation, the brain was quickly removed, placed in ice cold PBS (phosphate-buffered saline) and then positioned on a brain mold, where 1.5-mm slices were taken containing the PFC. The slices were placed on dry ice, and the prelimbic area of PFC was dissected out and stored at 80 °C. Frozen tissue blocks were lysed in a solution containing (in mM) HEPES 50, NaCl 150, MgCl2 1.5, EGTA 5, Glycerol 1%, Triton-X100 1%, 1:1000 Protease inhibitors cocktail. Proteins ran on 8.5% bis acrylamide gel and transferred onto nitrocellulose membrane. The membrane was blocked, incubated in rabbit polyclonal anti-NR2A (Alomone Labs, Jerusalem, Israel, 1:2000), rabbit polyclonal anti-NR2B (Alomone labs, Jerusalem, Israel, 1:2000) or rabbit monoclonal anti-GAPDH (Cell Signaling Technology Europe BV, Leiden, Netherlands, 1:1000), washed, incubated in secondary goat antirabbit IgG Horseradish Peroxidase Conjugate antibody (Invitrogen, 1:5000), and exposed to film. Analysis of NR2A, NR2B and GAPDH expression was performed with ImageJ software, and the raw values of NR2A and NR2B from each sample were normalized to their respective GAPDH values.

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Nissl staining

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Brains of heterozygous and Rac1 cKO mice (PD70) were removed, and placed in 4% PFA. After 24 hours, the brains were place in PBS with azide (at 4 °C), for maintenance until slicing. Brains were glued onto vibratome stage and 40-lm-thick slices were acquired from adult (>60 days old) heterozygous and Rac1 cKO animals (VT1000S, Leica Microsystems, Wetzlar, Germany). For each animal, 3–4 sections were used, corresponding to different rostrocaudal levels of the brain (2.22–1.70), all including the PFC. Sections were incubated in xylene for 5 min and then for 3 min in 90%, 70% ethanol solutions and dH2O, followed by a 10-min incubation in 0.1% Cresyl Violet solution. Sections were then dehydrated with increasing concentrations of ethanol (70%, 90%, 100%), incubated in xylene for 5 min and coverslipped with permount. Images from whole sections were obtained in 5 magnification of a light microscope (Axioskop 2FS, Carl Zeiss AG, Oberkochen, Germany) and merged using Adobe Photoshop software. Cortical thickness was measured manually in Adobe Photoshop 14.2 using stitched photos of the whole PFC section. The width was measured from the midline to the beginning of the white matter, in 3–4 different sections from each animal. In order to count the cells, a region that includes the prelimbic region of the PFC was selected (1.2 * 105 lm2) and cropped. The background color of each cropped image was converted to black, while the cells were colored blue. The images were loaded into Matlab, where the number of ‘blue’ pixels was counted. Each cell was assumed to be composed of four pixels. Therefore, the number of cells was measured as the total number of ‘blue’ pixels divided by four. An average rostrocaudal number was calculated for the number of

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neuronal cells for Rac1 heterozygous and Rac1 cKO animals.

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Immunohistochemistry

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For YFP and PV immunohistochemistry, adult mice, 2–3 months old, were perfused with 4% PFA, following fixation with the same solution for 1 h at 4 °C. They were subsequently processed as previously described in Vidaki et al., 2012. Rat monoclonal anti-GFP (Nacalai Tesque, Kyoto, Japan, 1:500) and rabbit polyclonal antiparvalbumin (PV) (Swant, Bellinzona, Switzerland; 1:1000) were used as primary antibodies. Goat anti-ratAlexa Fluor-488 and goat anti-rabbit-Alexa Fluor-555 (Molecular Probes, Eugene, OR, USA 1:800) were used as secondary antibodies. For quantification of YFP and PV/YFP interneurons in adult mice, at least three pairs of littermate animals were used (heterozygous and Rac1 cKO). After PV and YFP immunostaining, images were obtained with a confocal microscope (Leica TCS SP2, Leica, Nussloch, Germany). For each pair, three sections corresponding to distinct bregma along the rostrocaudal axis (in between 2.22 and 1.70) were selected, all including the PFC. YFP- and PV/YFP-positive cells in the PFC were counted and an average rostrocaudal number was calculated for the interneuron subpopulations of heterozygous and Rac1 cKO animals.

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Diazepam treatment

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For the rescue experiments, diazepam was acquired from the Pharmacy of the University General Hospital in Heraklion as a 5 mg/ml solution, and was diluted in sterile saline solution. Each animal was injected with 0.5 mg/kg diazepam, intraperitoneally (i.p.), once a day, from PD11–20. Mice were allowed to reach adulthood and their brains were removed at PD70 according to the Golgi–Cox staining procedure described above.

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Statistical analysis

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Appropriate statistical analysis was performed in Microsoft Office Excel 2007, GraphPad Prism6 or with IBM SPSS Statistics v.21, as indicated in the figure legends and the results section. Data in the graphs are presented as mean ± standard error of mean (SEM).

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RESULTS

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Characterization of interneurons in the adult PFC in the mutant mice

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(Vidaki et al., 2012). As a result, the GABAergic interneuron population in the barrel cortex is significantly reduced, by 50%, due to a defect in the cell cycle exit of interneuron progenitors as well as a delay in migration and not due to a differentiation defect (Vidaki et al., 2012). Here, we examined the long-term consequences of Rac1 absence in MGE-derived interneurons in the adult mouse cortex, and specifically the PFC, a brain area in which GABAergic inhibition is critical for its neuronal function and cognitive behavior (Sohal et al., 2009; Yizhar et al., 2011; Marı´ n, 2012). Nissl staining of brain slices from heterozygous and Rac1 cKO mice revealed no differences in the number of cells present within the prelimbic area of the PFC, as well as in the PFC thickness between heterozygous and Rac1 cKO mice (Fig. 1). Therefore, the gross anatomy of adult PFC was not altered between heterozygous and Rac1 cKO mice. Next, the number and distribution of the interneurons in the adult PFC were investigated, using equivalent cryosections for the heterozygous and Rac1 cKO mouse PFC at different levels throughout the rostrocaudal axis (Fig. 2A–C). We quantified the positive cells for the lineage marker YFP, which marks all Nkx2.1-expressing cells, and PV, which marks a major subpopulation of

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We used the Rac1 cKO mice in order to study the effect of embryonic deficits of GABAergic interneuron progenitors in the adult PFC. In these mice, the Rac1 protein was deleted from Nkx2.1-expressing cells, using Cre/loxP technology. In the nervous system, Nkx2.1 is expressed in MGE-cells from embryonic day (E)9 (Sussel et al., 1999). In the Rac1 cKO mice, the Rac1 protein is absent from MGE-derived cells by E12 (Vidaki et al., 2012). Therefore, Rac1 cKO mice do not express the Rac1 protein in MGE-derived interneurons that are destined to become PV- and SST-positive interneurons in the cortex

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Fig. 1. Gross anatomical features of the PFC are not different between heterozygous and Rac1 cKO mice. Representative images of Nissl-stained brain slices containing the PFC. The outlined shape indicates the PFC area measured. (A) Bar graph showing that there is no difference in the PFC thickness (n = 3 mice from each genotype, ttest, p > 0.1). (B) Bar graph showing that there is no difference in the number of cells stained within the PFC, within the enclosed area indicated in A (n = 3 mice from each genotype, ttest, p > 0.1).

Please cite this article in press as: Konstantoudaki X et al. Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.02.048

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Fig. 2. The number of MGE-derived cortical interneurons in the Rac1 cKO adult is severely reduced. (A–A0 ). Coronal sections of the PFC from heterozygous (top) and Rac1 cKO (bottom) adult mice immunostained using antibodies: anti-GFP and anti-PV. (B–B0 ). Representative areas of PFC from heterozygous (top) and Rac1 cKO (bottom) mice. Scale bar = 300 lm. (C–C0 ). Zoom in of PFC representative areas from heterozygous (top) and Rac1 cKO (bottom) mice. Scale bars = 75 lm. (D–E) Graphs showing the numbers of YFP-positive and YFP/PV-positive interneurons were reduced in the Rac1 cKO mice compared to heterozygous mice (t-test, ****p < 0.0001; ***p = 0.0003). (F) Graph showing the percentage of YFP/PV-positive interneurons/total YFP-positive interneurons was the same in the Rac1 cKO and heterozygous mice (p = 0.4).

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cortical interneurons originating in the MGE. Our analysis showed that the number of Nkx2.1-derived YFP-positive interneurons and the number of PV/YFP-positive interneurons are significantly decreased in the PFC of adult Rac1 cKO mice (Fig. 2D–E). After calculating the ratio of PV/ YFP-positive cells over total YFP-positive cells, we found the same ratio for both heterozygous and Rac1 cKO mice (Fig. 2F). This finding revealed that the reduction of PV/YFP-positive cells in the PFC of Rac1 cKO mice does not result from the inability of mutant precursors to differentiate into PV+ interneurons but from a migratory impairment of total YFP and PV/YFP cells to the PFC, as is the case in the barrel cortex (Vidaki et al., 2012).

Changes in basal synaptic transmission and shortterm synaptic plasticity in the adult PFC

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We, then, investigated the properties of the mature PFC glutamatergic system in heterozygous and Rac1 cKO mice using the acute brain slice preparation. To study basal synaptic transmission, the evoked synaptic response of the Rac1 cKO PFC was analyzed compared to heterozygous PFC (n = 17 slices from 12 heterozygous mice and n = 11 from 7 Rac1 cKO mice). We delivered current pulses of increasing intensity through the stimulating electrode positioned in layer II of the PFC and recorded fEPSPs from layer II PFC (Fig. 3A). The fEPSP responses to increasing amplitude

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Fig. 3. Changes in basal synaptic transmission, PPR and LTP in Rac1 cKO compared to heterozygous mice. (A) Schematic showing the position of the electrodes in PFC brain slices (R: recording electrode, S: stimulating electrode). (B) The fEPSP responses to increasing amplitude of stimulation currents were the same in Rac1 cKO mice (n = 7) and heterozygous mice (n = 12) (2-way repeated measures ANOVA, F(1,18) = 0.5, p > 0.1). (C) (Top) Indicative traces of paired-pulse recordings from heterozygous and Rac1 cKO mice at 20 Hz paired-pulse frequency. Scale bars: 1 mV (vertical), 40 ms (horizontal). (Bottom) Graph showing the PPR of the heterozygous and Rac1 cKO mice at 10 Hz, 20 Hz and 50 Hz frequencies. Paired-pulse facilitation is observed in heterozygous mice (n = 12) when the paired-pulse frequency was 20 Hz, but not in Rac1 cKO mice (n = 7) (2-way repeated measures ANOVA, F(1,18) = 3.94, p < 0.05 (*)). No paired-pulse facilitation or depression was observed in heterozygous and Rac1 cKO mice at the 10 Hz or 50 Hz frequencies. (D) In heterozygous mice (n = 12), tetanic stimulation results in enhanced fEPSP for at least 45 min. In Rac1 cKO mice (n = 7), the enhanced response following the tetanus is significantly smaller (2-way repeated measures ANOVA, F(1,18) = 7.31, p < 0.01). (E) We tested whether NMDA receptor activation could underlie the LTP observed in heterozygous mice. The presence of 50 lM AP-5 in the brain slice significantly decreased the LTP induced in heterozygous mice (n = 4) (2-way repeated-measures ANOVA F(1,12) = 73.7, p < 0.001), suggesting that the LTP we study is NMDA-dependent.

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of stimulation currents in the PFC were the same in Rac1 cKO mice compared to heterozygous mice (Fig. 3B). These results suggest that despite the decreased number of interneurons, the population synaptic response is unaltered. In order to further study the synaptic response properties, we delivered paired stimulations of different frequencies (10 Hz, 20 Hz and 50 Hz). We found that the paired-pulse ratio (PPR) was not different between heterozygous and Rac1cKO mice at 10-Hz and 50-Hz paired-pulse frequencies, mice, at which no facilitation nor depression is observed. At 20Hz paired-pulse frequency, the PPR showed facilitation in heterozygous mice, compared to the other frequencies, in agreement with other studies (Hernan et al., 2013). However, the PPR at 20 Hz was significantly decreased in the Rac1 cKO mice compared to heterozygous mice, and showed no facilitation in comparison with the PPR at the other frequencies (Fig. 3C). Such pairedpulse facilitation is thought to contribute to the emergence

of persistent activity, the cellular correlate of working memory, a cognitive function mediated by the PFC (Wang, 1999; Wang et al., 2006). Therefore, decreasing paired-pulse facilitation at 20 Hz could disrupt the emergence of persistent activity in the PFC of Rac1 cKO mice and consequently their working memory performance.

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LTP in the PFC

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Next, we studied the Rac1 cKO mice for possible defects in long-term plasticity. For this, we employed tetanic stimulation, which in heterozygous brain slices (n = 17) resulted in 50% increase of baseline fEPSP responses for at least 45 min. However, in PFC slices from Rac1 cKO mice (n = 11), the same tetanic stimulation did not result in fEPSP potentiation (2-way repeated measures ANOVA, F1,18 = 8.31, p < 0.01) (Fig. 3D). LTP is known to require NMDA receptor activation in both the hippocampus (Bliss and Collingridge, 2013) and the

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PFC (Goto et al., 2009). Indeed, LTP in layer II PFC is also dependent on NMDA receptor activation, since no facilitation of the fEPSP is observed in the presence of AP5, ((2R)-amino-5-phosphonovaleric acid), an NMDA receptor antagonist (Fig. 3E). Therefore, NMDAdependent LTP was found decreased in mice with reduced numbers of interneurons in the PFC. Decreased synaptic plasticity in the PFC has also been identified in several animal models of pathological states (Goto et al., 2009).

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NMDA receptor subunit expression

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NMDA receptors are heteromers of the NR1 subunit and either NR2A and NR2B subunits (Paoletti et al., 2013) and mediate the emergence of LTP (Malenka and Nicoll, 1993). Therefore, we examined whether changes in NMDA receptor subunit expression are present in Rac1 cKO mice by performing western blots from PFC tissue of Rac1 cKO mice and heterozygous littermates. We found a significant decrease in the expression of both NR2A and NR2B subunits (normalized to GAPDH expression) in the PFC of Rac1 cKO mice (n = 9) compared to heterozygous mice (n = 11) (Kruskal–Wallis test, p = 0.03) (Fig. 4). These results suggest that the decreased expression of NMDA receptors could partially mediate the reduction in LTP in Rac1 cKO mice.

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Dendritic morphology

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It is well-established that dendritic structure, particularly dendritic spine number and shape, is positively

A.

heterozygous

3.5

Rac1 cKO

correlated with LTP expression (Matsuzaki et al., 2004; Zuo et al., 2005; Alvarez and Sabatini, 2007). Specifically, the mushroom type spines are mostly related to LTP while the thin spines are thought to mediate persistent activity in the PFC (Kasai et al., 2003). Therefore, we analyzed Golgi–Cox-stained slices and investigated the PFC pyramidal neuron dendritic morphology in heterozygous and Rac1 cKO mice. No major changes were observed in the length and total spine density of pyramidal neuron dendrites. However, a more detailed analysis of the different types of dendritic spines uncovered a significant decrease in the density of mushroom type spines (Kruskal–Wallis test, p < 0.05) on secondary dendrites, but no change in the density of thin or stubby spines (n = 11 cells from four heterozygous mice and n = 9 from 4 Rac1 cKO mice) (Fig. 5 and Table 1).These results suggest that alterations in the dendritic structure could also explain, in part, the decrease in LTP induction in the PFC.

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Effects of GABA enhancement during early postnatal development on dendritic morphology

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We next aimed to determine whether enhancing GABA receptor function during early development could alleviate some of the changes observed in pyramidal neurons (Fig. 6A). Heterozygous and Rac1 cKO mice were treated i.p. with 1 mg/kg diazepam, once a day for 10 days, from PD11–20. Another group of heterozygous mice was treated with saline, during the same postnatal period. No Rac1 cKO mice survived to adulthood in the saline-treated group, therefore, the direct comparison

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B. 5.0 4.5

3.0 2.5

NR2B relative density

4.0

NR2A relative density

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7

*

2.0 1.5 1.0

*

3.5 3.0 2.5 2.0 1.5 1.0

0.5 0.5 0.0

HET

0.0

cKO

NR2A 165kDa

NR2B 165kDa

HET

cKO

GAPDH ~40kDa Fig. 4. NMDA receptor expression and dendritic structure in heterozygous and Rac1 cKO mice. (A) (Top) Graphs showing that the density of the NR2A subunit, normalized to GAPDH density, is significantly reduced in the PFC of Rac1 cKO mice (n = 9) compared to heterozygous mice (n = 11) (Kruskal–Wallis test, *p < 0.05). (Bottom) Representative blots of NR2A and GAPDH for Het (heterozygous) and Rac1 cKO mice. (B) (Top) Graphs showing that the density of the NR2B subunit, normalized to GAPDH density, is significantly reduced in the PFC of Rac1 cKO mice (n = 9) compared to heterozygous mice (n = 11) (Kruskal–Wallis test, *p < 0.05). (Bottom) Representative blots of NR2B and GAPDH for Het (heterozygous) and Rac1 cKO mice. Please cite this article in press as: Konstantoudaki X et al. Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.02.048

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Spine Density (per 10µm)

A.

C.

1.8

Heterozygous

1.6 1.4 1.2 1.0

2µm

0.8

Rac1 cKO

0.6 0.4 0.2 2µm

0 All spines

B. heterozygous

Spine Density (per 10µm)

2.5

Rac1 cKO

2.0 1.5 1.0

*

0.5 0.0

Thin

Stubby

Mushroom

Fig. 5. Dendritic structure in PFC pyramidal neurons in Rac1 cKO and heterozygous mice. (A) The density of dendritic spines in Rac1 cKO mice is not significantly different from that of heterozygous mice (Kruskal–Wallis test, p > 0.1). (B) The density of mushroom type spines on the apical dendrites of PFC pyramidal neurons of Rac1 cKO mice (n = 9) is significantly reduced compared to the heterozygous mice (n = 11) (Kruskal–Wallis test, *p < 0.05), but not the thin and stubby type of dendritic spines. (C) Representative photographs showing the decreased number of mushroom type dendritic spines in the PFC of Rac1 cKO mice (right) compared to the heterozygous mice (left).

Table 1. Measurements of parameters of dendritic morphology in PFC pyramidal neurons in heterozygous and Rac1 cKO mice

# of secondary dendrites Dendritic length (lm) # of thin spines # of stubby spines # of mushroom spines Total # of spines Density of thin spines (per 10 lm) Density of stubby spines (per 10 lm) Density of mushroom spines (per 10 lm) Total dendritic spine density

485 486 487 488 489 490 491

Heterozygous mice

Rac1 cKO

Kruskal–Wallis test

3.13 ± 0.31 93.94 ± 18.1 13.52 ± 2.63 22.14 ± 3.47 11.86 ± 2.51 45 ± 8 1.31 ± 0.16 1.75 ± 0.16 0.85 ± 0.05 4.94 ± 0.76

2.40 ± 0.43 71.09 ± 10.13 10.11 ± 3.26 15.56 ± 4.75 5.33 ± 1.48 31 ± 9 1.15 ± 0.2 1.67 ± 0.3 0.67 ± 0.17 3.45 ± 0.67

0.2 0.6 0.3 0.45 0.02* 0.2 0.5 0.2 0.03* 0.2

between saline-treated and diazepam-treated Rac1 cKO mice was not possible. Mice were then allowed to grow into adulthood (PD70), at which time their brains were removed and subjected to the Golgi–Cox staining procedure. The Golgi–Cox analysis showed no difference between saline-treated heterozygous (n = 11), diazepam-treated heterozygous (n = 11) and

Rac1 cKO mice (n = 7) (one-way ANOVA, F(2,17) = 0.051, p = 0.9), with regard to the number of secondary dendrites and the dendritic spine density for thin, stubby or mushroom-type spines (Table 2 and Fig. 6B). When comparing the untreated Rac1 cKO mice, as shown in Fig. 5, with the diazepam-treated Rac1 cKO mice, a difference in the mushroom spine

Please cite this article in press as: Konstantoudaki X et al. Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.02.048

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A. PD11-20 DZP treatment i.p. once daily

Birth

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

heterozygous - saline treated heterozygous - DZP treated Rac1 cKO - DZP treated

C.

Rac1 cKO - untreated Rac1 cKO - DZP treated

2.5

Spine Density (per 10µm)

Spine Density (per 10µm)

B. 2.0

PD70, Brains place in Golgi-Cox solution

2.0

*

1.5 1.0 0.5 0.0

thin

stubby

mushroom

thin

stubby

mushroom

D. Heterozygous -non-treated

Heterozygous - DZP treated Rac1 cKO - DZP treated

2µm

Fig. 6. Dendritic structure in PFC pyramidal neurons in Rac1 cKO and heterozygous mice that were treated with diazepam in early postnatal life. (A) Schematic diagram of the experimental timeline. The injections with diazepam (1 mg/kg/day) were done in mice PD11-20, and their brains were removed and placed in Golgi-Cox solution at 70 days old. (B) The spine density of thin, stubby or mushroom-type spine density on the apical dendrites was not significantly different between the three groups of mice examined (heterozygous saline-treated n = 11, heterozygous diazepamtreated n = 11 and Rac1 cKO diazepam-treated, n = 7, one-way ANOVA, F(2,28) = 2.4, p > 0.1). (C) The mushroom spine density is increased in diazepam-treated Rac1 cKO mice (n = 7) compared to untreated Rac1 cKO mice (n = 9). (D) Representative photographs of dendritic spines of the three groups examined: heterozygous mice, saline-treated, heterozygous mice – diazepam treated, and Rac1 cKO mice – diazepam treated. Table 2. Measurements of parameters of dendritic morphology in PFC pyramidal neurons in heterozygous and Rac1 cKO mice that were treated with diazepam during early postnatal life (PD 11–20)

# of secondary dendrites Total dendritic length (lm) # of thin spines # of stubby spines # of mushroom spines Total # of spines Dendritic spine density (thin) (per 10 lm) Density of stubby spines (per 10 lm) Density of mushroom spines (per 10 lm) Total dendritic spine density

499 500 501 502 503 504 505 506 507

Heterozygous mice – saline treated (n = 11)

Heterozygous mice – diazepam-treated (n = 11)

Rac1 cKO– diazepamtreated (n = 7)

One-way ANOVA test

3.5 ± 0.3 87 ± 12 11.9 ± 2 13.2 ± 1.9 10.5 ± 2 39.9 ± 2 1.37 ± 0.15 1.61 ± 0.15 1.17 ± 0.18 4.4 ± 0.3

3.1 ± 0.4 61 ± 9 7.7 ± 1 7.4 ± 0.9 6.6 ± 1 18.5 ± 2.2 2.94 ± 0.13 1.73 ± 0.15 0.35 ± 0.03 3.9 ± 0.5

3.3 ± 0.4 87 ± 13 8.4 ± 1.3 11.7 ± 3.3 10.1 ± 2.9 30.2 ± 7 1.3 ± 0.17 1.6 ± 0.3 1.5 ± 0.3 5.2 ± 0.9

0.8 0.2 0.1 0.1 0.3 0.1 0.9 0.4 0.6 0.3

density, but not in the thin or stubby spine density, is present. Specifically, untreated Rac1 cKO mice have significantly decreased mushroom spine density (Fig. 6C). Since this comparison does not take into account the 10-day i.p. injection procedure, we compared heterozygous untreated mice to heterozygous saline-treated mice in order to determine whether the injection protocol by itself had an effect on dendritic morphology. Several reports suggest that chronic mild

stress during the early postnatal period affects dendritic morphology in adulthood (Chocyk et al., 2013; Yang et al., 2013). Although repeated injections for 10 days could simulate repeated mild stress, most reports showing defects in dendritic morphology use more intense stress paradigms, such as restrain stress or social stress (Chocyk et al., 2013). Our analysis shows that saline treatment in heterozygous mice does not result in dendritic spine density changes (Table 3), suggesting that

Please cite this article in press as: Konstantoudaki X et al. Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.02.048

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Table 3. Comparison of parameters of dendritic morphology between saline-treated with untreated heterozygous mice

# of secondary dendrites Total dendritic length (lm) # of thin spines # of stubby spines # of mushroom spines Total # of spines Dendritic spine density (thin) (per 10 lm) Density of stubby spines (per 10 lm) Density of mushroom spines (per 10 lm) Total dendritic spine density

Heterozygous mice

Heterozygous mice – saline treated (n = 11)

Kruskal–Wallis test

3.13 ± 0.31 93.94 ± 18.1 13.52 ± 2.63 22.14 ± 3.47 11.86 ± 2.51 45 ± 8 1.31 ± 0.16 1.75 ± 0.16 0.85 ± 0.05 4.94 ± 0.76

3.5 ± 0.3 87 ± 12 11.9 ± 2 13.2 ± 1.9 10.5 ± 2 39.9 ± 2 1.37 ± 0.15 1.61 ± 0.15 1.17 ± 0.18 4.4 ± 0.3

0.7 0.3 0.4 0.09 0.6 0.6 0.6 0.2 0.8 0.6

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the exposure to the mild stress of i.p. injections does not have a deleterious effect on dendritic morphology. Collectively, our results suggest that the decrease seen in the mushroom spine density of Rac1 cKO mice could be reversed in the Rac1 cKO mice treated with diazepam.

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DISCUSSION

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In this study, we report that a significant decrease in the cortical interneuron number causes adaptations in several features of the mature glutamatergic transmission in the adult PFC, such as the PPR at 20 Hz, LTP induction, dendritic spines and NMDA receptor subunit expression. Furthermore, we find that increasing GABA receptor function during early postnatal life reverses the defect in dendritic morphology.

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GABAergic inhibition and PFC function

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The PFC mediates several higher order cognitive processes, such as attention, behavioral inhibition, and social behavior (Etkin, 2012). GABAergic function is necessary for proper PFC functioning. Inhibiting GABA synthesis increases impulsivity and locomotor activity in rats, while it transiently impairs attention (Paine et al., 2015). In addition, inactivating PV interneurons with optogenetics disrupts proper social behavior (Yizhar et al., 2011). Furthermore, GABAergic function interferes with network activity in the PFC, since decreasing the activity of PV interneurons disrupts the gamma oscillations, which are necessary for proper cognitive function and related behaviors (Sohal et al., 2009). Interfering with migration of interneurons by deleting the Dlx4/5 transcription factors also reveals a pathological phenotype with impairments in gamma frequency oscillations in PFC (Cho et al., 2015). Our data reinforce the findings that the inhibitory and excitatory systems are intricately connected; therefore, deficits in the inhibitory system affect the properties of the glutamatergic system. Specifically, our study demonstrates that in the presence of decreased inhibition throughout the postnatal life of the animal, several parameters of the glutamatergic system, such as PPR responses, synaptic plasticity, NMDA receptor expression and dendritic spine density, are reduced. These changes collectively decrease the ability of the glutamatergic system to potentiate its responses and possibly reflect homeostatic mechanisms aiming to limit

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the augmented excitability initiated by the decreased interneurons. Homeostatic plasticity has been observed when excitatory transmission is decreased or increased (Turrigiano and Nelson, 2004). Both the glutamatergic and GABAergic systems adapt to changes in excitatory drive. In our study, we found that changes in the GABAergic interneurons affects properties of the glutamatergic system. The mechanisms involved in this regulation are not known, but could involve changes of GABAergic receptors on pyramidal neurons (Vertkin et al., 2015).

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The role of LTP in PFC function

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The PFC is considered the neuroanatomical substrate of working memory (Goldman-Rakic, 1996), a type of short-term memory that allows for on-line representation of information for a few seconds before an action takes place. Because of the short-term nature of working memory, physiological and cellular mechanisms that could underlie working memory have been extensively studied in the PFC. Persistent activity (or delay-period activity) has been uncovered as the cellular correlate of working memory (Goldman-Rakic, 1995). Cellular mechanisms that underlie persistent activity include the NMDA current and calcium-activated non-selective cation current (Wang, 1999; Sidiropoulou et al., 2009; Sidiropoulou and Poirazi, 2012; Wang et al., 2013). The role of LTP, the cellular correlate of long-term memory, in PFC function is not well understood. However, protein synthesis is required for proper performance in working memory tasks (Touzani et al., 2007). Moreover, although persistent activity is recorded in monkeys before training in working memory tasks (Meyer et al., 2007), the properties of persistent activity change after training (Qi and Constantinidis, 2013). Furthermore, deficits in synaptic plasticity are observed in animal models of psychiatric disorders with deficits in PFC function (Goto et al., 2009). Therefore, it seems that longer-term plasticity mechanisms are likely to be involved in shaping and/or modulating short-term memory processes, such as working memory. LTP in the PFC has been studied either in the connections between layer II and layer V or in the ventral hippocampal input to layer V PFC neurons (Goto et al., 2009). In addition, spike timing-dependent plasticity has also been observed in layer V of the PFC (Couey

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et al., 2007). Several neurotransmitter systems are involved in prefrontal cortical LTP, such as the NMDA receptor, dopamine and metabotropic glutamate receptors (Otani et al., 1999, 2003; Matsuda, 2006; Kolomiets et al., 2009). Our study has identified that layer II synapses exhibit LTP, which is NMDA dependent. Furthermore, our results demonstrate that the degree of potentiation is correlated with NMDA receptor expression and mushroom-type spine density in PFC neurons. Similar mechanisms for LTP have been found in the hippocampus and other cortical regions.

Effect of inhibition on LTP induction and maintenance It is well known that GABA regulates the induction and maintenance of LTP. GABA blockade produces synaptic potentiation in vivo (Matsuyama et al., 2008), or enhances the EPSP-spike coupling in vitro (Staff and Spruston, 2003). Decreased GABAergic transmission usually results in increased network excitability and could have a ceiling or saturating effect for LTP, decreasing the degree of potentiation of synaptic responses. Furthermore, changes in GABAergic transmission could alter the rules for LTP induction (Meredith et al., 2003). Pathological conditions in mice that result in long-term decreased GABAergic transmission also affect the emergence of LTP (Jedlicka et al., 2009; Wang et al., 2014). Our data also demonstrate that a chronic pathological condition, such as the significant developmental decrease in interneuron numbers, results in impaired LTP in the PFC. In addition, our study reveals that the decreased inhibition leads to network adaptations, such as reduced dendritic length and spine density and decreased NMDA receptor subunit expression. Therefore, it is possible that chronic alterations in GABAergic transmission trigger network-wide adaptations that eventually change synaptic plasticity.

Recovery of structural plasticity after GABAergic enhancement Throughout the organism’s development, critical periods have been identified during which environmental or pharmacological manipulations alter cortical function and behavior. For example, treatment with GABAergic agonists/antagonists shifts the critical period duration for ocular dominance column development in the primary visual cortex (Hensch, 2004, 2005). Diazepam treatment during the early postnatal period results in anxiety defects in adulthood (Shen et al., 2012). In our study, we did not find any effect of diazepam treatment in adult heterozygous mice, suggesting that dendritic morphology is not significantly affected by changes in GABAergic signaling during the early postnatal period in mice without any defects. However, diazepam treatment during the early postnatal period did reverse the dendritic morphology defect in Rac1 cKO mice, which display significantly reduced GABAergic interneuron numbers. Recently, the idea that a critical period exists for the development of neuropsychiatric disorders has surfaced. This critical per-

11

iod is very likely to be modulated by changes in GABAergic function (Meredith, 2014). Our data show that treating Rac1 cKO mice with diazepam during the early postnatal life (11–20 days of age) does not cause a significant decrease in dendritic spine density in PFC neurons compared to diazepamtreated heterozygous mice, and at the same time, increases the dendritic spine density compared to untreated Rac1 cKO mice (Fig. 6). Although no direct comparison could be made between saline-treated and diazepam-treated Rac1 cKO mice (because no Rac1 cKO mice survived to adulthood in the saline-treated dams), our data show that the diazepam treatment does not allow the structural plasticity deficit to occur. This reversal effect reinforces the idea of such a critical period during the early postnatal life. The 10-day i.p. treatment could be regarded as a mild stressor. Social and repeated restrain stress have been shown to affect dendritic morphology (Chocyk et al., 2013; Yang et al., 2013); however, in our study, the dendritic spine density was not altered between untreated and saline-treated heterozygous mice (Table 3). Future experiments could further investigate the effects of diazepam treatment on various animal models of diseases with defects in the GABAergic system, in order to determine whether such a treatment could alleviate some of the pathological symptoms.

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CONCLUSIONS

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In conclusion, our data contribute to the understanding of PFC synaptic physiology and demonstrate that the capacity for potentiation in the PFC glutamatergic system is reduced when the number of interneurons populating the PFC is significantly decreased. Thus, our data suggest that interneuron defects can result in glutamatergic synaptic deficits, which could be relevant for several neuropsychiatric disorders with interneuron dysfunction.

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Acknowledgments—We would like to thank Katerina Kalemaki and Zouzana Kounoupa for their assistance with genotyping of mice, Juan Varela and Donald Cooper for guidance with setting up the electrophysiological recordings, Nikolitsa Stathakopoulou and Kalliopi Lambraki for help with Golgi–Cox analysis, Lydia Pavlidi for help with Western blot experiments and analysis. This study was supported by the Empirikion Foundation and NARSAD Young Investigator award (KS), a Marie Curie Fellowship (MC PERG7-GA-2010-268292) and Postdoctoral-Fellowship LS7629 (ST and DK).

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(Accepted 22 February 2016) (Available online xxxx)

Please cite this article in press as: Konstantoudaki X et al. Impaired synaptic plasticity in the prefrontal cortex of mice with developmentally decreased number of interneurons. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.02.048

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