Neuropeptides 62 (2017) 27–35

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Neuropeptides

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Neuropeptide Y neuronal network dysfunction in the frontal lobe of agenetic mouse model of schizophrenia

Shunsuke Morosawa a,⁎, Shuji Iritani a, Hiroshige Fujishiro a, Hirotaka Sekiguchi a, Youta Torii a,Chikako Habuchi a, Keisuke Kuroda b, Kozo Kaibuchi b, Norio Ozaki a

a Department of Psychiatry, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japanb Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan

⁎ Corresponding author.E-mail addresses: smorosawa1@gmail.com (S.Morosaw

(S. Iritani), fujishiro1975@gmail.com (H. Fujishiro), S-guc(H. Sekiguchi), youtat@med.nagoya-u.ac.jp (Y. Torii), hab(C. Habuchi), k-kuroda@med.nagoya-u.ac.jp (K. Kuroda),(K. Kaibuchi), ozaki-n@med.nagoya-u.ac.jp (N. Ozaki).

http://dx.doi.org/10.1016/j.npep.2016.12.0100143-4179/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 August 2016Received in revised form 18 November 2016Accepted 22 December 2016Available online 5 January 2017

Neuropeptide Y (NPY) has been found to play a critical role in variousmental functions as a neurotransmitter andis involved in the development of schizophrenia, a particularly intractable psychiatric disease whose precise eti-ology remains unknown. Recent molecular biological investigations have identified several candidate geneswhich may be associated with this disease, including disrupted-in-schizophrenia 1 (DISC1). The role of DISC1would involve neurogenesis and neuronal migration. However, the functional consequences of this gene defecthave not yet been fully clarified in neuronal systems. In the present study, to clarify the neuropathological chang-es associated with the function of DISC1, we explored how DISC1 dysfunction can induce abnormalities in theNPY neuronal network in the central nervous system.We performed immunohistochemical analyses (includingthe observation of the distribution and density) of prefrontal cortex specimens from DISC1-knockout (KO)mice,which are considered to be a novel animal model of schizophrenia. We then evaluated the number and size ofNPY-immunoreactive (NPY-IR) neurons and the length of NPY-IR fibers. The number of NPY-IR neurons andthe length of the fibers were decreased in the prefrontal cortex of DISC1-KOmice. The decrease was particularlyprominent in the superficial regions, and the distribution of NPY-IR neurons differed between wild-type andDISC1-KOmice. However, the size of the neurons in the cortices of the DISC1-KO and wild-type mice did not dif-fer markedly. Our findings suggest that dysfunction of DISC1may lead to the alteration of NPY neurons and neu-rotransmission issues in NPY-containing neuron systems, which seem to play important roles in both thementalfunction and neuronal development. DISC1 dysfunction may be involved in the pathogenesis of schizophreniathrough the impairment of the NPY neuronal network.

© 2017 Elsevier Ltd. All rights reserved.

Keywords:Animal modelImmunohistochemistryPrefrontal cortexSchizophreniaDISC1 (disrupted-in-schizophrenia 1)

1. Introduction

Schizophrenia is a common and complex psychiatric disease that oc-curs in approximately 1% of the population, and elucidating its etiologyhas proven to be quite difficult. It has been widely thought to be aneurodevelopment disorder. Various etiologies, including genetic andepigenetic factors, seem to be inextricably linked to or to contribute toits pathophysiology (Harrison, 2007; Harrison and Weinberger, 2005).

Recent molecular biological investigations have identified severalputative candidate genes that may be responsible for schizophrenia(Harrison, 2007; Iritani, 2007). One of the major candidate genes is

a), iritani@med.nagoya-u.ac.jphi@amber.plala.or.jpuchi@hm9.aitai.ne.jpkaibuchi@med.nagoya-u.ac.jp

disrupted-in-schizophrenia 1 (DISC1), which was first described as astrong candidate gene in a large Scottish family in which balanced chro-mosomal translocation segregateswith schizophrenia and other psychi-atric disorders (Blackwood et al., 2001; Brandon et al., 2009). DISC1appears to involved with neurogenesis and neuronal migration inneurodevelopment (Brandon et al., 2009); it has been suggested thatthe loss of the DISC1 functionmay underlie the neurodevelopment dys-function observed in patients with schizophrenia (Kamiya et al., 2005).

Several types of mutant mice with an impaired DISC1 function havebeen generated and analyzed to elucidate the role of DISC1 in brain de-velopment and the disease (Li et al., 2007; Shen et al., 2008; Wong andJosselyn, 2016). Thesemousemodels show deficits in neurogenesis andbrain development (Clapcote et al., 2007; Wong and Josselyn, 2016).They display variable phenotypes, including sensorimotor gating distur-bance, prepulse inhibition and working memory, which are relevant toschizophrenia (Cash-Padgett and Jaaro-Peled, 2013). The investigationof alterations in the neuronal development of DISC1 mutant mice maybe useful for understanding the changes that were observed to occur

Fig. 1. The areas examined in the mouse prefrontal cortex. This figure shows general viewof a coronal section of the anterior mouse brain. The three boxes indicate the ROIs, whichwere placed on the ACC, m-PFC and OFC.

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in schizophrenia patients in previous studies (Hikida et al., 2007; Holleyet al., 2013).

Various types of neurotransmitters andmodulators within the braintissue are presumed to be associated with a range of mental functions(Malavolta and Cabral, 2011; van den Pol, 2012). Therefore, dysfunc-tions in these elements are expected to be involved in the etiology ofpsychiatric disease; however, few studies have so far investigated therelationship between these elements and the etiology of psychiatric dis-ease, especially schizophrenia. Neuropeptide Y (NPY) is one of theseneurotransmitters. NPY is a 36-amino acid peptide that is widelyexpressed in the central and peripheral nervous systems of humansand many animals (Holzer et al., 2012; Lin et al., 2004), and exhibits ahigh degree of phylogenetic conservation across species (Gilpin, 2012;Sajdyk et al., 2004). NPY is expressed mainly in ɤ-aminobutyric acid(GABA) interneurons and acts as a neurotransmitter or neuromodulatorin the inhibitory synaptic function (Baraban and Tallent, 2004; van denPol, 2012) and neuronal development (Benarroch, 2009). As NPY affectsthe regulation of many physiological functions, including the stimula-tion of food intake and cognitive functions (Qi et al., 2016; Reichmannand Holzer, 2016; White, 1993), studies have investigated the relation-ship between NPY dysfunction and the pathogenesis of major psychiat-ric disorders (Karl et al., 2010; Kormos and Gaszner, 2013; Obuchowiczet al., 2004; Tasan et al., 2016). Reduced NPY peptide or precursormRNA levels (Gabriel et al., 1996; Kuromitsu et al., 2001; LaCrosse andOlive, 2013) and an altered distribution of NPY-containing neurons(Ikeda et al., 2004) have been observed in the frontal cortices of schizo-phrenic patients. As NPY is expressed in GABAergic interneurons, defi-cient cortical NPY expression is congruent with the proposedGABAergic pathology of schizophrenia spectrum disorder (Hashimotoet al., 2008; Lewis et al., 2005). Thus, the NPY disturbance is consideredto be involved in the etiology of schizophrenia and the aberrant inhibi-tory synaptic function in the prefrontal cortex (LaCrosse and Olive,2013; Stalberg et al., 2014). However, details regarding the involvementof NPY in the etiology of schizophrenia remain unclear.

The altered expression of the NPY systems in the brain tissue causedby DISC1 deficiency might help clarify how DISC1 deficiency influencesneuronal development, which induces the pathological changes in thebrain tissue that contribute to the pathogenesis of schizophrenia.

In the present study,we usedDISC1-knockout (KO)mice, which lackexons 2 and 3 of theDISC1 gene (Kuroda et al., 2011). Thesemice exhib-it mild anxiety and severe impulsivity and altered synaptic plasticity.These behavioral abnormalities are consistent with the clinical featuresof patients with schizophrenia (Kuroda et al., 2011). Recent studies ofthe brains of these mice have shown the presence of alterations in thecatecholamine system (Iritani et al., 2016) and GABA-ergic system,such as in the levels of parvalbumin and calbindin (Umeda et al.,2016). In this study, we performed immunohistochemical analyses, in-cluding observation of the distribution and density of NPY at the pre-frontal cortices of these DISC1-KO mice, using rabbit anti-NPYdelipidized whole antiserum to clarify the NPY expression in thebrain.We also determinedwhether or not the expression of somatostat-in (SOM), a neuropeptide partly co-localized with NPY in interneurons(Sakai et al., 1995),was altered inDISC1 KOmice. Hypofrontality has be-come one of themost widely cited and influential findings in the litera-ture on schizophrenia (Hill et al., 2004) and frontal lobe impairmentsare important targets for the treatment of the disease (McAllister etal., 2015). In the present study,we focused on the anterior cingulate cor-tex (ACC) and the medial prefrontal cortex (m-PFC) and orbitofrontalcortex (OFC). The ACC is related with the self-reflection processing im-pairment observed in patientswith schizophrenia (Tan et al., 2015). Thedysfunction of the dorsolateral prefrontal cortex is associated with theimpairments in working memory that are observed in patients withschizophrenia (Goldman-Rakic, 1994; Hashimoto et al., 2008). Theseareas in humans are considered to correspond to the rodent m-PFC(Gass and Chandler, 2013; Seamans et al., 2008). Neuroimaging studieshave revealed that the OFC plays an important role in the development

of severe negative symptoms in schizophrenic patients (Kanahara et al.,2013).

2. Materials and methods

2.1. Subjects (animal model)

Six 12-week-old DISC1-KO (−/−) mice (male: 4, female: 4) and thesame number of age and sex-matched Disc1 (+/+) littermate (wild-type)micewere used. The number of animals was set in order to ensurean appropriate range of power (0.7–0.9) (Mann et al., 1991). The micewere bred under the same conditions. The background of this animalmodel has been described in a previous study (Kuroda et al., 2011).

2.2. Preparation

The animals were placed under deep anesthesia and then perfusedwith a tissuefixative solution (4% paraformaldehyde in 0.1Mphosphatebuffer, pH 7.4). Their brains were immediately removed, and tissueblocks were immersed in a 20% sucrose-0.05 M phosphate buffer solu-tion for N3 days at 4 °C. The sections (40 μm) were cut using a freezingcryostat and treated as free-floating sections.

2.3. Immunohistochemistry

The sections were rinsed twice in 0.1 M Tris-Cl buffered saline (TBS;pH 7.4, 0.9%NaCl) containing 0.3% TritonX-100 (TX) and2%normal goatserum (NGS) for 15 min at room temperature. The sections were incu-batedwith the primary antibodies for 48 h at 4 °C. The primary antibodyused in this study was rabbit anti-NPY delipidized whole antiserum(product number: N9528, lot: 096 K4761; 1:5000; Sigma, Saint Louis,USA) and rabbit anti-SOM lyophilized whole serum (catalog number:20067 lot number: 216002; 1:1600; Immunostar, Hudson, Wisconsin,USA). The sectionswere then rinsed in NGS-TX TBS twice and incubatedin medium containing biotinylated anti-universal (rat and/or rabbit)IgG (Vecstain; 1:100) for 30 min at room temperature. After rinsingthe sections in NGS-TX TBS twice again and TBS solution once, the sec-tions were incubated with an avidin-biotin peroxidase complex (ABCmethod) for 30 min. Finally, the sections were rinsed in TBS solutiontwice and reacted with 0.05% 3,3diaminobenzine-HCl in 0.05 M Tris-HCl buffer (pH 7.6) for 2 or 3 min and mounted on slides.

Fig. 3. The numbers of NPY-IR neurons. These graphs show the number of NPY-IR neurons in each segment of the ROIs. Therewas a significant difference in the number of NPY-IR neuronsin total and in the upper segment in the wild-type and DISC1-KO mice (⁎P b 0.05).

Fig. 2. Photomicrographs of NPY-IR neurons in the anterior cingulate cortex. (a) Lowmagnification photomicrographs of NPY-IR neurons captured in the anterior cingulate cortex of wild-type (left) andDISC1-KOmice (right). The regionwas subdivided into three equal segments (the upper,middle, and lower segments). (b)Highmagnification photomicrographs of NPY-IRneurons captured in anterior cingulate cortex of wild-type mice (top) and DISC1-KO mice (bottom).

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Table 1The size of the NPY-IR neurons.

The area of NPY-IR neurons (μm2)

Wild-type mice DISC1-KO mice

Median (IQR) Median (IQR)

ACC Upper segment 130 (123−131) 121 (108–128)Middle segment 125 (107–133) 127 (104–136)Lower segment 130 (123–139) 130 (114–144)Full ROI 127 (118–140) 127 (111–134)

m-PFC Upper segment 127 (120–148) 138 (129–164)Middle segment 135 (131–156) 136 (120–162)Lower segment 139 (116–152) 123 (111–146)Full ROI 137 (134–144) 140 (128–156)

OFC Upper segment 127 (119–160) 128 (114–149)Middle segment 132 (117–137) 122 (112–134)Lower segment 139 (134–148) 128 (115–149)Full ROI 135 (128–144) 129 (117–145)

This table shows the average area of the NPY-IR neurons in each of segment in the frontalcortices. Therewas no significant difference in the area of the NPY-IR neurons in thewild-type and KO mice.

Fig. 4. Photomicrographs of NPY-IR neurons in the anterior cingulate cortex. High-magnification photomicrographs of NPY-IR neurons captured in the anterior cingulate cortex of wild-type (top) and DISC1-KO mice (bottom).

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2.4. Observation and analysis

Weobserved the specimens using a lightmicroscope (BX53F; Olym-pus, Tokyo, Japan). For the PC-based image analysis of the specimens, adigital image of the specimenswas captured using a microscopic digitalcamera (Digital DP72; Olympus, Shinjuku, Tokyo, Japan). We used TheImage J 1.41 software package (free software provided by NIH, ImageJ, 2016) tomeasure the following described data. Amorphometric anal-ysis was performed; the analyst was blinded to the genetic information.

We observed and photographed the specimens in three regions ofinterest (ROIs) while referencing a mouse brain atlas (Bregma:1.7 mm by the Mouse Brain Atlas, 2016). The ROI was defined as a rect-angle of 600 μm in width that was vertical to the horizontal surface ofthe cortex in the coronal section of the ACC, m-PFC and OFC (Fig. 1).Each ROI was equally subdivided into three segments (upper, middleand lower segments) as estimates of the alteration in the distribution(Fig. 2). One assumed role of DISC1 is its involvement in the migrationof neurons. A disturbance in the migration may result in the disorgani-zation of the six-layer cortical structure. In this study, we considered itbest to perform our estimations independent of the six-layer corticalstructure. In the captured image, a neuronwith a clearly recognized nu-cleus was regarded as a NPY-immunoreactive (NPY-IR) neuron. There-fore, we counted the number of NPY-IR neurons in the three ROIsmanually. We also estimated the size of the NPY-IR neurons bymeasur-ing the area of each soma manually while referencing detailed micro-scopic observations. We also observed and counted the SOM-IRneurons in the ROIs. There was no significant difference in the heightof the gray matter of the prefrontal cortex among those specimens.

We alsomeasured the length of NPY-IR fibers. In a singlemicroscop-ic field (210 μmwide × 150 μmhigh) on the center of the upper, middleand lower segments, the length of eachNPY-IRfiberwasmanuallymea-suredwhile referencingdetailedmicroscopic observations. Subsequent-ly, the length of each NPY-IR fiber was summed up and we calculatedthe total length in the field of vision.

The groups were not large enough to decide normal distribution.Thus, the number and size of the NPY-IR cells and the length of theNPY-IR fibers were compared in wild-type mice and the KO miceusing the Mann-Whitney U test. P values of b0.05 were considered toindicate statistical significance.

3. Results

3.1. NPY-IR neurons

NPY-IR neurons were observed in both types of mice (Fig. 2). Therewas a significant decrease in the number NPY-IR neurons in all threeprefrontal cortices of the DISC1-KO mice in comparison to the wild-type mice (Fig. 3). The distribution of NPY-IR neurons was different,and there was a significant decrease in the number of NPY-IR neuronsin the upper segment of all three prefrontal cortices of the DISC1-KOmice in comparison to the wild-type mice. In contrast, the numbers ofNPY-IR neurons in the middle and lower segments did not differ to astatistically significant extent. There were no clear gender differencesin these results (data not shown).We noted no clearmorphological dif-ferences between thewild-type and KOmice (Fig. 4) and the size of theNPY-IR neurons did not differ in any of the segments of the wild-typeand DISC1-KO mice (Table 1).

Fig. 5. Photomicrographs of the NPY-IR fibers in the upper segment of themedial prefrontal cortex. Photomicrographs of the NPY-IR fibers in specimens obtained from the upper segmentof the medial prefrontal cortex in wild-type (left) and DISC1-KO mice (right).

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3.2. NPY-IR fibers

The NPY-IR fibers of the DISC1-KO mice appeared sparser and werenot well developed in comparison to those of wild-typemice, especiallyin the upper segment (Fig. 5). The sum of the length of the NPY-IR fibersin each segment of the DISC1-KOmice was significantly shorter than inthe wild-type mice, and similarly to the number of NPY-IR neurons, thelength of the NPY-IR fibers in the upper segment of the DISC1-KO wassignificantly shorter than in the wild-type mice, although the length ofthe NPY-IR fibers in the middle and lower segments did not differ

Fig. 6. The length of the NPY-IRfibers. These graphs show the sumof the length of theNPY-IRfiband in the upper segment of the wild-type and the DISC1-KO mice (⁎P b 0.05).

markedly between the models (Fig. 6). There were no clear gender dif-ferences in these results either (data not shown).

3.3. SOM-IR neurons

SOM-IR neurons were observed in both types of mice (Fig. 7). Thenumber of SOM-IR neurons in the total layers was significantly lowerin all three prefrontal cortices of the DISC1-KO mice than in those ofthe wild-type mice (Fig. 8). In contrast to the NYP-IR neuron findings,however, there was no significant difference in the number of SOM-IR

ers. Therewas a statistically significant difference in the length of theNPY-IR fibers in total

Fig. 7. Photomicrographs of SOM-IR neurons in the anterior cingulate cortex. (a) Low-magnification photomicrographs of SOM-IR neurons captured in the anterior cingulate cortex ofwild-type (left) and DISC1-KO mice (right). (b) High-magnification photomicrographs of SOM-IR neurons captured in the anterior cingulate cortex of wild-type mice (top) and DISC1-KO mice (bottom).

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neurons in the upper segment of any of the three prefrontal cortices ofthe DISC1-KOmice versus the wild-typemice. There were no clear gen-der differences in these results (data not shown).

4. Discussion

In the present study, the number of NPY- and SOM-IR neurons andthe length of the NPY-IR fibers in the prefrontal cortex of DISC1-KOmice differed significantly from the respective findings in wild-typemice. These differences were particularly prominent in the upper seg-ments. However, brain sections from DISC1-KOmice showed no signif-icant changes in the area of theNPY-IR neurons compared to those fromwild-typemice. There were also no clear gender differences in these re-sults. These decreases were found in all three of the regions that we ob-served. We confirmed that the power nearly exceeded 0.7 by post hoccalculations of the statistical power.

4.1. Abnormalities of the NPY system in the prefrontal cortex

We found decreased NPY-IR neurons in the prefrontal cortex ofDISC1-KO mice by the immunohistochemical investigation though anexamination of the brain structure using Nissl staining revealed a nearlynormal cytoarchitecture of the neocortex in the same DISC1-KO mousemodel that was used in a previous study (Kuroda et al., 2011). This find-ing suggested that the NPY system function may be dysregulated inDISC1-KO mice compared with wild-type mice. Some studies have re-ported that NPY peptide or precursor mRNA levels are reduced in thefrontal cortices of schizophrenic patients, suggesting a dysregulatedNPY system function in schizophrenia (LaCrosse and Olive, 2013). Ourfinding is in line with these previous results.

4.2. Impairment of NPY neurons by DISC1 KO

In this study, the decrease in the number of NPY-IR neuronswas par-ticularly prominent in the upper segment of the prefrontal cortex of

DISC1-KO mice, and the distribution of NPY-IR neurons differed be-tweenwild-type andDISC1-KOmice. These alterationswere not accom-panied by a reduction in cell size, which may suggest that DISC1dysfunction affects NPY neuronal network formation ormigration, rath-er thanmaturation. These suggestions seem tomatch previous findings.DISC1 has been thought to play a critical role in cortical developmentand neuronal migration (Singh et al., 2010). In addition, recent studieshave suggested that DISC1 is necessary for the proper migration of cor-tical interneurons (Steinecke et al., 2014; Steinecke et al., 2012), andsome studies have reported that the distribution of interneurons is al-tered and that interneuron migration is impaired in the prefrontal cor-tices of DISC1 mutant mice (Lee et al., 2013). Given these previousfindings, this decrease in the number of NPY-IR neurons might havebeen affected by dysfunction in the propermigration of NPY-containingneurons, although we cannot confirm this, as we did not observe thetemporal change in this study and cannot determine if therewas indeeda migration deficit. In addition to the previous reports that noted de-creased numbers of parvalbumin- and calbindin-IR neurons in the fron-tal cortex of model mice, as in this study (Umeda et al., 2016), ourfinding of decreased numbers of SOM-IR neurons supports the assump-tion that DISC1 dysfunction affects inhibitory neuronal network forma-tion broadly during development, rather than solely acting on asubpopulation of interneurons.

4.3. NPY deficiency and the mental function

NPY acts as a modulator of synaptic transmission and is involved inmental function (Benarroch, 2009). NPY-containing neurons havebeen found to construct synapses to the dendrites of pyramidal neurons(Hashimoto et al., 2010) and to modulate the release of GABA and glu-tamate (Malva et al., 2012; van den Pol, 2012). NPY seems to contributeto neuroprotection and the learning and memory functions that modu-late glutamatergic activity (Gotzsche and Woldbye, 2016; Malva et al.,2012; Smialowska et al., 2009). Aberrant of GABAergic activity is in-volved in various cognitive functions (Hashimoto et al., 2008; Rao et

Fig. 8. The numbers of SOM-IR neurons. These graphs show the number of SOM-IR neurons in each segment of the ROIs. There was a significant difference in the total number of SOM-IRneurons in the wild-type and DISC1-KO mice (⁎P ≤ 0.05).

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al., 2000) and NPY also seems to be involved in this aberrant activity(Baraban and Tallent, 2004). Thus NPY seems to affect various mentalfunctions that interact with GABAergic and glutamatergic neurotrans-mission. Therefore, NPY deficiency could be involved in neuronal andbehavioral abnormalities, including the synaptic plasticity and cognitivedysfunction observed in the DISC1-KOmice. A previous study using thesame DISC1-KO mouse model reported the dysfunction of theGABAergic neuronal system in the prefrontal cortex of DISC1-KO mice(Umeda et al., 2016), and our results correspond well with those ofthat study. Our study may therefore support the hypothesized dysfunc-tion of GABAergic and glutamatergic neurotransmission in schizophren-ic patients.

4.4. NPY and neurodevelopment

NPY has been thought to be involved in neurogenesis (Benarroch,2009; Malva et al., 2012). Recent study have reported that NPY modu-late neurotrophins and to be modulated by them, which influence syn-aptic plasticity and/or construction (Angelucci et al., 2014). Given theseprevious findings, NPY dysfunction through a DISC1 deficit might be animportant factor in neurodevelopmental disorders and in the etiology ofschizophrenia, though further investigation is needed as we did not in-vestigate neurotrophins in this study.

4.5. Gender differences

A previous study found that female mice exhibitedmore severe def-icits than males, and sex hormones were therefore thought to be

potentially involved in these results (Kuroda et al., 2011). However,we found no statistically significant gender differences in this study.These results might be due to the small sample size in our study, aswe observed only four male and four female mice. As such, further in-vestigation is needed.

4.6. Limitations

It will be necessary to demonstrate the role of DISC1, not only in thebrain of the animal model but also in the autopsied brains of patientswith schizophrenia to conclusively determine the pathophysiology ofthe illness.

5. Conclusions

Our study suggests that the dysfunction of DISC1 may lead to a de-crease in the numbers of NPY neurons in the prefrontal cortex of mice,especially in the superficial regions, and thereby result in deficient neu-rotransmission of the NPY-containing neuron system, which seems toplay an important role in both the mental function and neuronal devel-opment. Our findings suggest that NPY neuronal dysfunctionmay be in-duced through DISC1 deficiency. In the human brain, decreased NPYpeptide and altered NPY distribution in the frontal cortices of schizo-phrenic patients have been reported, and the dysregulated NPY systemfunction in schizophrenia has also been reported (Ikeda et al., 2004;LaCrosse and Olive, 2013). Our findings might support these previousresults, as DISC1 dysfunction may be involved in the pathogenesis of

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schizophrenia through the impairment of NPY neuronal networkdevelopment.

List of abbreviations

ACC anterior cingulate cortexDISC1 disrupted-in schizophrenia 1GABA γ-aminobutyric acidIR immunoreactiveKO knockoutm-PFC medial prefrontal cortexNPY neuropeptide YOFC orbitofrontal cortexROI regions of interestSOM somatostatin

Statement on ethics approval

All animal protocols were approved by the animal care and usecommittee of Nagoya University Graduate School of Medicine. Inaddition, the Principles for the Care and Use of Laboratory Animals,which were approved by the Japanese Pharmacological Society, andthe National Institutes of Health's (NIH) Guide for the Care and Useof Laboratory Animals were followed. All efforts were made tominimize the suffering of the animals used in this study and toreduce their number.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflicts of interest in association with thepresent study.

Funding

This study was supported by the Japanese Ministry of Education,Culture, Sports, Science, and Technology KAKENHI (Grant Numbers23591701 [2011−2013] and 26461742 [2014–2016]). This re-search is partially supported by the “Integrated research onneuropsychiatric disorders” carried out under the SRPBS fromAMED. The funder had no role in the study design, the data collec-tion or the analysis, the decision to publish, or the preparation ofthe manuscript.

Authors' contributions

S.M. drafted the manuscript, analyzed the data, and managed theexperimental procedures. S.I. developed the study concept anddesign. H.F. and H.S. prepared the brain tissue, analyzed the data,and performed the experiments. Y.T. and C.H. performed the experi-ments and developed the software-based technique used to analyzethe photomicrographs. K.K. and K.K. developed the animal model.N.O. coordinated the study and interpreted the results. All theauthors discussed the results and contributed to write the finalversion of the manuscript. All the authors approved the final versionof the manuscript.

Acknowledgments

The authors thankMs.MasamiMiyata for providing valuable techni-cal assistance.

References

Angelucci, F., Gelfo, F., Fiore, M., Croce, N., Mathe, A.A., Bernardini, S., et al., 2014. The effectof neuropeptide Y on cell survival and neurotrophin expression in in-vitro models ofAlzheimer's disease. Can. J. Physiol. Pharmacol. 92, 621–630.

Baraban, S.C., Tallent, M.K., 2004. Interneuron diversity series: interneuronalneuropeptides – endogenous regulators of neuronal excitability. TrendsNeurosci. 27, 135–142.

Benarroch, E.E., 2009. Neuropeptide Y: its multiple effects in the CNS and potential clinicalsignificance. Neurology 72, 1016–1020.

Blackwood, D.H., Fordyce, A., Walker, M.T., St Clair, D.M., Porteous, D.J., Muir, W.J., 2001.Schizophrenia and affective disorders – cosegregation with a translocation at chro-mosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 find-ings in a family. Am. J. Hum. Genet. 69, 428–433.

Brandon, N.J., Millar, J.K., Korth, C., Sive, H., Singh, K.K., Sawa, A., 2009. Understanding therole of DISC1 in psychiatric disease and during normal development. J. Neurosci. 29,12768–12775.

Cash-Padgett, T., Jaaro-Peled, H., Sep 3 2013. DISC1 mouse models as a tool to deciphergene-environment interactions in psychiatric disorders. Front. Behav. Neurosci. 7,113.

Clapcote, S.J., Lipina, T.V., Millar, J.K., Mackie, S., Christie, S., Ogawa, F., et al., 2007. Behav-ioral phenotypes of Disc1 missense mutations in mice. Neuron 54, 387–402.

Gabriel, S.M., Davidson, M., Haroutunian, V., Powchik, P., Bierer, L.M., Purohit, D.P., et al.,1996. Neuropeptide deficits in schizophrenia vs. Alzheimer's disease cerebral cortex.Biol. Psychiatry 39, 82–91.

Gass, J.T., Chandler, L.J., 2013. The plasticity of extinction: contribution of the prefrontalcortex in treating addiction through inhibitory learning. Front. Psych. 4, 46.

Gilpin, N.W., 2012. Neuropeptide Y (NPY) in the extended amygdala is recruited duringthe transition to alcohol dependence. Neuropeptides 46, 253–259.

Goldman-Rakic, P.S., 1994. Working memory dysfunction in schizophrenia.J. Neuropsychiatry Clin. Neurosci. 6, 348–357.

Gotzsche, C.R., Woldbye, D.P., 2016. The role of NPY in learning and memory. Neuropep-tides 55, 79–89.

Harrison, P.J., 2007. Schizophrenia susceptibility genes and neurodevelopment. Biol. Psy-chiatry 61, 1119–1120.

Harrison, P.J., Weinberger, D.R., 2005. Schizophrenia genes, gene expression, and neuro-pathology: on thematter of their convergence. Mol. Psychiatry 10, 40–68 (image 45).

Hashimoto, T., Arion, D., Unger, T., Maldonado-Aviles, J.G., Morris, H.M., Volk, D.W., et al.,2008. Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortexof subjects with schizophrenia. Mol. Psychiatry 13, 147–161.

Hashimoto, T., Matsubara, T., Lewis, D.A., 2010. Schizophrenia and cortical GABA neuro-transmission. Psychiatr. Neurol. Jpn. (Seishin Shinkeigaky Zasshi) 112, 439–452.

Hikida, T., Jaaro-Peled, H., Seshadri, S., Oishi, K., Hookway, C., Kong, S., et al., 2007. Domi-nant-negative DISC1 transgenic mice display schizophrenia-associated phenotypesdetected by measures translatable to humans. Proc. Natl. Acad. Sci. U. S. A. 104,14501–14506.

Hill, K., Mann, L., Laws, K.R., Stephenson, C.M., Nimmo-Smith, I., McKenna, P.J., 2004.Hypofrontality in schizophrenia: a meta-analysis of functional imaging studies. ActaPsychiatr. Scand. 110, 243–256.

Holley, S.M., Wang, E.A., Cepeda, C., Jentsch, J.D., Ross, C.A., Pletnikov, M.V., et al., 2013.Frontal cortical synaptic communication is abnormal in Disc1 genetic mouse modelsof schizophrenia. Schizophr. Res. 146, 264–272.

Holzer, P., Reichmann, F., Farzi, A., 2012. Neuropeptide Y, peptide YY and pancreatic poly-peptide in the gut-brain axis. Neuropeptides 46, 261–274.

Ikeda, K., Ikeda, K., Iritani, S., Ueno, H., Niizato, K., 2004. Distribution of neuropeptide Y in-terneurons in the dorsal prefrontal cortex of schizophrenia. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 28, 379–383.

Image J, 2016. http://rsb.info.nih.gov/ij/ Accessed 17 August 2016.Iritani, S., 2007. Neuropathology of schizophrenia: a mini review. Neuropathology 27,

604–608.Iritani, S., Sekiguchi, H., Habuchi, C., Torii, Y., Kuroda, K., Kaibuchi, K., et al., 2016. Catechol-

aminergic neuronal network dysfunction in the frontal lobe of a geneticmousemodelof schizophrenia. Acta Neuropsychiatr. 28, 117–123.

Kamiya, A., Kubo, K., Tomoda, T., Takaki, M., Youn, R., Ozeki, Y., et al., 2005. A schizophre-nia-associated mutation of DISC1 perturbs cerebral cortex development. Nat. CellBiol. 7, 1167–1178.

Kanahara, N., Sekine, Y., Haraguchi, T., Uchida, Y., Hashimoto, K., Shimizu, E., et al., 2013.Orbitofrontal cortex abnormality and deficit schizophrenia. Schizophr. Res. 143,246–252.

Karl, T., Chesworth, R., Duffy, L., Herzog, H., 2010. Schizophrenia-relevant behaviours in agenetic mouse model for Y2 deficiency. Behav. Brain Res. 207, 434–440.

Kormos, V., Gaszner, B., 2013. Role of neuropeptides in anxiety, stress, and depression:from animals to humans. Neuropeptides 47, 401–419.

Kuroda, K., Yamada, S., Tanaka, M., Iizuka, M., Yano, H., Mori, D., et al., 2011. Behavioralalterations associated with targeted disruption of exons 2 and 3 of the Disc1 genein the mouse. Hum. Mol. Genet. 20, 4666–4683.

Kuromitsu, J., Yokoi, A., Kawai, T., Nagasu, T., Aizawa, T., Haga, S., et al., 2001. Reducedneuropeptide Y mRNA levels in the frontal cortex of people with schizophrenia andbipolar disorder. Brain Res. Gene Expr. Patterns 1, 17–21.

LaCrosse, A.L., Olive, M.F., 2013. Neuropeptide systems and schizophrenia. CNS Neurol.Disord. Drug Targets 12, 619–632.

Lee, F.H., Zai, C.C., Cordes, S.P., Roder, J.C., Wong, A.H., 2013. Abnormal interneurondevelopment in disrupted-in-schizophrenia-1 L100P mutant mice. Mol. Brain6, 20.

Lewis, D.A., Hashimoto, T., Volk, D.W., 2005. Cortical inhibitory neurons and schizophre-nia. Nat. Rev. Neurosci. 6, 312–324.

35S. Morosawa et al. / Neuropeptides 62 (2017) 27–35

Li, W., Zhou, Y., Jentsch, J.D., Brown, R.A., Tian, X., Ehninger, D., et al., 2007. Specific devel-opmental disruption of disrupted-in-schizophrenia-1 function results in schizophre-nia-related phenotypes in mice. Proc. Natl. Acad. Sci. U. S. A. 104, 18280–18285.

Lin, S., Boey, D., Herzog, H., 2004. NPY and Y receptors: lessons from transgenic andknockout models. Neuropeptides 38, 189–200.

Malavolta, L., Cabral, F.R., 2011. Peptides: important tools for the treatment of central ner-vous system disorders. Neuropeptides 45, 309–316.

Malva, J.O., Xapelli, S., Baptista, S., Valero, J., Agasse, F., Ferreira, R., et al., 2012. Multifacesof neuropeptide Y in the brain – neuroprotection, neurogenesis and neuroinflamma-tion. Neuropeptides 46, 299–308.

Mann, M.D., Crouse, D.A., Prentice, E.D., 1991. Appropriate animal numbers in biomedicalresearch in light of animal welfare considerations. Lab. Anim. Sci. 41, 6–14.

McAllister, K.A., Mar, A.C., Theobald, D.E., Saksida, L.M., Bussey, T.J., 2015. Comparing theeffects of subchronic phencyclidine and medial prefrontal cortex dysfunction on cog-nitive tests relevant to schizophrenia. Psychopharmacology 232, 3883–3897.

Mouse Brain Atlas, 2016. http://www.mbl.org/atlas165/atlas165_start.html Accessed 17August 2016.

Obuchowicz, E., Krysiak, R., Herman, Z.S., 2004. Does neuropeptide Y (NPY) mediate theeffects of psychotropic drugs? Neurosci. Biobehav. Rev. 28, 595–610.

Qi, Y., Fu, M., Herzog, H., 2016. Y2 receptor signalling in NPY neurons controls bone for-mation and fasting induced feeding but not spontaneous feeding. Neuropeptides55, 91–97.

Rao, S.G., Williams, G.V., Goldman-Rakic, P.S., 2000. Destruction and creation of spatialtuning by disinhibition: GABA(A) blockade of prefrontal cortical neurons engagedby working memory. J. Neurosci. 20, 485–494.

Reichmann, F., Holzer, P., 2016. Neuropeptide Y: a stressful review. Neuropeptides 55,99–109.

Sajdyk, T.J., Shekhar, A., Gehlert, D.R., 2004. Interactions between NPY and CRF in theamygdala to regulate emotionality. Neuropeptides 38, 225–234.

Sakai, K., Maeda, K., Chihara, K., Kaneda, H., 1995. Increases in cortical neuropeptide Y andsomatostatin concentrations following haloperidol-depot treatment in rats. Neuro-peptides 29, 157–161.

Seamans, J.K., Lapish, C.C., Durstewitz, D., 2008. Comparing the prefrontal cortex of ratsand primates: insights from electrophysiology. Neurotox. Res. 14, 249–262.

Shen, S., Lang, B., Nakamoto, C., Zhang, F., Pu, J., Kuan, S.L., et al., 2008. Schizophrenia-re-lated neural and behavioral phenotypes in transgenic mice expressing truncatedDisc1. J. Neurosci. 28, 10893–10904.

Singh, K.K., Ge, X., Mao, Y., Drane, L., Meletis, K., Samuels, B.A., et al., 2010. Dixdc1 is a crit-ical regulator of DISC1 and embryonic cortical development. Neuron 67, 33–48.

Smialowska, M., Domin, H., Zieba, B., Kozniewska, E., Michalik, R., Piotrowski, P., et al.,2009. Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists invitro and in vivo. Neuropeptides 43, 235–249.

Stalberg, G., Ekselius, L., Lindstrom, L.H., Larhammar, D., Boden, R., 2014. Neuropeptide Y,social function and long-term outcome in schizophrenia. Schizophr. Res. 156,223–227.

Steinecke, A., Gampe, C., Valkova, C., Kaether, C., Bolz, J., 2012. Disrupted-in-schizophrenia1 (DISC1) is necessary for the correct migration of cortical interneurons. J. Neurosci.32, 738–745.

Steinecke, A., Gampe, C., Nitzsche, F., Bolz, J., Jul 8 2014. DISC1 knockdown impairs thetangential migration of cortical interneurons by affecting the actin cytoskeleton.Front. Cell. Neurosci. 8:190. http://dx.doi.org/10.3389/fincel.2014.00190.

Tan, S., Zhao, Y., Fan, F., Zou, Y., Jin, Z., Zen, Y., et al., 2015. Brain correlates of self-evalua-tion deficits in schizophrenia: a combined functional and structural MRI study. PLoSOne 10, e0138737.

Tasan, R.O., Verma, D., Wood, J., Lach, G., Hormer, B., de Lima, T.C., et al., 2016. The role ofneuropeptide Y in fear conditioning and extinction. Neuropeptides 55, 111–126.

Umeda, K., Iritani, S., Fujishiro, H., Sekiguchi, H., Torii, Y., Habuchi, C., et al., Jul 16 2016. Im-munohistochemical evaluation of the GABAergic neuronal system in the prefrontalcortex of a DISC1 knockout mouse model of schizophrenia. Synapse. http://dx.doi.org/10.1002/syn.21924.

van den Pol, A.N., 2012. Neuropeptide transmission in brain circuits. Neuron 76, 98–115.White, J.D., 1993. Neuropeptide Y: a central regulator of energy homeostasis. Regul. Pept.

49, 93–107.Wong, A.H., Josselyn, S.A., 2016. Caution when diagnosing your mouse with schizophre-

nia: the use and misuse of model animals for understanding psychiatric disorders.Biol. Psychiatry 79, 32–38.