Adult conditional knockout of PGC-1α in GABAergic neurons causes exaggerated startle reactivity, impaired short-term habituation and hyperactivity

Brain Research Bulletin 157 (2020) 128–139

Contents lists available at ScienceDirect

Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull

Research report

Adult conditional knockout of PGC-1α in GABAergic neurons causes exaggerated startle reactivity, impaired short-term habituation and hyperactivity

T

Jia Wang*,1, Huang-Rong Song1, Mei-Na Guo, Si-Fei Ma, Qi Yun, Wei-Ning Zhang* School of Medicine, Jiangsu University, Zhenjiang, Jiangsu Province 212013, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: PGC-1α Habituation Sensitization Hyperactivity Parvalbumin Interneurons

Interneurons not only contribute to the global balance of activity in cortical networks but also mediate the precise gating of information through specific proteins. Accumulating evidence demonstrates that peroxisomeproliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α) is concentrated in inhibitory interneurons and that it plays an important role in neuropsychiatric diseases. However, the functions of the transcriptional coactivator PGC-1α in sensorimotor gating, short-term habituation and spatial reference memory are still not entirely clear. To investigate the precise involvement of PGC-1α in the progression of psychiatric disorders, we first generated PGC-1α conditional knockout mice through transgenic expression of Cre recombinase under the control of dlx5/6 promoter, Cre-mediated excision events occurred specifically in γ-amino-butyric-acid-(GABA) ergic neurons. Short-term habituation and spatial reference memory in Dlx5/6-Cre::PGC-1αfl/fl mice were evaluated using the novel object recognition test and the Morris water maze test, and sensorimotor gating was measured by prepulse inhibition of the acoustic startle reflex. Protein expression of parvalbumin (PV) in specific brain regions was studied by western blotting, immunofluorescence and immunohistochemistry. Here, we show that mice lacking the PGC-1α gene in GABAergic neurons exhibit deficits in short-term habituation, hyperactivity, reduced prepulse inhibition and exaggerated startle reactivity but normal associative spatial reference memory. In particular, these mice display aberrant salience, whereby more attention is paid to a further copy of the original object (now familiar) (relative to the first presentation of the original object, and relative to the presentation of the novel object). These behavioral dysfunctions were associated with decreased PV expression in the cortex (including somatosensory and motor cortex) as well as in the hippocampus, especially in its CA1 and CA3 regions. Together, these findings draw attention to a hyper-response phenotype of PGC-1α conditional knockout mice and indicate that PGC-1α is a novel regulator of gene expression and function in PV-positive interneurons and a potential therapeutic target for psychiatric disorders associated with PGC-1α dysregulation.

1. Introduction GABAergic neurons synthesize and release the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which controls the excitation of neurons in the brain. Although GABAergic inhibitory neurons constitute just 10–25 % of total cortical and hippocampal neurons, the synaptic inhibition exerted by these neurons plays a crucial role in regulating synaptic transmission, synaptic plasticity, and synchronization of neurons through maintaining an appropriate excitation and inhibition balance (E/I balance) (Lin et al., 2005a). Recently, multiple lines of evidence have suggested that dysfunction of GABAergic neurons is involved in various brain disorders, including epilepsy, autism, and

schizophrenia (Kim et al., 2019). Locally projecting interneurons that release the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are arguably the most diverse cell population in the brain. More than 20 categories of inhibitory GABAergic interneurons have been described in the cortex and the hippocampus (Tremblay et al., 2016), while certain subsets of interneurons are preferentially affected in psychiatric disorders (Marin, 2012). In the cortex, hippocampus, and striatum, parvalbumin (PV) is expressed in a particular subset of interneurons. PV+ interneurons help maintain the oscillatory activity of networks in the gamma frequency range (30−80 Hz) (Bartos et al., 2007) that help coordinate neuronal communication among circuits and brain regions (Uhlhaas et al., 2010).

⁎

Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (W.-N. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.brainresbull.2020.02.005 Received 30 October 2019; Received in revised form 5 February 2020; Accepted 9 February 2020 Available online 11 February 2020 0361-9230/ © 2020 Elsevier Inc. All rights reserved.

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

water were available ad libitum. All procedures were conducted in accordance with the policies established by Jiangsu University, the Chinese Council on Animal Care and National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) regarding the ethical treatment of animals used for research.

The extent of GABAergic cellular diversity has only recently begun to be appreciated thanks to single-cell transcriptomic analysis (Tasic et al., 2018), and only a limited number of transcriptional factors that control the maturation and function of specific interneuron populations during early postnatal brain development have been identified(Butt et al., 2007). As a transcriptional coactivator, peroxisome proliferator activated receptor PPARγ coactivator-α (PGC-1α) regulates gene expression by binding to specific transcription factors and recruiting histone acetyltransferases to initiate transcription. PGC-1α has been coined the "master regulator of metabolism" based on its ability to induce gene programs controlling mitochondrial biogenesis and antioxidant production (Lin et al., 2005b). Recent studies show that PGC1α localizes to neurons that express high levels of the 67 -kDa enzyme glutamic acid decarboxylase (GAD67) during early postnatal development, GAD67 is responsible for the conversion of glutamate, an excitatory neurotransmitter, to GABA, an inhibitor neurotransmitter (Dougherty et al., 2014). The results of these studies are not consistent with the view that PGC-1α only acts as a generic regulator of metabolism in the brain (Lin et al., 2002) and have led us to propose that this transcriptional coactivator functions primarily to regulate gene programs specifically within GABAergic neurons. The overarching goal of the research was to elucidate the functions of PGC-1α in the central nervous system. The present study can be divided into two parts: ① observation of short-term habituation, startle reactivity, sensorimotor gating and spatial reference memory function in PGC-1α conditional knockout mice; ② elucidation of the possible relevance of PGC-1α to PV expression and function. Here, we show that genetic deletion of PGC-1α in GABAergic neurons causes a hyper-response phenotype of schizophrenia in mice. Hence, we suggest that the real value of PGC-1α genetically modified mice is not as "models of neuropsychiatric diseases" but as experimental tools to help elucidate the underlying neural mechanisms.

2.2. Genotype identification Ear-tip DNA was collected from offspring 2 d after birth, and genotyping was performed using two separate polymerase chain reaction (PCR) assays. PGC-1α+/+, PGC-1αfl/+, and PGC-1αfl/fl mice were genotyped using primer F (5′-TCCAGTAGGCAGAGATTTATGAC-3′) and primer R (5′-TGTCTGGTTTGACAATCTGCTAGGTC-3′). Dlx5/6-Cre mice were genotyped using primer F (5′-GCGGTCTGGCAGTAAAAACT ATC-3′) and primer R (5′-GTGAAACAGCATTGCTGTCACTT-3′). 2.3. Experimental design 2.3.1. Behavioral test battery The order in which the tests were administered was Novel object recognition (NOR), Prepulse inhibition (PPI) of the acoustic startle reflex, and Morris water maze (MWM) test. One test was conducted per animal per day, and the tests were administered in the ordered of least disruptive (absence of noxious stimulation: e.g., NOR) to most disruptive (e.g., PPI and the MWM test, which involved physical stimuli such as loud noises and cool water). Test batteries of this type are frequently used in behavioral studies, especially in studies of animals with targeted mutations. Although it is well known that behavioral tests can induce stress and that this should be considered in the analysis of behavioral effects, a sufficient number of studies have clearly demonstrated the validity of using such batteries of tests to identify patterns of phenotypic change related to neuropsychiatric pathology and treatment (Glass et al., 2019).

2. Materials and methods 2.1. Animals

2.3.2. Novel object learning The novel object recognition task was tested in 3 phases: habituation, sample and test, as described by Southwell, 2013 (Leger et al., 2013). During the habituation phase, the animal was allowed to explore an empty arena (40 cm × 40 cm × 40 cm) for 10 min. A short habituation time was used to reduce novelty responses to the open-field apparatus and to allow the mouse to explore novel stimuli in a familiar environment. During the sample phase (phase 1), the mouse was placed in the arena with two sample objects for a period of 5 min, during which it was allowed to investigate the objects and familiarize itself with them. The mouse was then returned to its home cage for 5 min. A test phase (phase 2) was then performed in which the mouse was presented with one of the familiarized sample objects and a novel object for 5 min. Object exploration was defined as sniffing, touching the object with the nose, or pointing the nose towards the object from a distance of less than 2 cm. The duration (time) or frequency of explorations of each object was recorded and expressed as a preference indexes (PI) for the test phase (Okuda et al., 2004). The PI defines the duration/frequency of exploration (D)/(F) of the novel object (DN)/(FN) by the total duration/frequency of exploration of the novel and familiar objects [Duration % for the novel object [% PIN=(DN)/(DN +DF)×100]; Frequency % for the novel object [% PIN=(FN)/(FN +FF)×100]] (Barkus et al., 2014a). After each session, the arena and objects were cleaned with 70 % ethanol to eliminate olfactory cues.

PGC-1αfl/fl mice were obtained from the Jackson Laboratory Jax mouse Cat No. 009666. To generate mouse strains deficient in PGC-1α by homologous recombination, a targeting plasmid was constructed in which exons 3–5 of the PGC-1α gene were flanked by loxP sites (Lin et al., 2004). GABAergic neuron-specific PGC-1α knockout mice were generated by crossing Dlx5/6-Cre-IRES-EGFP (Dlx5/6-Cre) mice (a generous gift from Dr. Chunjie Zhao, Medical School of Southeast University, China) with PGC-1αfl/fl mice. Dlx5/6-Cre was designed using a 0.5-kb fragment containing the id6/id5 enhancer and a betaglobin minimal enhancer placed immediately upstream of a Cre recombinase coding sequence and an internal ribosomal entry site (IRES)enhanced green fluorescent fusion protein. Dlx5 and Dlx6 (distal-less homeobox 5 and 6 genes) have been reported to be involved in the development and migration of GABAergic neurons (Kim et al., 2019; de Lombares et al., 2019). The PGC-1α mutant mice used in the experimental analysis were all second-generation offspring resulting from mating PGC-1αfl/fl mice to Dlx5/6-Cre mice. Dlx5/6-Cre::PGC-1α+/+ mice are referred to as controls (PGC-1α+/+, n = 10), Dlx5/6-Cre::PGC-1αfl/fl mice are referred to as homozygote mice (PGC-1α−/−, n = 9), and Dlx5/6Cre::PGC-1α+/fl mice are referred to as heterozygote mice (PGC-1α+/ − , n = 16). PGC-1α protein expression in the brain was low during the embryonic and early postnatal periods and peaked at postnatal day 28 (P28), with no significant difference between P28 and P90. Accordingly, behavioral experiments were conducted in 7-week-old mice (Wang et al., 2019). All animals were of C57BL/6 J genetic background and were housed two to five in a cage in a temperature-controlled colony room (21 ± 2℃) with a 12-h light-dark cycle (lights on at 7:00 am). Food and

2.3.3. Prepulse inhibition (Singer et al., 2013) 2.3.3.1. Apparatus. The apparatus consisted of four acoustic startle chambers designed for mice (SR-LAB, San Diego Instruments, San Diego, CA, USA). Each startle chamber comprised a nonrestrictive cylindrical enclosure made of clear Plexiglas and attached horizontally to a mobile platform that in turn rested on a solid base inside a sound129

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

day 7; in this test, the platform was removed, and the animals was placed in the MWM for 60 s. For the MWM test, three dependent measures, (1) swim distance (cm), (2) swim velocity (cm/sec) and (3) swim duration (sec) were tracked and digitized, and the information was stored for subsequent behavioral analysis. On day 8–10, the mice were trained to locate the position of the hidden platform, which was now located in the opposite quadrant SE. The procedure was the same as before. On day 11, the same probe test was conducted as on day 7. The parameters were the same as on day 7.

attenuated isolation cubicle. A high-frequency loudspeaker mounted directly above the animal enclosure inside each cubicle produced a continuous background noise of 65 dB (A-scale) and various acoustic stimuli in the form of white noise. The pulse and prepulse stimuli employed (sustained for 40 ms and 20 ms, respectively) were sudden elevations in the noise level over background with a rise time of 0.2–1.0 ms. Vibrations of the Plexiglas enclosure caused by the whole-body startle response of the animal were converted into analogue signals by a piezoelectric unit attached to the platform. These signals were digitized and stored by a computer.

2.3.4.3. Data collection. The entire behavioral pattern of the animals during the experimental trials was recorded by a video camera and a video monitor located in an adjacent room. The measurements and data analysis were performed using Ethovision 8.5 software (Noldus, the Netherlands). The Startle Reflex software system (SR-LAB, San Diego Insutrument, CA, USA) was used to assess PPI.

2.3.3.2. Test procedure. In the demonstration of PPI of the acoustic startle reflex, subjects were presented with a pulse stimulus and prepulses. The pulse stimulus was 120 dBA in intensity. Prepulses of various intensities (69, 73, 77, 81, and 85 dBA, corresponding to 4, 8, 12, 16, and 20 dBA above background, respectively) were employed. The pulse-alone trial could be considered as having a prepulse of 0 dBA above background. The SOA (stimulus onset asynchrony) of the prepulse and pulse stimuli on prepulse-plus-pulse trials was 100 ms. A session began with placement of the animal into the Plexiglas enclosure. After a 5-min acclimatization period, 6 consecutive pulsealone trials were presented to habituate and stabilize the animals' startle response. Subsequently, the animal was subjected to 12 blocks of discrete test trials. Each block consisted of one type of trial: pulse-alone trials, prepulse-pulse trials at each of the five levels of prepulse, prepulse-alone trials at each of the five levels of prepulse, and nostimulus trials. The trials in each block were presented in pseudorandom order. The session concluded with a final block of six consecutive startle-alone trials. The interval between successive trials was variable, with a mean of 15 ± 5 s. The enclosures were cleansed and dried after each session.

2.4. Western blot After the completion of behavioral testing, the mice (n = 3–6 in each group) were decapitated. The entire brain was removed, and samples of the prefrontal cortex, cerebellum and hippocampus (right and left, separately) were immediately frozen in liquid nitrogen and homogenized in radio-Immunoprecipitation assay (RIPA) tissue protein extraction reagent (Beyotime, Shanghai, China) containing appropriate protease inhibitors (PMSF, Beyotime, Shanghai, China). The samples were electrophoresed on 10 % BisTris gels (Bio-Rad) and transferred to PVDF membranes. The blots were blocked for 1.5 h in Tris-buffered saline containing 0.1 % Tween-20 and 5 % BSA or powdered milk. The membranes were incubated with primary antibodies against PGC-1α diluted 1: 1000, Abcam, Cambridge, UK or PV diluted 1: 1000; Millipore, New York, USA overnight at 4℃ and then incubated with horseradish peroxidase-linked antibodies diluted 1: 5000, Beyotime, Shanghai, China at room temperature for 1 h. Bands were detected by enhanced chemiluminescence Tanon, Shanghai, China and exposed to X-ray film. Protein band intensities were quantified by using the Image J software. Uniform loading was verified based on immunoreactivity of β-tubulin (1: 2,000, Abm, Vancouver, Canada).

2.3.3.3. Prepulse-elicited reactivity. The mean reactivity score obtained on prepulse-alone trials for trials at all prepulse intensities was used to evaluate prepulse-elicited reactivity. 2.3.3.4. % PPI. The reactivity scores obtained on the middle 12 pulsealone trials and prepulse-plus-pulse trials were used to evaluate PPI. When measuring PPI, it is customary to normalize the reduction of startle reaction (i.e., the reactivity score obtained on prepulse-pluspulse trials at different prepulse intensities) to each subject's average reactivity score across the pulse-alone trials, and to express the result as percent inhibition. The individual animal’s mean startle reactivity thus served as the baseline; against this baseline, the effectiveness of the prepulse at inhibiting the startle response to the subsequent pulse stimulus was expressed in percent, that is, % PPI=[(pulse alone(prepulse plus pulse))/ pulse alone×100 %].

2.5. Immunofluorescence Mice (n = 3–6 in each group) were anesthetized with ethyl carbamate, perfused transcardially with PBS and then with 4 % paraformaldehyde (PFA) in 0.01 M PBS, pH 7.4, Brain tissue was removed and placed in 4 % PFA for 24 h, and transfer to 30 % sucrose for 24 h. Frozen coronal Sections (7 μm) were then cut on a sliding microtome and collected serially. The sections were washed in PBS for 30 min and blocked with 10 % goat serum for 1.5 h at 25℃. The sections were then incubated with primary antibodies (rabbit anti-PGC-1α, Abcam, Cambridge, UK) (1: 300 in incubation buffer) and mouse anti-PV (1: 400, Millipore, New York, USA) overnight at 4℃. After being rinsed in PBT, the sections were incubated for 2 h at 25℃ with Alexa Fluor 647 goat anti-rabbit IgG (H + L) (diluted 1: 500, Abcam, Cambridge, UK) and FITC goat anti-mouse IgG (H + L) (1: 400, Multi Sciences, Hangzhou, China). After several rinses in PBT, the sections were incubated for 15 min at 25℃ with DAPI (1: 1000, BBI Life sciences, Shanghai, China). Free-floating sections were mounted on dry gelatincoated slides, and fluorescence was visualized using a fluorescence microscope (BX41, Olympus, Tokyo, Japan).

2.3.4. Water maze (Vloeberghs et al., 2006) 2.3.4.1. Apparatus. The maze was filled with water made opaque by the addition of white latex paint. The temperature of the water was 21 ± 2℃. A round perspex platform (10 cm diameter) was placed in a fixed position inside the pool in one of the four quadrants. The platform was submerged 2 cm below the surface of the water and was therefore hidden from the animals’ view; it remained in the same position during all the 4 training trials of each day. 2.3.4.2. Test procedure. During the first six days (Day 1–6), the mice were trained to locate the position of the hidden platform, which was located in quadrant NW. Each animal received four training trials per day, and each trial started from a different position (north, east, south, west), in random order. Animals that failed to escape within 60 s were guided to the platform by the experimenter and were assigned an escape latency of 60 s. They were allowed to spend 15 s on the platform (Pouzet et al., 2002) and were then placed in a heated holding cage for 15 s before the next trial commenced. A probe test was conducted on

2.6. Immunohistochemistry Mice (n = 3–6 in each group) were anesthetized with ethyl carbamate and perfused as described above. Their brains were removed and fixed in 4 % paraformaldehyde for at least 24 h before tissue dehydration and embedding in a transparent wax block. The tissue blocks 130

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

were sectioned at 6 μm. The sections were incubated in 0.05 M trisodium citrate (pH 6.0) in a microwave oven to enhance antigen recognition and then in 3 % hydrogen peroxide to quench endogenous peroxidase activity. After blocking in 10 % serum for 1 h, the sections were incubated with an antibody against PV (MAB1572, 1: 500, Millipore, Germany) overnight at 4 °C. The sections were washed in PBS, incubated for 1 h at room temperature with the corresponding secondary antibody, and washed again in PBS. After washing, DAB was used for dyeing, and hematoxylin was used for retaining. The sections were dehydrated in a graded series of ethanol and xylenes and mounted using non-aqueous mounting medium. Selected images were captured using a SPOT camera and software system (Diagnostic Instruments, Sterling Heights, MI, USA) attached to a Leica DM5000B microscope (Leica, Bannockburn, IL, USA) with 4x and 10x objectives.

Fig. 2. PGC-1α−/− mice display lower body weight. Diagram illustrating the body weight, * P < 0.05, ** P < 0.01, and * ** P < 0.001 indicate differences between PGC-1α−/− (n = 9) and PGC-1α+/+ (n = 10) mice. #P < 0.05 and ### P < 0.001 indicate differences between PGC-1α−/− and PGC-1α+/− (n = 16) mice.

2.7. Statistical analyses All statistical analyses were performed using StatView 5.01 software (Abacus Concepts, Inc., Berkeley, CA, USA, 1992). The data were first subjected to ANOVA. Post hoc comparisons were conducted using Fisher's protected least significant difference test. Significant differences were accepted at P < 0.05. All values are presented as means ± S.E.M. The possible effect of gender on the results of each of the tests was analyzed using mice of each of the three genotypes. The (Genotype × Gender) ANOVA analysis showed no differences between female and male mice of the same genotypes; therefore, we presented only data from female mice.

and male [F2,15 = 6, P = 0.011] mice. Body weight was significantly lower in PGC-1α−/− (11 ± 1) mice than in PGC-1a +/−(13 ± 1, P = 0.023) and PGC-1a +/+ (14 ± 1, P = 0.004) mice. 3.3. Mortality rate We next evaluated the mortality rate (calculated as percentage of animals that died before behavioral experiments were performed at around 7 weeks) in mice of the three genotypes. As shown in Table 1, no differences in mortality were found among PGC-1α−/− mice (11.11 %), PGC-1α+/− mice (15.63 %) and the PGC-1α+/+ (Control, ctr) mice (12.66 %).

3. Results 3.1. Induction of mutation

3.4. Behavioral results

To evaluate the role of PGC-1α in behavior and determine its potential mechanism, we generated inducible, GABAergic neuron-specific PGC-1α KO mice. Conditional heterozygous PGC-1αfl/+ were genotyped by PCR and subsequently mated to generate homozygous PGC1αfl/fl knockout mice. These matings produced floxed PGC-1α mice in the expected Mendelian ration, and the floxed mice appeared normal, and were fertile. To achieve GABAergic neuron-specific PGC-1α deletion (Fig. 1), mice carrying the floxed PGC-1α alleles were crossed with Dlx5/6-Cre mice; Dlx5/6-Cre::PGC-1αfl/fl (−/−), Dlx5/6-Cre::PGC1α+/fl (+/−) and Dlx5/6-Cre:: PGC-1α+/+ (+/+) mice were obtained.

3.4.1. Deficiency of object-related short-term habituation in PGC-1α conditional knockout mice in the NOR test We first analyzed the effect of gender in mice of the three genotypes. A 3 × 2 (Genotype × Gender) two-way ANOVA for novel object showed no significant differences between female and male mice of the same genotype [F1,66 = 1, P = 0.317]. Fig. 3B-C presents the mean time spent exploring the object A and B during the sample phase among the mice of the three genotypes. Post hoc Fisher's LSD tests indicated that no significance was found across the three genotypes for exploring the object A [F2,30 = 1, P = 0.448] and B [F2,30 = 0.1, P = 0.894] during the sample phase. While noted difference of the time spent exploring the familiar object A [F2,32 = 9, P = 0.001] (Fig. 3D) and the novel object C [F2,32 = 4, P = 0.040] (Fig. 3E) during the test phase was obtained by Post hoc Fisher's LSD tests. PGC-1α−/− mice showed a significant increase in time spent exploring the familiar object A (54 ± 19 & 4 ± 1, P = 0.001) and a significant reduction in time spent exploring the novel object C (3 ± 1 & 8 ± 1, P = 0.012) compared to PGC-1α+/+ mice during the test phase. As shown in Fig. 3F-G, in female mice, genotype had a marked effect on % Preference index for exploring the novel object based on duration [F2,32 = 4, P = 0.031] and on frequency [F2,32 = 4, P = 0.048] during the test phase. PGC-1α−/− mice showed reduced % Preference index based on duration (32 ± 11& 65 ± 11, P = 0.028) and reduced %

3.2. Body weight The body weights of all mice were measured every three days and recorded from the 3rd to the 42nd day of age. The body weight of the female mice are shown in Fig. 2. The presence of the mutation had a notable effect on body weight in both female [F2,19 = 5, P = 0.014]

Table 1 Mortality rates of genotyped mice. Fig. 1. PCR products obtained from genomic DNA from Dlx5/6-Cre::PGC1αfl/fl (−/−), Dlx5/6-Cre::PGC-1αfl/+ (+/−), Dlx5/6-Cre::PGC-1α+/+ (+/+) and PGC-1αfl/fl (f/f) mice. The 400- and 360-bp bands result from amplification of the PGC-1αfl/fl and wild-type alleles, respectively. The 100-bp band results from amplification of the Dlx5/6-Cre allele.

WT PGC-1a PGC-1a PGC-1a

131

+/+ +/− −/−

Surviving mice

Dead mice

Total mice

Mortality rate

34 69 27 40

0 10 5 5

34 79 32 45

0% 12.66 % 15.63 % 11.11 %

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

Fig. 3. PGC-1α−/− mice display impaired short-term habituation in the novel object recognition test. (A) The top panel shows the design of the standard novel object recognition task. During the habituation phase, the animal was allowed to explore an empty arena for 10 min. 24 h later, the animals were exposed to two objects A and B (Sample phase) and then after a 5 min interval they received a 5 min test in which they were allowed to explore a duplicate of the familiar object A and a novel object C (Test phase). The time spent exploring the object A (B) and B (C) during the sample phase among the mice of the three genotypes is shown. Again, the time spent exploring the duplicate of the familiar object A (D) and the novel object C (E) during the test phase is also illustrated. The time/frequency spent exploring the novel object during the test phase is shown as % Preference index (PI) for duration (F) and frequency (G), respectively. PI allows discrimination between the novel and familiar objects, i.e., it uses the difference in recognition duration (D)/frequency (F) for novel (DN)/(FN) object but then divides this value by the total duration/ frequency of exploration of the novel and familiar objects [Duration % for the novel object: [% PIN=(DN)/(DN+DF)×100]; Frequency % for the novel object: [% PIN=(FN)/(FN+FF)×100]]. (H) % Duration spent exploring object A during the sample phase (phase 1) was calculated using the formula [(time at object A)/(time at object A + time at object B)], and % Duration for object A during the test phase (phase 2) was calculated using the formula [(time at object A)/(time at object A + time at object C)] for mice of the three genotypes. (I) % Duration for object B was calculated using the formula [(time at object B)/(time at object A + time at object B)] during sample phase (phase 1), and % Duration for object C was calculated using the formula (time at object C)/[(time at object A + time at object C)] during the test phase (phase 2) across the three genotypes. The distances moved by mice of the three genotypes during the habituation phase are shown in (J). The one-sample ttest was applied to evaluated the duration in PGC-1α+/+ mice (K) and in PGC-1α−/− mice (L) during the test phase, as illustrated in the bar plot. The dashed line at 50 % indicates chance performance. The error bars indicate S. E. M. *P < 0.05, **P < 0.01.

during phase 1 than for object C during phase 2, indicating that PGC1−/−mice exhibited a pronounced deficit in short-term habituation. The above results were supported by two-way (Genotype × Phase) ANOVA, which yielded a main effect of Genotype [F2,58 = 3, P = 0.043] and of Phase [F1,58 = 7, P = 0.013] and an interaction of Genotype × Phase [F2,58 = 5, P = 0.011]. Fig. 3J presents the mean distance moved by the PGC-1α genotype mice during the habituation phase. Post hoc Fisher's LSD tests [F2,32 = 7, P = 0.002] indicated that PGC-1α−/− mice showed a significant increase in distance moved during habituation compared to PGC-1α+/ + mice (2827 ± 249 & 1885 ± 129, P = 0.009). In the test phase, the mouse was exposed to a familiar object (A) and a novel object (B). The one-sample t-test was applied to evaluate the difference of time spent in exploring the familiar and in exploring the novel object during the test phase in PGC-1α+/+ (Fig. 3K) and in PGC1α−/− (Fig. 3L) mice. PGC-1α+/+ mice preferentially chose to explore the novel alternative, reflecting their stimulus-specific habituation to

Preference index based on frequency (40 ± 7& 65 ± 7, P = 0.028) compared to PGC-1α+/+ mice. Fig. 3H shows the difference in % duration for object A calculated using the formula [(time at object A)/(time at object A + time at object C)] in mice across the three genotypes. As can be observed, PGC-1α+/− and PGC-1α+/+ mice never showed any preference for object A when the sample phase (phase 1) was compared with the test phase (phase 2), but PGC-1α−/− mice showed significantly increased preference for object A in phase 2 compared to phase 1. Fig. 3I presents the difference in % duration for object B calculated using the formula [(time at object B)/(time at object A + time at object B)] during the sample phase (phase 1), and % Duration for object C calculated using the formula (time at object C)/[(time at object A + time at object C)] during the test phase (phase 2) for mice of the three genotypes. As can be observed, PGC-1α+/− and PGC-1α+/+ mice never showed any preference for object B or C when phase 1 was compared with phase 2, but PGC-1α−/ − mice showed significantly increased exploring preference for object B 132

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

Fig. 4. PGC-1α−/− mice display reduced prepulse inhibition and exaggerated startle reactivity. (A) The mean startle magnitude obtained in the first six and last six pulse-alone (120 dBA) trials are shown in the bar graph. (B) Evaluation of auditory habituation in mice of the three genotypes using the formula (last 6 pulses)/(first 6 pulses). (C) % PPI is expressed as a function of prepulse intensity (69, 73, 77, 81, and 85dBA) for mice of the three genotypes. % PPI=[(pulse alone-(prepulse plus pulse))/pulse alone×100 %]. All posthoc comparisons are based on Fisher's LSD, *P < 0.05, **P < 0.01, and ***P < 0.001 indicate differences between PGC-1α−/− and PGC-1α+/+ mice; #P < 0.05 and ##P < 0.01 indicate differences between PGC-1α−/− and PGC-1α+/− mice.

the familiar object [P = 0.031]. In contrast, PGC-1α−/− mice displayed a deficit in short-term habituation, as shown by their lower preference for a novel object compared to a familiar object that was presented recently [P = 0.023].

mice showed prominently disrupted PPI with prepulse stimuli of varying intensities, including 73 (37 ± 10 & 5 ± 12, P = 0.023), 81 (53 ± 7 & 20 ± 13, P = 0.029) and 85 dBA (59 ± 6 & 30 ± 10, P = 0.032), corresponding to 8, 16, and 20 dBA above background, respectively.

3.4.2. Exaggerated startle reactivity and reduced prepulse inhibition were exhibited by PGC-1α conditional knockout mice in the PPI test 3.4.2.1. Reactivity scores. We first analyzed the effect of gender. Twoway (Genotype × Gender) ANOVA of startle reactivity [F2,64 = 1, P = 0.644] and % PPI [F2,64 = 0.2, P = 0.844] showed no significant differences between female and male mice of the same genotype. Fig. 4A-B present the startle habituation. Two-way (Genotype × Pulse-alone trial) mixed ANOVA yielded a main effect of Genotype [F2,64 = 9, P < 0.001] but not of Trial [F1,64 = 1, P = 0.328] or of interaction between Genotype × Trial [F2,64 = 0.1, P = 0.900]. Oneway ANOVA of the mean habituation, calculated using the formula [(Last 6 pulses (End))/(First 6 pulses (Start)], showed no significant differences among the mice of the three genotypes [F2,32 = 0.1, P = 0.900].

3.4.3. Hyperactivity and normal spatial reference memory were exhibited by PGC-1α conditional knockout mice in the MWM test Spatial training occurred over a period of nine days. Two probe trials, each with a ceiling time of 60 s, were administered the day after spatial training was completed (Fig. 5A). 3.4.3.1. Acquisition. We first analyzed the effect of gender. Two-way (Genotype × Gender) mixed ANOVA of distance [F2,64 = 0.006, P = 0.994], latency [F2,64 = 0.3, P = 0.760] and velocity [F2,64 = 0.5, P = 0.626] showed no significant differences between female and male mice of the same genotype. Fig. 5B presents the mean path length to reach the hidden platform for mice of each of the three genotypes. As can be seen, none of the mice showed significant differences in path length during acquisition training on day 1–6. Genotype had no effect on the escape path length (F2,37 = 1, P = 0.562). Fig. 5C presents the mean latency to reach the hidden platform over 6 days of training for mice of each of the three genotypes. Two-way (Genotype × Days) repeated ANOVA of escape latency yielded an approaching effect of Genotype (F2,32 = 4, P = 0.052). To further analyze the contradictory results of the acquisition performance indexed by the weaken efficiency of escape latency and by the escape path length, the velocity to reach the hidden platform was recoded (Fig. 5D). Two-way (Genotype × Days) repeated ANOVA of escape velocity yielded a significant Genotype effect (F2,32 = 5, P = 0.0171) (Fig. 5D). Thus, PGC1α gene deletion increased locomotor activity.

3.4.2.2. Startle reactivity. Fig. 4C shows the startle magnitude Two-way (Genotype × Prepulse intensity) mixed ANOVA of startle reactivity yielded a main effect of Genotype [F2,32 = 8, P = 0.002]. Post hoc comparisons showed that PGC-1α−/− mice showed significantly elevated startle reactivity at all prepulse intensities relative to the PGC-1α+/+ group, including prepulse intensities of 69 (114 ± 38 & 25 ± 3, P = 0.002), 73 (117 ± 42 & 20 ± 3, P = 0.002), 77 (104 ± 37 & 17 ± 3, P = 0.002), 81 (98 ± 36 & 14 ± 2, P = 0.002), and 85 dBA (90 ± 33 & 12 ± 1, P = 0.001), corresponding to 4, 8, 12, 16, and 20 dBA above background, respectively. 3.4.2.3. Prepulse inhibition (% PPI). A 3 × 5 (Genotype × Prepulse intensity) mixed ANOVA of percent PPI yielded a main effect of Genotype [F2,32 = 4, P = 0.026]. As shown in Fig. 4D, PGC-1α gene deletion disrupted PPI in an intensity-dependent manner, as shown by the analysis of percent PPI. Compared to PGC-1α+/+ mice, PGC-1α−/−

3.4.3.2. Probe test 1. The MWM was divided into four sectors: NW, NE, SW, and SE. The NW sector, in which the platform was positioned during the acquisition training phase, was used as the target sector. We 133

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

Fig. 5. PGC-1α−/− mice display normal spatial reference memory and hyperactivity. (A) shows the schedule for training in the MWM. The acquisition phase is performed from day 1 to day 6, and on day 7 a probe test is performed in which the escape platform is removed. The reversal learning phase is conducted from day 8 to day 10, followed by a second probe test 24 h later (day 11). Path length (B), escape latency (C) and velocity (D) to reach the hidden platform during the acquisition and reversal phase for animals are illustrated as the mean values obtained in 4 trials. Distance (E), duration (F) and velocity (G) of swimming in the target zone (NW) of the water maze pool during probe test 1 are shown in the bar graphs. Distance (H), duration (I) and velocity (J) spent in the target zone (SE) of the water maze pool during probe test 2 are also displayed. The location of the escape platform (the platform was removed for the probe test) is indicated by a solid circle. All post-hoc comparisons are based on Fisher's LSD. Values are expressed as means ± S. E. M. *P < 0.05 and **P < 0.01 indicate differences between PGC-1α−/− and PGC-1α+/ + mice.

first analyzed the gender effect in mice of the three genotypes. Two-way (Genotype × Gender) mixed ANOVA of swimming distance [F2,64 = 2, P = 0.151], duration [F2,64 = 0.1, P = 0.861] and velocity [F2,64 = 2, P = 0.198] in the target zone showed no significant differences between female and male mice of the same genotype. As shown in Fig. 5E, the distance in target zone was analyzed using one-way ANOVA with genotype as a main factor. There were no significant differences in swimming distance in the target zone among mice of the three genotypes[F2,64 = 2, P = 0.135]. The duration of genotype effect [F2,32 = 0.5, P = 0.075] was, however, not uniformly related to the distance in all groups (Fig. 5F). A possible reason for this is that PGC1α−−/− mice displayed hyperactivity, as indicated by elevated velocity

[F2,32 = 4, P = 0.040] (Fig. 5G). 3.4.3.3. Reversal task. Fig. 5B also shows the mean path length to reach the hidden platform on successive days; in the later tests, the hidden platform was located in quadrant SE rather than in quadrant NW as in acquisition phase. We first analyzed the gender affect across the three genotypes. Two-way (Genotype × Gender) mixed ANOVA of distance [F2,64 = 0.007, P = 0.993], latency [F2,64 = 0.1, P = 0.884] and velocity [F2,64 = 1, P = 0.442] showed no significant differences between female and male mice of the same genotype. Genotype had no effect on escape path length [F2,37 = 2, P = 0.132] (Fig. 5B); a similar result was obtained in the escape latency analysis (Fig. 5C) [F2,32 = 1, P 134

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

recombinase under the control of the ZP3 (zona pellucida 3) promoter, which is a transiently activated during oocyte development. These PGC1α null mice were reported to exhibit progressive vacuolization in various brain region within the first 3 months of age as well as exhibit abnormal behavioral characteristics, including stimulus-induced myoclonus, dystonic posturing, and frequent limb clasping, after homologous recombination, only half of PGC-1α null pups survived the early postnatal period (Lin et al., 2004). The broad deficits exhibited by PGC-1α null mice make it impossible to specifically explore behaviors related to locomotors activity and cognitive function. To overcome this issue, the current study used mice in which the mutation was limited to GABAergic neurons This was achieved by using a Dlx5/6 promoter that was activated only in GABAergic interneuron. This restricted mutation resulted in mice that survived the postnatal period at the same rate as control mice. Hence, these conditional knockout mice can be used to evaluated fine behavioral changes. Despite reports of PGC-1α deficiency in multiple neurological disorders and the documented ability of PGC-1α to drive transcriptional pathways in energy-demanding issues (Agudelo et al., 2014), little is known about the behavior of mice lacking in GABAergic neurons in the novel object recognition test, the prepulse inhibition test and the Morris water maze test. This study is the first to explore the impact of the PGC-1α gene on short-term habituation, startle reactivity, prepulse inhibition and spatial reference memory. The results of the novel object recognition test revealed a specific and striking impairment in short-term habituation in PGC-1α−/− animals. Habituation refers to the decreased tendency to respond to a stimulus that has become familiar due to prior exposure (Barkus et al., 2014b). It is likely to be an adaptive response that helps ensure that attentional resources are allocated to novel and potentially important stimuli (Barkus et al., 2014a). PGC-1α−/− mice exhibited a pronounced deficit in short-term habituation as shown by their slower habituation to a would-be familiar object (Fig. 3). In this task, the mice were first exposed to two familiar objects during a sample "Exposure" phase (phase 1) and allowed to explore them freely. When a PGC-1α+/+ animal was presented with novel objects, it began to explore them, but its exploratory activity gradually decreased or habituated as the objects became familiar. In a subsequent "Test" phase (phase 2), the mouse was exposed to a copy of the original object (now familiar) and a novel object. PGC-1α+/+ animals preferentially chose to explore the novel object, reflecting their stimulus-specific habituation to the original object (Fig. 3K). In contrast, PGC-1α−/− mice displayed a deficit in short-term habituation; they failed to decrease the amount of attention paid to recently presented stimuli (Fig. 3L). Consequently, PGC-1α−/− mice showed less preference than PGC-1α+/+ mice for a novel object compared to a familiar object that had been recently presented. Notably, we have also shown that PGC-1α−/− mice can exhibit sensitization, whereby more attention is paid to a recently presented stimulus than to a non-recent stimulus (Sanderson et al., 2011). In both PGC-1α+/+ and PGC-1α−/− mice, if the two objects they exposed to were different then animals spent an equal amount of time in exploring each object (Fig. 3B-C). In contrast, if repeated exposure with the same object, then PGC-1α+/+ mice spent less time in exploration on its second exposure (Fig. 3D), reflecting short-term habituation to the familiar object. However, when a further copy of the original object was presented to PGC-1α−/− mice, they actually spent more time in exploring the object (familiar object) on its second exposure (relative to the first presentation of the original object (Fig. 3H), and relative to the presentation of the novel object) (Fig. 3D-G, 3 L). That means PGC1α−/− mice do have a memory of the object that they have just experienced. However, they express the memory in a very special way, as described by Barkus laboratory, under some conditions the attention being paid to a recently presented neutral stimulus can actually increase rather than decrease (sensitization) (Barkus et al., 2014b). The water maze test was originally chosen to evaluate learning and memory function in this study. PGC-1α+/+ and PGC-1α−/− mice

= 0.353]. To better identify the critical factors involved, we plotted the difference scores contrasting the velocity. PGC-1α−/− mice showed a significantly higher velocity than PGC-1α+/− mice. This was supported by two-way (Genotype × Days) repeated ANOVA, which yielded a main effect of Genotype (F2,37 = 7, P = 0.003) (Fig. 5D). 3.4.3.4. Probe test 2. We first analyzed the gender effect in mice of the three genotypes. Two-way (Genotype × Gender) mixed ANOVA of swimming distance [F2,64 = 0.5, P = 0.621], duration [F2,64 = 3, P = 0.084] and velocity [F2,64 = 1, P = 0.398] in the target zone showed no significant differences between female and male mice of the same genotype. As shown Fig. 5H, the distance in the target zone was analyzed using one-way ANOVA with genotype as a main factor. There were no significant differences in swimming distance in the target zone in mice of different genotypes [F2,32 = 1, P = 0.508]. The duration of genotype effect [F2,32 = 4, P = 0.040] was, however, not uniformly related to the distance in all groups (Fig. 5I). A possible reason for this is that PGC-1α−/− mice displayed hyperactivity, as depicted by elevated velocity [F2,32 = 3, P = 0.046] (Fig. 5J). 3.5. Decrease in PGC-1α protein levels in the brains of PGC-1α−/− mice As shown in Fig. 6, significant reduction in the expression of PGC-1α was found in the brains of PGC-1α−/− mice compared with PGC-1α+/ + mice. Reduced expression was observed in the hippocampus [F2,6 = 8, P = 0.018] (Fig. 6A-A′) and prefrontal cortex [F2,6 = 11, P = 0.011] (Fig. 6B-B′) but not in the cerebellum [F2,6 = 0.31, P = 0.745] (Fig. 6CC′) . 3.6. Morphology and PV levels in the brains of PGC-1α−/− mice PGC-1α regulates PV expression in a tissue-specific manner (McMeekin et al., 2016). The gross morphology of the brains of PGC1α+/+ and PGC-1α−/− animals, as revealed by PV staining, was studied by immunofluorescence and immunohistochemistry (Fig. 7). Decreased levels of PV were observed in the motor cortex [F1,6 = 15, P < 0.01], the somatosensory cortex [F1,5 = 8, P = 0.037] (Fig. 7A-B and I) and the hippocampus, especially in its CA1 [F1,5 = 8, P = 0.037] and CA3 [F1,5 = 25, P < 0.01] regions (Fig. 7E-F and H), but not in the cerebellum [F1,6 = 3, P = 0.130] (Fig. 7C-D) of PGC-1α−/− animals. Furthermore, when PGC-1α+/+ and PGC-1α−/−cortical and hippocampal brain lysates were immunoblotted using an antibody against PV, decreased levels of PV were found in the cortex [F1,4 = 30, P < 0.01] and the hippocampus [F1,4 = 8, P < 0.05] of PGC-1α−/− mice compared to PGC-1α+/+ mice (Fig. 7J-L). 4. Discussion PGC-1α is a master regulator of metabolism in peripheral tissues. However, the detailed molecular functions of PGC-1α in the brain in vivo are not fully understood. Recent evidence demonstrates that PGC1α localizes to the nuclei of GABAergic interneurons by the second week of postnatal life during the maturation of the rodent brain (Dougherty et al., 2014; Wang et al., 2019). Because GABAergic neurons play essential roles in shaping excitatory synaptic information and regulating neuronal activity across the brain, it is likely that PGC-1α in GABAergic neurons is also involved in the regulation of GABAergic inhibition in the brain. In this study, we aimed to determine the cell type-specific roles of PGC-1α in neuronal functions, focusing especially on GABAergic neurons. To achieve this goal and to identify the function of PGC-1α in vivo, we generated GABAergic neuron-specific PGC-1α KO mice by crossing Dlx5/6-Cre mice with mice harboring the floxed PGC-1α allele. In a previous attempt to determine the role of PGC-1α in a variety of processes, the Spiegelman laboratory generated PGC-1α null mice. These PGC-1α null mice were generated through transgenic expression of Cre 135

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

Fig. 6. Expression of the PGC-1α gene in different brain regions of mice of the three genotypes. Immunoblots and quantification of PGC-1α expression in the hippocampus (A)/(A'), prefrontal cortex (B)/(B'), and cerebellum (C)/(C') are shown. Values are expressed as means ± S. E. M. *P < 0.05 and **P < 0.01 indicate differences between PGC-1α−/− and PGC-1α+/+ mice; #P < 0.05 and ##P < 0.01 indicate differences between PGC-1α−/− and PGC-1α+/− mice. n = 3 per genotype.

intense nature of the stimulus that leads to a defensive response (Heesink et al., 2017). Impairments in PPI could also be considered to indicate failure to reduce the attention paid to a stimulus (the startle stimulus) based on recent prior experience (the prepulse (Braff et al., 1992)). It has been well documented that patients with schizophrenia exhibit deficits in habituation over a range of timescales, both behaviorally (e.g., in terms of habituation of the startle response (Geyer and Braff, 1982)) and physiologically (e.g., reduction in evoked responses to auditory stimuli after repeated presentation (Freedman et al., 1996)). Therefore, a link between habituation deficits and schizophrenia has been made previously. What is novel here is the link between deficits in short-term habituation that can lead to sensitization and the notion that patients may experience aberrant salience, with greater attention being paid to recently presented stimuli (Spitzer, 1995; Kapur, 2003), which suggesting the link between short-term habituation and aberrant salience (Barkus et al., 2014b). Along with the remarkable increase in swimming velocity in the MWM test and in the distance moved during the habituation phase in the NOR test observed in PGC-1α−/− mice, let

displayed normal spatial reference memory, as demonstrated by the lack of a significant difference in the distance moved in the target zone in which the platform was located (Fig. 5E, 5 H). The swimming duration of PGC-1α−/− mice in the target zone was also analyzed. A notable reduction in swimming duration in the target zone was found in PGC-1α−/− animals (Fig. 5F). This reduction was probably a result of the approximately 2-fold higher swimming velocity of the PGC-1α−/− mice compared to PGC-1α+/+ mice (Fig. 5G and J) rather than of a deficiency in spatial reference memory. These results are consistent with the observed elevation in the locomotor activity of PGC-1α−/− mice during the habituation phase of the NOR test (Fig. 3B). A similar conclusion that PGC-1α conditional knockout mimic a hyper-response phenotype was reported in our previous study (Wang et al., 2019). This study is also the first to explore acoustic startle reactivity in PGC-1α conditional knockout mice. In the prepulse inhibition test, we observed that PGC-1α−/− mice displayed exaggerated sensitive startle responses. Due to the sudden and intense nature of the stimulus that leads to a defensive response, an exaggerated sensitive startle reflex can indicate a lowered threshold of perceiving threat, due to the sudden and 136

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

Fig. 7. Decreased PV protein expression throughout the cortex and hippocampus in PGC-1α−/− mice. Immunofluorescence using an antibody specific to PV was conducted on 7-μm coronal brain sections from PGC-1α−/− (Cre-fl/fl) and PGC-1α+/+ (Cre-WT) mice. The expression patterns and quantification of PV in the prefrontal cortex (A-B), cerebellum (C-D), and hippocampus (E-F) are shown in the bar graphs. Immunohistochemistry using an antibody specific to PV was conducted on 20-μm coronal brain sections from Cre-fl/fl and Cre-WT mice. The expression patterns and quantification of PV in the prefrontal cortex (G, I) and hippocampus (G, H) are shown in the bar graphs. Cortical and hippocampal brain lysates of PGC-1α+/+ and PGC-1α−/− mice were immunoblotted using an antibody against PV. Decreased expression (J-K) and levels (L) of PV were found in the brains of PGC-1α−/− mice. n = 3/genotype. Scale bars = 100 μm. Green = PV; Purple = DAPI; Red = PGC-1α. *P < 0.05 and **P < 0.01 indicate differences between PGC-1α−/− and PGC-1α+/+ mice.

us anticipate that PGC-1α−/− mice present a hyper-response phenotype of schizophrenia. Given the transcriptional coactivator role of PGC-1α and the unique function of PV-positive neurons, we hypothesize that PGC-1α is required for protein expression of the Ca2+-binding protein PV

throughout the cerebrum. Abnormalities in inhibitor signaling are consistent with decreased PV from GABAergic neuron. As well as extending the evidence that PV positive interneurons play a key role for the brain region of hippocampus (Caputi et al., 2012) and cortex (Lewis et al., 2012) in regulating attentional processes like short-term 137

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

habituation. We postulate that the absence of PGC-1α causes dysregulated proliferation or migration of these specific PV interneuron populations, resulting in a less density of such cells in defined regions of the hippocampus and cortex. Further investigation is needed to determine the underlying molecular mechanism. PV interneurons in the hippocampus and cortex are thought to arise from progenitors in the medial ganglionic eminence during embryonic development and to tangentially migrate to these regions (Wonders and Anderson, 2006). However, due to the postnatal induction of PGC-1α expression, it is unlikely that PGC-1α mediates the fate determination of interneurons. Interestingly, in the present study, decreased PV expression was limited to the cortex and hippocampus and was not observed in the cerebellum. Because PGC-1α does not directly bind to DNA, we speculate that the regional specificity of PV deficiency is due to differential expression of transcription factors activated by PGC-1α. Here, we investigated the cell-type-specific role of PGC-1α in the brain by targeting GABergic neurons. GABAergic neuron-specific PGC1α conditional knockout mice (Dlx5/6-Cre::PGC-1αfl/fl) exhibited hyperactivity, exaggerated startle reactivity, reduced prepulse inhibition, and impaired short-term habituation. We propose that this mouse phenotype represents a cause of aberrant salience and, in turn, that aberrant salience (and the resulting positive symptoms) in schizophrenia might arise, at least in part, from a deficit in short-term habituation. This abnormal behavior was accompanied by decreased PV expression in the cortex, including the somatosensory cortex and the motor cortex, as well as in the hippocampus, especially in its CA1 and CA3 regions. This proposal links an established risk gene with a psychological process central to psychosis and is supported by findings of a marked reduction in the density of parvalbumin interneurons in the frontal cortex of patients with schizophrenia (Kaar et al., 2019). Taken together, these findings reveal that PGC-1α plays a unique role in interneuron gene expression and function and suggest that disruption of PGC-1α expression could have profound consequences for interneuron function. Future studies are required to determine whether the reduction in PV is based on transcriptional inhibition or on loss of PV-expressing interneuron mediated by PGC-1α gene deletion.

2014b. What causes aberrant salience in schizophrenia? A role for impaired shortterm habituation and the GRIA1 (GluA1) AMPA receptor subunit. Mol. Psychiatry 19, 1060–1070. Bartos, M., Vida, I., Jonas, P., 2007. Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks, Nature reviews. Neuroscience 8, 45–56. Braff, D.L., Grillon, C., Geyer, M.A., 1992. Gating and habituation of the startle reflex in schizophrenic patients. Arch. Gen. Psychiatry 49, 206–215. Butt, S.J., Cobos, I., Golden, J., Kessaris, N., Pachnis, V., Anderson, S., 2007. Transcriptional regulation of cortical interneuron development. J. Neurosci. 27, 11847–11850. Caputi, A., Fuchs, E.C., Allen, K., Le Magueresse, C., Monyer, H., 2012. Selective reduction of AMPA currents onto hippocampal interneurons impairs network oscillatory activity. PLoS One 7. de Lombares, C., Heude, E., Alfama, G., Fontaine, A., Hassouna, R., Vernochet, C., de Chaumont, F., Olivo-Marin, C., Ey, E., Parnaudeau, S., Tronche, F., Bourgeron, T., Luquet, S., Levi, G., Narboux-Neme, N., 2019. Dlx5 and Dlx6 expression in GABAergic neurons controls behavior, metabolism, healthy aging and lifespan. Aging 11, 6638–6656. Dougherty, S.E., Bartley, A.F., Lucas, E.K., Hablitz, J.J., Dobrunz, L.E., Cowell, R.M., 2014. Mice lacking the transcriptional coactivator PGC-1alpha exhibit alterations in inhibitory synaptic transmission in the motor cortex. Neuroscience 271, 137–148. Freedman, R., Adler, L.E., Myles-Worsley, M., Nagamoto, H.T., Miller, C., Kisley, M., McRae, K., Cawthra, E., Waldo, M., 1996. Inhibitory gating of an evoked response to repeated auditory stimuli in schizophrenic and normal subjects. Human recordings, computer simulation, and an animal model. Arch. Gen. Psychiatry 53, 1114–1121. Geyer, M.A., Braff, D.L., 1982. Habituation of the Blink reflex in normals and schizophrenic patients. Psychophysiology 19, 1–6. Glass, R., Norton, S., Fox, N., Kusnecov, A.W., 2019. Maternal immune activation with staphylococcal enterotoxin A produces unique behavioral changes in C57BL/6 mouse offspring. Brain Behav. Immun. 75, 12–25. Heesink, L., Kleber, R., Hafner, M., van Bedaf, L., Eekhout, I., Geuze, E., 2017. Anger and aggression problems in veterans are associated with an increased acoustic startle reflex. Biol. Psychol. 123, 119–125. Kaar, S.J., Angelescu, I., Marques, T.R., Howes, O.D., 2019. Pre-frontal parvalbumin interneurons in schizophrenia: a meta-analysis of post-mortem studies. J. Neural Transm. Vienna (Vienna) 126, 1637–1651. Kapur, S., 2003. Psychosis as a state of aberrant salience: a framework linking biology, phenomenology, and pharmacology in schizophrenia. Am. J. Psychiatry 160, 13–23. Kim, H.Y., Yang, Y.R., Hwang, H., Lee, H.E., Jang, H.J., Kim, J., Yang, E., Kim, H., Rhim, H., Suh, P.G., Kim, J.I., 2019. Deletion of PLCgamma1 in GABAergic neurons increases seizure susceptibility in aged mice. Sci. Rep. 9, 17761. Leger, M., Quiedeville, A., Bouet, V., Haelewyn, B., Boulouard, M., Schumann-Bard, P., Freret, T., 2013. Object recognition test in mice. Nat. Protoc. 8, 2531–2537. Lewis, D.A., Curley, A.A., Glausier, J.R., Volk, D.W., 2012. Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci. 35, 57–67. Lin, J., Puigserver, P., Donovan, J., Tarr, P., Spiegelman, B.M., 2002. Peroxisome proliferator-activated receptor gamma coactivator 1beta (PGC-1beta), a novel PGC-1related transcription coactivator associated with host cell factor. J. Biol. Chem. 277, 1645–1648. Lin, J., Wu, P.H., Tarr, P.T., Lindenberg, K.S., St-Pierre, J., Zhang, C.Y., Mootha, V.K., Jager, S., Vianna, C.R., Reznick, R.M., Cui, L., Manieri, M., Donovan, M.X., Wu, Z., Cooper, M.P., Fan, M.C., Rohas, L.M., Zavacki, A.M., Cinti, S., Shulman, G.I., Lowell, B.B., Krainc, D., Spiegelman, B.M., 2004. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell 119, 121–135. Lin, J.D., Handschin, C., Spiegelman, B.M., 2005a. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 1, 361–370. Lin, J.D., Handschin, C., Spiegelman, B.M., 2005b. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 1, 361–370. Marin, O., 2012. Interneuron dysfunction in psychiatric disorders, Nature reviews. Neuroscience 13, 107–120. McMeekin, L.J., Lucas, E.K., Meador-Woodruff, J.H., McCullumsmith, R.E., Hendrickson, R.C., Gamble, K.L., Cowell, R.M., 2016. Cortical PGC-1alpha-Dependent transcripts are reduced in postmortem tissue from patients with schizophrenia. Schizophr. Bull. 42, 1009–1017. Okuda, S., Roozendaal, B., McGaugh, J.L., 2004. Glucocorticoid effects on object recognition memory require training-associated emotional arousal. Proc. Natl. Acad. Sci. U.S.A. 101, 853–858. Pouzet, B., Zhang, W.N., Feldon, J., Rawlins, J.N., 2002. Hippocampal lesioned rats are able to learn a spatial position using non-spatial strategies. Behav. Brain Res. 133, 279–291. Sanderson, D.J., Sprengel, R., Seeburg, P.H., Bannerman, D.M., 2011. Deletion of the GluA1 AMPA receptor subunit alters the expression of short-term memory. Learn. Mem. 18, 128–131. Singer, P., Hauser, J., Llano Lopez, L., Peleg-Raibstein, D., Feldon, J., Gargiulo, P.A., Yee, B.K., 2013. Prepulse inhibition predicts working memory performance whilst startle habituation predicts spatial reference memory retention in C57BL/6 mice. Behav. Brain Res. 242, 166–177. Spitzer, M., 1995. A neurocomputational approach to delusions. Compr. Psychiatry 36, 83–105. Tasic, B., Yao, Z.Z., Graybuck, L.T., Smith, K.A., Nguyen, T.N., Bertagnolli, D., Goldy, J., Garren, E., Economo, M.N., Viswanathan, S., Penn, O., Bakken, T., Menon, V., Miller, J., Fong, O., Hirokawa, K.E., Lathia, K., Rimorin, C., Tieu, M., Larsen, R., Casper, T., Barkan, E., Kroll, M., Parry, S., Shapovalova, N.V., Hirschstein, D., Pendergraft, J., Sullivan, H.A., Kim, T.K., Szafer, A., Dee, N., Groblewski, P., Wickersham, I., Cetin, A., Harris, J.A., Levi, B.P., Sunkin, S.M., Madisen, L., Daigle, T.L., Looger, L., Bernard, A., Phillips, J., Lein, E., Hawrylycz, M., Svoboda, K., Jones, A.R., Koch, C., Zeng, H.K.,

Author contributions Conceived and designed the experiments: JW WNZ. Performed the experiments: HRS MNG SFM QY. Analyzed the data: JW HRS. Wrote the paper: JW. Declaration of Competing Interest The authors have no conflict of interest to declare. Acknowledgements This work was supported by Clinic Development Fund of Jiangsu (BE2016724), Talent Project funding of Jiangsu Province (BRA2019171, LGY2017025), Zhenjiang Social Development Project (SH2019059) and National Natural Science Foundation of China (81671053). The language was edited by Elsevier Language Editing Services. References Agudelo, L.Z., Femenia, T., Orhan, F., Porsmyr-Palmertz, M., Goiny, M., MartinezRedondo, V., Correia, J.C., Izadi, M., Bhat, M., Schuppe-Koistinen, I., Pettersson, A.T., Ferreira, D.M.S., Krook, A., Barres, R., Zierath, J.R., Erhardt, S., Lindskog, M., Ruas, J.L., 2014. Skeletal muscle PGC-1alpha1 modulates kynurenine metabolism and mediates resilience to stress-induced depression. Cell 159, 33–45. Barkus, C., Sanderson, D.J., Rawlins, J.N.P., Walton, M.E., Harrison, P.J., Bannerman, D.M., 2014a. What causes aberrant salience in schizophrenia? A role for impaired short-term habituation and the GRIA1 (GluA1) AMPA receptor subunit. Mol Psychiatr 19, 1060–1070. Barkus, C., Sanderson, D.J., Rawlins, J.N., Walton, M.E., Harrison, P.J., Bannerman, D.M.,

138

Brain Research Bulletin 157 (2020) 128–139

J. Wang, et al.

mice display working memory impairment in the plus-shaped water maze. Neurosci. Lett. 407, 6–10. Wang, J., Yun, Q., Qian, J.J., Song, H.R., Wang, L., Inkabi, S.E., Xu, R.J., Hu, Y.M., Zhang, W.N., Einat, H., 2019. Mice lacking the transcriptional coactivator PGC-1alpha exhibit hyperactivity. Neuropsychobiology 1–7. Wonders, C.P., Anderson, S.A., 2006. The origin and specification of cortical interneurons. Nat. Rev. Neurosci. 7, 687–696.

2018. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563 72-+. Tremblay, R., Lee, S., Rudy, B., 2016. GABAergic interneurons in the neocortex: from cellular properties to circuits. Neuron 91, 260–292. Uhlhaas, P.J., Roux, F., Rodriguez, E., Rotarska-Jagiela, A., Singer, W., 2010. Neural synchrony and the development of cortical networks. Trends Cogn. Sci. (Regul. Ed.) 14, 72–80. Vloeberghs, E., Van Dam, D., D’Hooge, R., Staufenbiel, M., De Deyn, P.P., 2006. APP23

139