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Sensorimotor gating, working and social memory deficits in mice with reduced expression of the vesicular glutamate transporter VGLUT1
Behavioural Brain Research 228 (2012) 328–332
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Research report
Sensorimotor gating, working and social memory deficits in mice with reduced expression of the vesicular glutamate transporter VGLUT1 Dragos Inta a,∗,1 , Miriam A. Vogt a,1 , Stephanie Perreau-Lenz b , Miriam Schneider b , Natascha Pfeiffer a , Sonja M. Wojcik c , Rainer Spanagel b , Peter Gass a a Department for Psychiatry and Psychotherapy, RG Animal Models in Psychiatry, Central Institute of Mental Health Mannheim, Medical Faculty Mannheim/Heidelberg University, J5, 68159 Mannheim, Germany b Institute for Psychopharmacology, Central Institute of Mental Health Mannheim, Medical Faculty Mannheim/Heidelberg University, J5, 68159 Mannheim, Germany c Department of Molecular Neurobiology, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany
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Article history: Received 24 August 2011 Received in revised form 2 December 2011 Accepted 6 December 2011 Available online 16 December 2011 Keywords: Glutamate Vesicular transporter VGLUT1 Schizophrenia Mice
a b s t r a c t Glutamate is the main excitatory neurotransmitter in the central nervous system. A hypoglutamatergic state is believed to play an important role in the pathophysiology of schizophrenia. The release of glutamate in the brain is modulated by a class of vesicular glutamate transporters, VGLUT1–3. Among them, VGLUT1 represents the isoform predominantly expressed in the neocortex and hippocampus. Here we investigated the potential involvement of VGLUT1 deficiency in generating schizophrenia-like abnormalities by testing mice with diminished expression of VGLUT1 in several behavioural tests relevant for schizophrenia. We found behavioural alterations in these mice resembling correlates of schizophrenia, such as working- and social memory impairments and deficits in prepulse inhibition (PPI) of the acoustic startle reflex (ASR), but normal locomotor behaviour under basal conditions. Our data may be important for a better understanding of the contribution of reduced VGLUT1-mediated presynaptic glutamatergic neurotransmission in the generation of several behavioural abnormalities associated with schizophrenia. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Vesicular packaging and release of glutamate is a major step in excitatory neurotransmission in the brain, controlled by a family of three different subtypes of vesicular glutamate transporters (named VGLUT1–3) [33]. These molecules accumulate glutamate in presynaptic vesicles before its release. All three isoforms show strong structural and functional similarities and a largely complementary expression pattern with minimal overlap in the brain. VGLUT1 is localized in the neocortex, hippocampus and amygdala, whereas VGLUT2 predominates in the thalamus, hypothalamus and brain stem [13,14]. VGLUT2 is present only in layer IV of the cortex and in the granular layer of the dentate gyrus, areas devoid of VGLUT1 [15]. The third isoform, VGLUT3, does not co-localize with VGLUT1 or VGLUT2 [7], being expressed preferentially in cholinergic and serotonergic, as well as in GABAergic neurons [17,21]. At the functional level, expression of VGLUT1 and VGLUT2 has been correlated with distinct synaptic properties, VGLUT1 is
∗ Corresponding author. Tel.: +49 0 621 1703 2932; fax: +49 0 621 1703 6205. E-mail address: [email protected] (D. Inta). 1 These authors contributed equally to this work. 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.12.012
expressed mainly in glutamatergic synapses associated with low release probability, which exhibit long-term potentiation (LTP), whereas VGLUT2 is present in synapses with high release probability, associated with long-term depression (LTD) [13]. VGLUT2 is expressed at high levels during development, whereas VGLUT1 is present at low levels at birth and strongly increases postnatally, becoming the predominant isoform in the adult cortex [28]. The crucial importance of these transporters is revealed by the fact that in mice, the conventional knockout of either VGLUT1 or VGLUT2 is lethal: VGLUT2 knockout (KO) mice die immediately after birth, due to respiratory failure [29], whereas VGLUT1 KO mice show after the third postnatal week a progressive neurological phenotype, with incoordination, blindness and enhanced startle response [14,38]. Therefore, only heterozygous mice lacking these VGLUTs are suitable for behavioural analysis. Protein levels of VGLUT1 in heterozygous mice (VGLUT1+/−) are reduced by 40–60% compared to wild-type levels [35], and VGLUT1+/− mice show a reduction in the reserve pool of synaptic vesicles and an impairment in hippocampal LTP and learning [3,35]. Decreased expression of VGLUT1 in these mice is accompanied by reduced levels of GABA in the frontal cortex and hippocampus, enhanced anxiety, a depressivelike behaviour [35] and a higher vulnerability to stress [16]. In contrast, VGLUT2+/− mice have normal locomotor and fear-related
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behaviour, and show no social interaction abnormalities, as well as no learning and memory deficits [29]. Ablation of VGLUT3 results in a hypocholinergic striatal [18] and an anxiety-like phenotype [1]. Taken together, these data reveal the specific roles of different isoforms of VGLUT in specific brain functions and in several psychiatric disorders such as anxiety and depression. Schizophrenia is also one of the psychiatric diseases where glutamatergic dysfunction is thought to play an important role [27,36]. The glutamatergic hypothesis of schizophrenia emerged from clinical studies demonstrating a psychotogenic effect of NMDA receptor antagonists in humans [25] and the worsening of psychotic symptoms in schizophrenic patients [26]. Meanwhile, a large number of mouse models with mutations in several subtypes of glutamate receptors provided useful insight into the different components of the glutamatergic system that are thought to be implicated in the generation of schizophrenia-like abnormalities [22]. However, whereas the role of postsynaptic deficits in glutamatergic transmission for inducing schizophrenia-like abnormalities is welldocumented, the influence of presynaptic glutamatergic deficits is less well understood. In particular, the contribution of VGLUT1 deficiency in generating schizophrenia-like abnormalities has not been yet established. It appears particularly important to clarify this aspect, considering that VGLUT1 is the main vesicular glutamate transporter in cortical areas, including those associated with alterations seen in schizophrenia (e.g. prefrontal cortex, hippocampus) [15]. This motivated us to use a standard battery of behavioural tests to reveal possible schizophrenia-like alterations in VGLUT1+/− mice. 2. Materials and methods 2.1. Animals All experiments were performed in approximately 3-month-old male VGLUT1 heterozygous mice and wildtype littermate controls on a C57BL/6N background (>10 generations backcrossing to C57BL/6N background) [38]. Heterozygous mice showed no apparent phenotypic abnormalities during development and adulthood. The animals were bred and maintained in the animal facility of the Central Institute of Mental Health, Mannheim. Mice were supplied with food and tap water ad libitum. All experiments were approved by German animal welfare authorities (Regierungspräsidium Karlsruhe). 2.2. Behavioural testing The animals were subjected to behavioural test paradigms relevant to schizophrenia including the Open Field test, prepulse inhibition (PPI) of the acoustic startle reflex (ASR), a test of sociability and social memory and T-Maze as a test for working memory (n = 11–13 per genotype). All behavioural experiments were performed during the dark phase after 2 weeks of acclimation to a reversed dark–light cycle (12 h/12 h, lights on at 7 pm, kept during the whole phase of testing). Prior to each test, mice were acclimated to the experimental room for at least 30 min. Bodyweight was assessed weekly when the cages were changed. 2.2.1. Open Field Exploration and activity monitoring was performed in a square, white Open Field, measuring 50 cm × 50 cm and illuminated from above by 25 lux. Mice were placed individually into the arena and monitored for 10 min by a Video camera (Sony CCD IRIS). The resulting data were analysed using the image processing system EthoVision 3.1 (Noldus Information Technology, Wageningen, The Netherlands). For each sample, the system recorded position and the occurrence of defined events. Parameters assessed in the present study were total distance moved, velocity, and thigmotaxis (i.e. the percentage of time spent in a corridor with a maximal distance of 10 cm to the walls).
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preference of the new arm was determined (ratio (time or entries) new vs. old minus 1) to circumvent possible activity differences masking the results. Light intensity was 25 lux.
2.2.3. Startle reaction and sensorimotor gating measured by PPI PPI is a measure of sensorimotor gating both in humans and animals, such that a weak pre-stimulus (prepulse) attenuates the startle response to a loud noise presented immediately after. Deficient PPI is a measure of the loss of sensorimotor gating, leading to sensory flooding and cognitive fragmentation in schizophrenia patients. The ASR and PPI testing occurred in a startle chamber (SR-LAB; San Diego Instruments, San Diego, USA), in which a loudspeaker inside the box produced a continuous background noise of 60 dB of sound pressure level (SPL) as well as the acoustic startle pulses. A white noise pulse was used as the startle stimulus, which had an intensity of 115 dB SPL and duration of 40 ms; four different white noise intensities (65, 70, 75 and 80 dB SPL, duration 20 ms) were used as prepulses. An acclimatization time of 5 min, during which the mice received no stimulus except the background noise, was followed by the presentation of 5 initial startle stimuli. After this habituation program the test program was started with six different trial types presented in a pseudorandom order: (1) pulse alone (assessing ASR), (2) control (no stimulus), (3) pulse with preceding prepulse (prepulse 65 dB, 100 ms before pulse), (4) pulse with preceding prepulse (prepulse 70 dB, 100 ms before pulse), (5) pulse with preceding prepulse (75 dB, 100 ms before pulse) and (6) pulse with preceding prepulse (prepulse 80 dB, 100 ms before pulse). A total of 10 presentations of each trial type were given with an interstimulus interval randomized between 10 s and 20 s. PPI was calculated as the percent decrease of the ASR magnitude in trials when the startle stimulus was preceded by a prepulse [100 × (mean ASR amplitude on pulse alone trials − mean ASR amplitude on prepulse-pulse trials)/mean ASR amplitude on pulse alone trials].
2.2.4. Sociability and social memory test The sociability and social memory test was carried out as described by Jamain et al. [23]. The social testing arena was a rectangular, three-chambered box. Each chamber was 20 × 30 × 30 cm3 in size. Dividing walls were made of clear Plexiglas, with rectangular openings (5 × 6 cm2 ) allowing access into each chamber. The test mouse was first placed in the middle chamber and allowed to explore. The openings into the two side chambers were obstructed by Plexiglas panels during this first habituation phase. After 5 min, the doors were opened and the animal was allowed to explore the 2 outer compartments (equipped with one empty wire cage in each compartment, size 7 × 7 × 8 cm3 ). After the habituation period, an unfamiliar C57BL/6N male mouse (“stranger 1”) that had no prior contact with the subject mouse was placed in one of the side chambers. The location of stranger 1 in the left vs. right side chamber was systematically alternated between trials. The stranger mouse was enclosed in a small wire cage that allowed nose contact through the bars but prevented fighting. The animals serving as strangers had previously been habituated to placement in the small cage. A new identical empty wire cage was placed in the opposite chamber. Openings to the side chambers were then unblocked and the subject mouse was allowed to explore the entire social test arena for a 10 min session (“Sociability”). For the social memory test, the empty wire cage was exchanged with a new unfamiliar male C57BL/6N mouse (“stranger 2”) and the subject mouse was again allowed to explore the arena for another 10 min. The amount of time spent in each chamber and the number of entries into each chamber during both sessions were measured. An entry was defined as all four paws in one chamber. The percentage of time in each side chamber in comparison to the time spent in the both chambers was analysed.
2.3. Statistical analysis Statistical analysis was performed using the statistical program PASW 18 for Windows. Inter-group comparisons were calculated by Student’s t-tests. Where appropriate, the analysis was complemented by within subject factors to explore the dependence of treatment effects on time (e.g. bodyweight development). The significance threshold was set at p < 0.05.
3. Results 2.2.2. T-Maze The T-Maze test is a spatial working memory paradigm, in which the animals’ ability to recognize and differentiate between a new unknown and a familiar compartment is analysed. The T-shaped maze was made of white plastic with two 20 cm long arms, which extended at a right angle from a 40 cm long alley. The arms and the alley had a width of 10 cm and were surrounded by 25 cm high walls. The test consisted of two trials with an intertrial interval of 1 h, during which the animals were returned to their home cage. During an 8 min acquisition trial, one of the short arms was closed. In a second 3 min retention trial, mice had access to all three arms. Number of visits and time spent in either of short arms were assessed and the
3.1. General observations VGLUT1+/− mice did not display altered home cage behaviour or bodyweight differences in comparison to wild-type mice (data not shown). All mice significantly gained weight during the testing phase (repeated measurement ANOVA: factor time: F(4,96) = 94.396, p = 0.000).
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Fig. 1. Locomotor behaviour of VGLUT1+/− mice and littermate controls in the Open Field. VGLUT1+/− and control mice exhibit comparable locomotor activity as demonstrated by (a) total distance moved (cm), (b) velocity (cm/s) and (c) time in the centre (%). n = 13 per genotype. All data are means + SEM.
3.2. VGLUT1+/− mice display regular locomotor behaviour in the Open Field test We found no significant difference between VGLUT1+/− mice and littermate controls in total distance moved (Fig. 1A), velocity (Fig. 1B), distance to the walls (data not shown) and time spent in the centre of the Open Field (Fig. 1C). 3.3. VGLUT1+/− mice show sensorimotor gating and startle reaction alterations VGLUT1+/− mice vs. littermate controls showed a significantly enhanced amplitude of the ASR (p < 0.001) (Fig. 2A). Regarding PPI, VGLUT1+/− mice demonstrated significant PPI deficits at all prepulse intensities applied (prepulse intensity: 65 dB: p < 0.05; 70 dB: p < 0.05; 75 dB: p < 0.01; 80 dB: p < 0.01) (Fig. 2B). 3.4. VGLUT1+/− mice show a working memory deficit in the T-Maze test Working memory, analysed by the ability to recognize (and prefer) a new, unfamiliar arm of the T-Maze in contrast to a familiar arm, already explored 1 h earlier, was altered in VGLUT1 heterozygous mice. VGLUT1+/− mice showed a significantly reduced preference to enter the new unfamiliar arm compared to their littermate controls (p < 0.05). However, they stayed in the new arm as long as the control mice (Fig. 3). 3.5. Sociability and social memory in VGLUT1+/− mice Sociability and social memory tests in mice are established methods to measure interactional deficits that may correspond to social withdrawal in schizophrenia patients. VGLUT1 heterozygous mice showed no statistically significant difference in sociability, preferring to a comparable extent as control mice the contact with an unfamiliar mouse (“stranger 1”) in comparison to an empty wire cage (paired Student’s t-test “stranger 1” vs. empty cage: VGLUT1: p < 0.05, controls: p < 0.01) (Fig. 4A). In contrast, VGLUT1+/− mice showed a distinct phenotype in the social memory session: they did not significantly differentiate between strangers 1 and 2, in contrast to their littermate controls which clearly preferred the unfamiliar stranger 2 (paired Student’s t-test “stranger 1” vs. “stranger 2” controls: p < 0.05) (Fig. 4B). 4. Discussion Here we show that mice with reduced expression of the VGLUT1 transporter exhibit several behavioural abnormalities associated with schizophrenia, such as abnormal working and social memory
and sensorimotor gating deficits. These data suggest a potential contribution of a deficient cortical presynaptic glutamatergic transmission mediated by VGLUT1, in the generation of several abnormalities associated with schizophrenia. Our data may be of relevance for humans, since several postmortem studies indicated a direct connection between VGLUT1 and schizophrenia. Reduced VGLUT1 mRNA expression in the prefrontal cortex (PFC) [10] and VGLUT1 protein levels in the anterior cingulate cortex [31] were reported in patients with schizophrenia. A strong decrease, by 79%, in the density of VGLUT1-immunoreactive axonal boutons, selectively in layer 3 of the PFC has been found in post-mortem tissue from schizophrenia patients [5]. Other studies reported negative results with regard to a direct involvement of VGLUT1 in schizophrenia [11,30]. The different outcomes of these studies in comparison to those revealing a positive correlation between VGLUT1 and schizophrenia (the latter done mostly in older schizophrenic patients) [11,30], may suggest an accelerated age-dependent decrease in VGLUT1 in schizophrenia [11]. Harrison et al. proposed that VGLUT1-mediated presynaptic glutamatergic abnormalities may be progressive in schizophrenia, worsening with aging and leading to persistent cognitive alterations associated with the disease [19] Cognitive deficits are considered core features of schizophrenia [2]. Previous studies already investigated VGLUT1+/− mice in several learning assays, reporting deficits in spatial reversal learning and impaired hippocampal LTP [3] and recognition memory [35]. In our analysis of VGLUT1+/− mice, we observed cognitive alterations as assessed in the T-Maze test. Additionally, we identified significant social memory abnormalities. The novelty discrimination in a social context constitutes a valuable tool for revealing attentional deficits used in several animal models of schizophrenia [12,34]. Therefore, this result appears particularly relevant to the disease, since a selective attention deficit, the inability to differentiate relevant from irrelevant information, is considered to underlie many cognitive deficits of schizophrenia [4]. Taken together, these data point out the importance of presynaptic mechanisms controlling glutamate release in inducing cognitive alterations associated with schizophrenia. In addition to abnormalities in working and social memory, VGLUT1+/− mice displayed sensorimotor gating deficits. PPI of the startle reflex is an established measure of sensorimotor gating that is reduced in schizophrenia patients [8]. The PPI deficit in VGLUT1+/− mice appears particularly relevant in the context of the high expression of VGLUT1 in the hippocampus and the known modulator role of the hippocampus on PPI [9]. NMDA receptors in the hippocampus, especially in its ventral part, as well as glutamatergic projections from the hippocampus to the nucleus accumbens, play a critical role in the regulation of PPI [24]. Our data suggest a regulatory role not only of the postsynaptic, but also
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Fig. 2. Acoustic startle response and prepulse inhibition in VGLUT1+/− mice and littermate controls. VGLUT1+/− mice show (a) an enhanced acoustic startle response and (b) deficits in prepulse inhibition (%). n = 11 per genotype. All data are means + SEM. Student’s t-test: *p < 0.05; **p > 0.01; ***p < 0.001.
Fig. 3. Preference of the new unfamiliar arm in the T-Maze test. VGLUT1+/− mice preferred (a) significantly less to enter the new arm but (b) spent comparable time in the new arm. n = 12–13 per genotype. All data are means + SEM. Student’s t-test: *p < 0.05.
of the presynaptic glutamatergic transmission of PPI. In addition to glutamatergic disturbances, the low GABA level in the frontal cortex and hippocampus of VGLUT1+/− mice [16,35] may also be implicated in the generation of sensorimotor gating abnormalities, since GAD65 KO mice show also a PPI deficit [20]. Of note, we found in VGLUT1+/− mice enhanced amplitude of the ASR, as in homozygous VGLUT1 KO mice [14]. A possible explanation for this increase is the enhanced anxiety of VGLUT1+/− mice [35] known to enhance the amplitude of the ASR [24]. Interestingly, some clinical studies reported as well increased amplitude of the ASR in schizophrenic patients when compared to normal subjects [6,32]. We did not find alterations in locomotor behaviour in the VGLUT1+/− mice under basal conditions. Of note, hyperlocomotion in the Open Field is considered a behavioural correlate for positive symptoms of schizophrenia [37]. Nevertheless, it was previously shown that VGLUT1+/− mice displayed an increase in the
total distance travelled after chronic mild stress [16]. This suggests that although locomotion is not affected in VGLUT1+/− mice under basal conditions, certain vulnerability towards developing schizophrenia-like abnormalities may exist in these mice after prolonged exposure to stress [16]. Interestingly, in contrast to the inconspicuous phenotype of VGLUT2+/− mice, inducible, restricted deletion of VGLUT2 in the cortex, hippocampus and amygdala of preadolescent mice induces several behavioural abnormalities associated with schizophrenia (hyperlocomotion, cognitive dysfunctions, altered social behaviour, decreased sensorimotor gating) [39]. Taking into account the clear predominance of VGLUT1 over VGLUT2 in the cortex and hippocampus, these data strongly encourage future studies with inducible VGLUT1 KO mice during adolescence/adulthood (to overcome perinatal lethality) for analysing the possible role of VGLUT1 in the outbreak and chronification of schizophrenia.
Fig. 4. Sociability and social memory in VGLUT1+/− mice and littermate controls. VGLUT1+/− mice and littermate controls (a) both spent significantly more time in the compartment with stranger 1 in comparison with the compartment with an empty wire cage. (b) VGLUT1+/− mice did not prefer the new unfamiliar stranger 2 in the social memory test. n = 12 per genotype. All data are means + SEM. Paired Student’s t-test [(a) comparison between the compartment with stranger 1 and empty wire cage (a) and respectively with stranger 1/stranger 2 (b)] *p < 0.05; **p > 0.01.
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In conclusion, our data indicate that VGLUT1 may play an important role in some abnormalities associated with schizophrenia, particularly cognitive deficits. The understanding of the mechanisms underlying these disturbances appears important, since especially cognitive abnormalities show very limited response to current antipsychotic treatment. Future studies, using mice with selective deletion of VGLUT1 in specific brain areas, to avoid the lethality of the full knockout and in the same time providing a more restricted region-specific ablation of VGLUT1 may provide further useful insight in the role of this transporter in generating schizophrenia-like abnormalities. Conflict of interest statement None to declare. Acknowledgements We thank Andreas Meyer-Lindenberg from the Central Institute for Mental Health Mannheim, University of Heidelberg, for critically reading the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GA 427/11-1 to D.I. and P.G.). References [1] Amilhon B, Lepicard E, Renoir T, Mongeau R, Popa D, Poirel O, et al. VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety. J Neurosci 2010;30:2198–210. [2] Arguello PA, Gogos JA. Cognition in mouse models of schizophrenia susceptibility genes. Schizophr Bull 2010;36:289–300. [3] Balschun D, Moechars D, Callaerts-Vegh Z, Vermaercke B, Van Acker N, Andries L, et al. Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation and spatial reversal learning. Cereb Cortex 2010;20: 684–93. [4] Barch DM, Carter CS, Hachten PC, Usher M, Cohen JD. The “benefits” of distractibility: mechanisms underlying increased Stroop effects in schizophrenia. Schizophr Bull 1999;25:749–62. [5] Bitanihirwe BK, Lim MP, Kelley JF, Kaneko T, Woo TU. Glutamatergic deficits and parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia. BMC Psychiatry 2009;9:71. [6] Bolino F, Manna V, Di Cicco L, Di Michele V, Daneluzzo E, Rossi A, et al. Startle reflex habituation in functional psychoses: a controlled study. Neurosci Lett 1992;145:126–8. [7] Boulland JL, Qureshi T, Seal RP, Rafiki A, Gundersen V, Bergersen LH, et al. Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters. J Comp Neurol 2004;480:264–80. [8] Braff DL, Geyer MA. Sensorimotor gating and schizophrenia. Human and animal model studies. Arch Gen Psychiatry 1990;47:181. [9] Caine SB, Geyer MA, Swerdlow NR. Hippocampal modulation of acoustic startle and prepulse inhibition in the rat. Pharmacol Biochem Behav 1992;43:1201–8. [10] Eastwood SL, Harrison PJ. Decreased expression of vesicular glutamate transporter 1 and complexin II mRNAs in schizophrenia: further evidence for a synaptic pathology affecting glutamate neurons. Schizophr Res 2005;73:159–72. [11] Eastwood SL, Harrison PJ. Markers of glutamate synaptic transmission and plasticity are increased in the anterior cingulate cortex in bipolar disorder. Biol Psychiatry 2010;67:1010–6. [12] Feifel D, Mexal S, Melendez G, Liu PY, Goldenberg JR, Shilling PD. The brattleboro rat displays a natural deficit in social discrimination that is restored by clozapine and a neurotensin analog. Neuropsychopharmacology 2009;34: 2011–8. [13] Fremeau Jr RT, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 2001;31:247–60. [14] Fremeau Jr RT, Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm-Mathisen J, et al. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 2004;304:1815–9.
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Research report
Sensorimotor gating, working and social memory deficits in mice with reduced expression of the vesicular glutamate transporter VGLUT1 Dragos Inta a,∗,1 , Miriam A. Vogt a,1 , Stephanie Perreau-Lenz b , Miriam Schneider b , Natascha Pfeiffer a , Sonja M. Wojcik c , Rainer Spanagel b , Peter Gass a a Department for Psychiatry and Psychotherapy, RG Animal Models in Psychiatry, Central Institute of Mental Health Mannheim, Medical Faculty Mannheim/Heidelberg University, J5, 68159 Mannheim, Germany b Institute for Psychopharmacology, Central Institute of Mental Health Mannheim, Medical Faculty Mannheim/Heidelberg University, J5, 68159 Mannheim, Germany c Department of Molecular Neurobiology, Max-Planck Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany
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Article history: Received 24 August 2011 Received in revised form 2 December 2011 Accepted 6 December 2011 Available online 16 December 2011 Keywords: Glutamate Vesicular transporter VGLUT1 Schizophrenia Mice
a b s t r a c t Glutamate is the main excitatory neurotransmitter in the central nervous system. A hypoglutamatergic state is believed to play an important role in the pathophysiology of schizophrenia. The release of glutamate in the brain is modulated by a class of vesicular glutamate transporters, VGLUT1–3. Among them, VGLUT1 represents the isoform predominantly expressed in the neocortex and hippocampus. Here we investigated the potential involvement of VGLUT1 deficiency in generating schizophrenia-like abnormalities by testing mice with diminished expression of VGLUT1 in several behavioural tests relevant for schizophrenia. We found behavioural alterations in these mice resembling correlates of schizophrenia, such as working- and social memory impairments and deficits in prepulse inhibition (PPI) of the acoustic startle reflex (ASR), but normal locomotor behaviour under basal conditions. Our data may be important for a better understanding of the contribution of reduced VGLUT1-mediated presynaptic glutamatergic neurotransmission in the generation of several behavioural abnormalities associated with schizophrenia. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Vesicular packaging and release of glutamate is a major step in excitatory neurotransmission in the brain, controlled by a family of three different subtypes of vesicular glutamate transporters (named VGLUT1–3) [33]. These molecules accumulate glutamate in presynaptic vesicles before its release. All three isoforms show strong structural and functional similarities and a largely complementary expression pattern with minimal overlap in the brain. VGLUT1 is localized in the neocortex, hippocampus and amygdala, whereas VGLUT2 predominates in the thalamus, hypothalamus and brain stem [13,14]. VGLUT2 is present only in layer IV of the cortex and in the granular layer of the dentate gyrus, areas devoid of VGLUT1 [15]. The third isoform, VGLUT3, does not co-localize with VGLUT1 or VGLUT2 [7], being expressed preferentially in cholinergic and serotonergic, as well as in GABAergic neurons [17,21]. At the functional level, expression of VGLUT1 and VGLUT2 has been correlated with distinct synaptic properties, VGLUT1 is
∗ Corresponding author. Tel.: +49 0 621 1703 2932; fax: +49 0 621 1703 6205. E-mail address: [email protected] (D. Inta). 1 These authors contributed equally to this work. 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.12.012
expressed mainly in glutamatergic synapses associated with low release probability, which exhibit long-term potentiation (LTP), whereas VGLUT2 is present in synapses with high release probability, associated with long-term depression (LTD) [13]. VGLUT2 is expressed at high levels during development, whereas VGLUT1 is present at low levels at birth and strongly increases postnatally, becoming the predominant isoform in the adult cortex [28]. The crucial importance of these transporters is revealed by the fact that in mice, the conventional knockout of either VGLUT1 or VGLUT2 is lethal: VGLUT2 knockout (KO) mice die immediately after birth, due to respiratory failure [29], whereas VGLUT1 KO mice show after the third postnatal week a progressive neurological phenotype, with incoordination, blindness and enhanced startle response [14,38]. Therefore, only heterozygous mice lacking these VGLUTs are suitable for behavioural analysis. Protein levels of VGLUT1 in heterozygous mice (VGLUT1+/−) are reduced by 40–60% compared to wild-type levels [35], and VGLUT1+/− mice show a reduction in the reserve pool of synaptic vesicles and an impairment in hippocampal LTP and learning [3,35]. Decreased expression of VGLUT1 in these mice is accompanied by reduced levels of GABA in the frontal cortex and hippocampus, enhanced anxiety, a depressivelike behaviour [35] and a higher vulnerability to stress [16]. In contrast, VGLUT2+/− mice have normal locomotor and fear-related
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behaviour, and show no social interaction abnormalities, as well as no learning and memory deficits [29]. Ablation of VGLUT3 results in a hypocholinergic striatal [18] and an anxiety-like phenotype [1]. Taken together, these data reveal the specific roles of different isoforms of VGLUT in specific brain functions and in several psychiatric disorders such as anxiety and depression. Schizophrenia is also one of the psychiatric diseases where glutamatergic dysfunction is thought to play an important role [27,36]. The glutamatergic hypothesis of schizophrenia emerged from clinical studies demonstrating a psychotogenic effect of NMDA receptor antagonists in humans [25] and the worsening of psychotic symptoms in schizophrenic patients [26]. Meanwhile, a large number of mouse models with mutations in several subtypes of glutamate receptors provided useful insight into the different components of the glutamatergic system that are thought to be implicated in the generation of schizophrenia-like abnormalities [22]. However, whereas the role of postsynaptic deficits in glutamatergic transmission for inducing schizophrenia-like abnormalities is welldocumented, the influence of presynaptic glutamatergic deficits is less well understood. In particular, the contribution of VGLUT1 deficiency in generating schizophrenia-like abnormalities has not been yet established. It appears particularly important to clarify this aspect, considering that VGLUT1 is the main vesicular glutamate transporter in cortical areas, including those associated with alterations seen in schizophrenia (e.g. prefrontal cortex, hippocampus) [15]. This motivated us to use a standard battery of behavioural tests to reveal possible schizophrenia-like alterations in VGLUT1+/− mice. 2. Materials and methods 2.1. Animals All experiments were performed in approximately 3-month-old male VGLUT1 heterozygous mice and wildtype littermate controls on a C57BL/6N background (>10 generations backcrossing to C57BL/6N background) [38]. Heterozygous mice showed no apparent phenotypic abnormalities during development and adulthood. The animals were bred and maintained in the animal facility of the Central Institute of Mental Health, Mannheim. Mice were supplied with food and tap water ad libitum. All experiments were approved by German animal welfare authorities (Regierungspräsidium Karlsruhe). 2.2. Behavioural testing The animals were subjected to behavioural test paradigms relevant to schizophrenia including the Open Field test, prepulse inhibition (PPI) of the acoustic startle reflex (ASR), a test of sociability and social memory and T-Maze as a test for working memory (n = 11–13 per genotype). All behavioural experiments were performed during the dark phase after 2 weeks of acclimation to a reversed dark–light cycle (12 h/12 h, lights on at 7 pm, kept during the whole phase of testing). Prior to each test, mice were acclimated to the experimental room for at least 30 min. Bodyweight was assessed weekly when the cages were changed. 2.2.1. Open Field Exploration and activity monitoring was performed in a square, white Open Field, measuring 50 cm × 50 cm and illuminated from above by 25 lux. Mice were placed individually into the arena and monitored for 10 min by a Video camera (Sony CCD IRIS). The resulting data were analysed using the image processing system EthoVision 3.1 (Noldus Information Technology, Wageningen, The Netherlands). For each sample, the system recorded position and the occurrence of defined events. Parameters assessed in the present study were total distance moved, velocity, and thigmotaxis (i.e. the percentage of time spent in a corridor with a maximal distance of 10 cm to the walls).
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preference of the new arm was determined (ratio (time or entries) new vs. old minus 1) to circumvent possible activity differences masking the results. Light intensity was 25 lux.
2.2.3. Startle reaction and sensorimotor gating measured by PPI PPI is a measure of sensorimotor gating both in humans and animals, such that a weak pre-stimulus (prepulse) attenuates the startle response to a loud noise presented immediately after. Deficient PPI is a measure of the loss of sensorimotor gating, leading to sensory flooding and cognitive fragmentation in schizophrenia patients. The ASR and PPI testing occurred in a startle chamber (SR-LAB; San Diego Instruments, San Diego, USA), in which a loudspeaker inside the box produced a continuous background noise of 60 dB of sound pressure level (SPL) as well as the acoustic startle pulses. A white noise pulse was used as the startle stimulus, which had an intensity of 115 dB SPL and duration of 40 ms; four different white noise intensities (65, 70, 75 and 80 dB SPL, duration 20 ms) were used as prepulses. An acclimatization time of 5 min, during which the mice received no stimulus except the background noise, was followed by the presentation of 5 initial startle stimuli. After this habituation program the test program was started with six different trial types presented in a pseudorandom order: (1) pulse alone (assessing ASR), (2) control (no stimulus), (3) pulse with preceding prepulse (prepulse 65 dB, 100 ms before pulse), (4) pulse with preceding prepulse (prepulse 70 dB, 100 ms before pulse), (5) pulse with preceding prepulse (75 dB, 100 ms before pulse) and (6) pulse with preceding prepulse (prepulse 80 dB, 100 ms before pulse). A total of 10 presentations of each trial type were given with an interstimulus interval randomized between 10 s and 20 s. PPI was calculated as the percent decrease of the ASR magnitude in trials when the startle stimulus was preceded by a prepulse [100 × (mean ASR amplitude on pulse alone trials − mean ASR amplitude on prepulse-pulse trials)/mean ASR amplitude on pulse alone trials].
2.2.4. Sociability and social memory test The sociability and social memory test was carried out as described by Jamain et al. [23]. The social testing arena was a rectangular, three-chambered box. Each chamber was 20 × 30 × 30 cm3 in size. Dividing walls were made of clear Plexiglas, with rectangular openings (5 × 6 cm2 ) allowing access into each chamber. The test mouse was first placed in the middle chamber and allowed to explore. The openings into the two side chambers were obstructed by Plexiglas panels during this first habituation phase. After 5 min, the doors were opened and the animal was allowed to explore the 2 outer compartments (equipped with one empty wire cage in each compartment, size 7 × 7 × 8 cm3 ). After the habituation period, an unfamiliar C57BL/6N male mouse (“stranger 1”) that had no prior contact with the subject mouse was placed in one of the side chambers. The location of stranger 1 in the left vs. right side chamber was systematically alternated between trials. The stranger mouse was enclosed in a small wire cage that allowed nose contact through the bars but prevented fighting. The animals serving as strangers had previously been habituated to placement in the small cage. A new identical empty wire cage was placed in the opposite chamber. Openings to the side chambers were then unblocked and the subject mouse was allowed to explore the entire social test arena for a 10 min session (“Sociability”). For the social memory test, the empty wire cage was exchanged with a new unfamiliar male C57BL/6N mouse (“stranger 2”) and the subject mouse was again allowed to explore the arena for another 10 min. The amount of time spent in each chamber and the number of entries into each chamber during both sessions were measured. An entry was defined as all four paws in one chamber. The percentage of time in each side chamber in comparison to the time spent in the both chambers was analysed.
2.3. Statistical analysis Statistical analysis was performed using the statistical program PASW 18 for Windows. Inter-group comparisons were calculated by Student’s t-tests. Where appropriate, the analysis was complemented by within subject factors to explore the dependence of treatment effects on time (e.g. bodyweight development). The significance threshold was set at p < 0.05.
3. Results 2.2.2. T-Maze The T-Maze test is a spatial working memory paradigm, in which the animals’ ability to recognize and differentiate between a new unknown and a familiar compartment is analysed. The T-shaped maze was made of white plastic with two 20 cm long arms, which extended at a right angle from a 40 cm long alley. The arms and the alley had a width of 10 cm and were surrounded by 25 cm high walls. The test consisted of two trials with an intertrial interval of 1 h, during which the animals were returned to their home cage. During an 8 min acquisition trial, one of the short arms was closed. In a second 3 min retention trial, mice had access to all three arms. Number of visits and time spent in either of short arms were assessed and the
3.1. General observations VGLUT1+/− mice did not display altered home cage behaviour or bodyweight differences in comparison to wild-type mice (data not shown). All mice significantly gained weight during the testing phase (repeated measurement ANOVA: factor time: F(4,96) = 94.396, p = 0.000).
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Fig. 1. Locomotor behaviour of VGLUT1+/− mice and littermate controls in the Open Field. VGLUT1+/− and control mice exhibit comparable locomotor activity as demonstrated by (a) total distance moved (cm), (b) velocity (cm/s) and (c) time in the centre (%). n = 13 per genotype. All data are means + SEM.
3.2. VGLUT1+/− mice display regular locomotor behaviour in the Open Field test We found no significant difference between VGLUT1+/− mice and littermate controls in total distance moved (Fig. 1A), velocity (Fig. 1B), distance to the walls (data not shown) and time spent in the centre of the Open Field (Fig. 1C). 3.3. VGLUT1+/− mice show sensorimotor gating and startle reaction alterations VGLUT1+/− mice vs. littermate controls showed a significantly enhanced amplitude of the ASR (p < 0.001) (Fig. 2A). Regarding PPI, VGLUT1+/− mice demonstrated significant PPI deficits at all prepulse intensities applied (prepulse intensity: 65 dB: p < 0.05; 70 dB: p < 0.05; 75 dB: p < 0.01; 80 dB: p < 0.01) (Fig. 2B). 3.4. VGLUT1+/− mice show a working memory deficit in the T-Maze test Working memory, analysed by the ability to recognize (and prefer) a new, unfamiliar arm of the T-Maze in contrast to a familiar arm, already explored 1 h earlier, was altered in VGLUT1 heterozygous mice. VGLUT1+/− mice showed a significantly reduced preference to enter the new unfamiliar arm compared to their littermate controls (p < 0.05). However, they stayed in the new arm as long as the control mice (Fig. 3). 3.5. Sociability and social memory in VGLUT1+/− mice Sociability and social memory tests in mice are established methods to measure interactional deficits that may correspond to social withdrawal in schizophrenia patients. VGLUT1 heterozygous mice showed no statistically significant difference in sociability, preferring to a comparable extent as control mice the contact with an unfamiliar mouse (“stranger 1”) in comparison to an empty wire cage (paired Student’s t-test “stranger 1” vs. empty cage: VGLUT1: p < 0.05, controls: p < 0.01) (Fig. 4A). In contrast, VGLUT1+/− mice showed a distinct phenotype in the social memory session: they did not significantly differentiate between strangers 1 and 2, in contrast to their littermate controls which clearly preferred the unfamiliar stranger 2 (paired Student’s t-test “stranger 1” vs. “stranger 2” controls: p < 0.05) (Fig. 4B). 4. Discussion Here we show that mice with reduced expression of the VGLUT1 transporter exhibit several behavioural abnormalities associated with schizophrenia, such as abnormal working and social memory
and sensorimotor gating deficits. These data suggest a potential contribution of a deficient cortical presynaptic glutamatergic transmission mediated by VGLUT1, in the generation of several abnormalities associated with schizophrenia. Our data may be of relevance for humans, since several postmortem studies indicated a direct connection between VGLUT1 and schizophrenia. Reduced VGLUT1 mRNA expression in the prefrontal cortex (PFC) [10] and VGLUT1 protein levels in the anterior cingulate cortex [31] were reported in patients with schizophrenia. A strong decrease, by 79%, in the density of VGLUT1-immunoreactive axonal boutons, selectively in layer 3 of the PFC has been found in post-mortem tissue from schizophrenia patients [5]. Other studies reported negative results with regard to a direct involvement of VGLUT1 in schizophrenia [11,30]. The different outcomes of these studies in comparison to those revealing a positive correlation between VGLUT1 and schizophrenia (the latter done mostly in older schizophrenic patients) [11,30], may suggest an accelerated age-dependent decrease in VGLUT1 in schizophrenia [11]. Harrison et al. proposed that VGLUT1-mediated presynaptic glutamatergic abnormalities may be progressive in schizophrenia, worsening with aging and leading to persistent cognitive alterations associated with the disease [19] Cognitive deficits are considered core features of schizophrenia [2]. Previous studies already investigated VGLUT1+/− mice in several learning assays, reporting deficits in spatial reversal learning and impaired hippocampal LTP [3] and recognition memory [35]. In our analysis of VGLUT1+/− mice, we observed cognitive alterations as assessed in the T-Maze test. Additionally, we identified significant social memory abnormalities. The novelty discrimination in a social context constitutes a valuable tool for revealing attentional deficits used in several animal models of schizophrenia [12,34]. Therefore, this result appears particularly relevant to the disease, since a selective attention deficit, the inability to differentiate relevant from irrelevant information, is considered to underlie many cognitive deficits of schizophrenia [4]. Taken together, these data point out the importance of presynaptic mechanisms controlling glutamate release in inducing cognitive alterations associated with schizophrenia. In addition to abnormalities in working and social memory, VGLUT1+/− mice displayed sensorimotor gating deficits. PPI of the startle reflex is an established measure of sensorimotor gating that is reduced in schizophrenia patients [8]. The PPI deficit in VGLUT1+/− mice appears particularly relevant in the context of the high expression of VGLUT1 in the hippocampus and the known modulator role of the hippocampus on PPI [9]. NMDA receptors in the hippocampus, especially in its ventral part, as well as glutamatergic projections from the hippocampus to the nucleus accumbens, play a critical role in the regulation of PPI [24]. Our data suggest a regulatory role not only of the postsynaptic, but also
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Fig. 2. Acoustic startle response and prepulse inhibition in VGLUT1+/− mice and littermate controls. VGLUT1+/− mice show (a) an enhanced acoustic startle response and (b) deficits in prepulse inhibition (%). n = 11 per genotype. All data are means + SEM. Student’s t-test: *p < 0.05; **p > 0.01; ***p < 0.001.
Fig. 3. Preference of the new unfamiliar arm in the T-Maze test. VGLUT1+/− mice preferred (a) significantly less to enter the new arm but (b) spent comparable time in the new arm. n = 12–13 per genotype. All data are means + SEM. Student’s t-test: *p < 0.05.
of the presynaptic glutamatergic transmission of PPI. In addition to glutamatergic disturbances, the low GABA level in the frontal cortex and hippocampus of VGLUT1+/− mice [16,35] may also be implicated in the generation of sensorimotor gating abnormalities, since GAD65 KO mice show also a PPI deficit [20]. Of note, we found in VGLUT1+/− mice enhanced amplitude of the ASR, as in homozygous VGLUT1 KO mice [14]. A possible explanation for this increase is the enhanced anxiety of VGLUT1+/− mice [35] known to enhance the amplitude of the ASR [24]. Interestingly, some clinical studies reported as well increased amplitude of the ASR in schizophrenic patients when compared to normal subjects [6,32]. We did not find alterations in locomotor behaviour in the VGLUT1+/− mice under basal conditions. Of note, hyperlocomotion in the Open Field is considered a behavioural correlate for positive symptoms of schizophrenia [37]. Nevertheless, it was previously shown that VGLUT1+/− mice displayed an increase in the
total distance travelled after chronic mild stress [16]. This suggests that although locomotion is not affected in VGLUT1+/− mice under basal conditions, certain vulnerability towards developing schizophrenia-like abnormalities may exist in these mice after prolonged exposure to stress [16]. Interestingly, in contrast to the inconspicuous phenotype of VGLUT2+/− mice, inducible, restricted deletion of VGLUT2 in the cortex, hippocampus and amygdala of preadolescent mice induces several behavioural abnormalities associated with schizophrenia (hyperlocomotion, cognitive dysfunctions, altered social behaviour, decreased sensorimotor gating) [39]. Taking into account the clear predominance of VGLUT1 over VGLUT2 in the cortex and hippocampus, these data strongly encourage future studies with inducible VGLUT1 KO mice during adolescence/adulthood (to overcome perinatal lethality) for analysing the possible role of VGLUT1 in the outbreak and chronification of schizophrenia.
Fig. 4. Sociability and social memory in VGLUT1+/− mice and littermate controls. VGLUT1+/− mice and littermate controls (a) both spent significantly more time in the compartment with stranger 1 in comparison with the compartment with an empty wire cage. (b) VGLUT1+/− mice did not prefer the new unfamiliar stranger 2 in the social memory test. n = 12 per genotype. All data are means + SEM. Paired Student’s t-test [(a) comparison between the compartment with stranger 1 and empty wire cage (a) and respectively with stranger 1/stranger 2 (b)] *p < 0.05; **p > 0.01.
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In conclusion, our data indicate that VGLUT1 may play an important role in some abnormalities associated with schizophrenia, particularly cognitive deficits. The understanding of the mechanisms underlying these disturbances appears important, since especially cognitive abnormalities show very limited response to current antipsychotic treatment. Future studies, using mice with selective deletion of VGLUT1 in specific brain areas, to avoid the lethality of the full knockout and in the same time providing a more restricted region-specific ablation of VGLUT1 may provide further useful insight in the role of this transporter in generating schizophrenia-like abnormalities. Conflict of interest statement None to declare. Acknowledgements We thank Andreas Meyer-Lindenberg from the Central Institute for Mental Health Mannheim, University of Heidelberg, for critically reading the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GA 427/11-1 to D.I. and P.G.). References [1] Amilhon B, Lepicard E, Renoir T, Mongeau R, Popa D, Poirel O, et al. VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety. J Neurosci 2010;30:2198–210. [2] Arguello PA, Gogos JA. Cognition in mouse models of schizophrenia susceptibility genes. Schizophr Bull 2010;36:289–300. [3] Balschun D, Moechars D, Callaerts-Vegh Z, Vermaercke B, Van Acker N, Andries L, et al. Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation and spatial reversal learning. Cereb Cortex 2010;20: 684–93. [4] Barch DM, Carter CS, Hachten PC, Usher M, Cohen JD. The “benefits” of distractibility: mechanisms underlying increased Stroop effects in schizophrenia. Schizophr Bull 1999;25:749–62. [5] Bitanihirwe BK, Lim MP, Kelley JF, Kaneko T, Woo TU. Glutamatergic deficits and parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia. BMC Psychiatry 2009;9:71. [6] Bolino F, Manna V, Di Cicco L, Di Michele V, Daneluzzo E, Rossi A, et al. Startle reflex habituation in functional psychoses: a controlled study. Neurosci Lett 1992;145:126–8. [7] Boulland JL, Qureshi T, Seal RP, Rafiki A, Gundersen V, Bergersen LH, et al. Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters. J Comp Neurol 2004;480:264–80. [8] Braff DL, Geyer MA. Sensorimotor gating and schizophrenia. Human and animal model studies. Arch Gen Psychiatry 1990;47:181. [9] Caine SB, Geyer MA, Swerdlow NR. Hippocampal modulation of acoustic startle and prepulse inhibition in the rat. Pharmacol Biochem Behav 1992;43:1201–8. [10] Eastwood SL, Harrison PJ. Decreased expression of vesicular glutamate transporter 1 and complexin II mRNAs in schizophrenia: further evidence for a synaptic pathology affecting glutamate neurons. Schizophr Res 2005;73:159–72. [11] Eastwood SL, Harrison PJ. Markers of glutamate synaptic transmission and plasticity are increased in the anterior cingulate cortex in bipolar disorder. Biol Psychiatry 2010;67:1010–6. [12] Feifel D, Mexal S, Melendez G, Liu PY, Goldenberg JR, Shilling PD. The brattleboro rat displays a natural deficit in social discrimination that is restored by clozapine and a neurotensin analog. Neuropsychopharmacology 2009;34: 2011–8. [13] Fremeau Jr RT, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 2001;31:247–60. [14] Fremeau Jr RT, Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm-Mathisen J, et al. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 2004;304:1815–9.
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