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A t(1;11) balanced chromosomal translocation transects the Disc1 gene in a large Scottish family and produces genome-wide linkage to schizophrenia and recurrent major depressive disorder. This study describes our in vitro investigations into neurophysiological function in hippocampal area CA1 of a transgenic mouse (DISC1tr) that expresses a truncated version of DISC1 designed to reproduce aspects of the genetic situation in the Scottish t(1;11) pedigree. We employed both patch-clamp and extracellular recording methods in vitro to compare intrinsic properties and synaptic function and plasticity between DISC1tr animals and wild-type littermates. Patch-clamp analysis of CA1 pyramidal neurons (CA1-PNs) revealed no genotype dependence in multiple subthreshold parameters, including resting potential, input resistance, hyperpolarization-activated ‘sag’ and resonance properties. Suprathreshold stimuli revealed no alteration to action potential (AP) waveform, although the initial rate of AP production was higher in DISC1tr mice. No difference was observed in afterhyperpolarizing potentials following trains of 5–25 APs at 50 Hz. Patch-clamp analysis of synaptic responses in the Schaffer collateral commissural (SC) pathway indicated no genotype-dependence of paired pulse facilitation, excitatory postsynaptic potential summation or AMPA/NMDA ratio. Extracellular recordings also revealed an absence of changes to SC synaptic responses and indicated input–output and short-term plasticity were also unaltered in the temporoammonic (TA) input. However, in DISC1tr mice theta burst-induced long-term potentiation was enhanced in the SC pathway but completely lost in the TA pathway. These data demonstrate that expressing a truncated form of DISC1 affects intrinsic properties of CA1-PNs and produces pathway-specific effects on long-term synaptic plasticity.
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Since the discovery of a balanced chromosomal translocation (1;11) (q42.1;q14.3) segregating with psychiatric disorders in a large Scottish family (Jacobs et al., 1970; St Clair et al., 1990; Blackwood et al., 2001) and the subsequent identification of the disrupted-in-schizophrenia 1 (Disc1) gene (Millar et al., 2000), some genetic association studies in family samples and more diverse populations have provided evidence for Disc1 as a susceptibility gene for psychiatric illnesses including schizophrenia, bipolar disorder and recurrent major depression (Ekelund et al., 2004; Hodgkinson et al., 2004; Callicott et al., 2005; Sachs et al., 2005; Thomson et al., 2005; Hennah et al., 2009). However, other studies, including recent genome-wide association studies, have not identified Disc1 as a risk factor for psychiatric disorders (Zhang et al., 2005; Chen et al., 2007; Kim et al., 2008; Purcell et al., 2009; Mathieson et al., 2012) so although still of considerable interest in psychiatry, the role of Disc1 in such illnesses remains the subject of some debate (Sullivan, 2013).
The protein encoded by the Disc1 gene is known as DISC1. Numerous splice variants of this protein are likely to exist, although the full-length Disc1 gene encodes a protein consisting of 854 amino acids with a molecular mass of around 100 kDa. DISC1 expression is reported to be at its highest levels during development but expression persists into adulthood reducing somewhat as animals age. The protein is found in multiple brain regions in adults, including the hippocampus (Austin et al., 2004), the subject of this particular study. Functionally, DISC1 appears to be involved in a multitude of cellular processes, including processes that are critical for both brain development and the activity of the adult central nervous system (CNS) (Brandon & Sawa, 2011).
Motivated by its potential role in psychiatric disease, several DISC1-related genetically modified mice have been generated in recent years. These are reported to display a variety of schizophrenia-like phenotypes, both anatomically and behaviourally (Koike et al., 2006; Clapcote et al., 2007; Hikida et al., 2007; Li et al., 2007; Kvajo et al., 2008; Pletnikov et al., 2008; Shen et al., 2008). There are, by contrast, very few detailed studies describing neurophysiological phenotypes. Three reports have employed electrophysiological methods to examine intrinsic and synaptic properties in the mouse model developed by Koike et al. (2006). These animals express a truncated form of mouse Disc1 derived from a spontaneous mouse mutation. Findings include decreased early phase long-term potentiation (LTP) in the Schaffer collateral commissural (SC) pathway of the hippocampus (Kvajo et al., 2008) and modified short-term plasticity in the mossy fibre pathway (Kvajo et al., 2011), both observed with extracellular recording. Cellular level patch-clamp recordings in this mouse have also indicated decreased excitability of dentate granule cells (Kvajo et al., 2011), and alterations in the frequency of spontaneous synaptic currents in medial prefrontal cortex layer II/III pyramidal neurons (Holley et al., 2013).
The hippocampus plays a key role in cognitive function, and hippocampal CA1 pyramidal neurons (CA1-PNs) are probably the most widely studied neurons in the mammalian brain. Along with neurons in the subiculum, these cells represent a major output node of this brain area and make synapses with multiple target cells, including a monosynaptic projection to the prefrontal cortex implicated in psychiatric diseases. Additionally, high-resolution structural and functional magnetic resonance imaging studies suggest that the CA1 subregion may be differentially affected in schizophrenia patients (Narr et al., 2004; Schobel et al., 2009).
Various alterations in synaptic transmission and plasticity in area CA1 of the hippocampus have been reported in different rodent models related to schizophrenia. Deficits in basal synaptic transmission in the SC pathway have been described in the offspring of rats treated with the antimitotic agent methylazoxymethanol during pregnancy (Sanderson et al., 2012) and in a maternal immune activation rat model (Oh-Nishi et al., 2010). In a transgenic mouse model of the 22q11 deletion syndrome, whilst there were no deficits in basal synaptic transmission, increased excitatory postsynaptic current (EPSC) summation, paired-pulse ratios, and short- and long-term potentiation in the SC pathway have been reported (Earls et al., 2010). By contrast, impaired SC LTP has been shown both in rats subchronically dosed with the N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (Pollard et al., 2012), and in a maternal immune activation rat model (Oh-Nishi et al., 2010). There is much less information concerning synaptic alterations in DISC1 mouse models. One report describes decreased post-tetanic potentiation with no changes in basal synaptic transmission, paired-pulse ratios or LTP in the SC pathway (Kvajo et al., 2008). A more recent study shows decreased short-term potentiation, without changes to basal synaptic transmission or LTP in the mossy fibre pathway (Kvajo et al., 2011).
Studies in different mouse models related to schizophrenia have also described alterations to a variety of intrinsic neuronal properties in the hippocampus. Examples include decreased input resistance (Ri) and excitability in granule cells of the dentate gyrus in a DISC1 transgenic mouse model (Kvajo et al., 2011), depolarized resting membrane potential (RMP), increased action potential (AP) firing frequency and decreased AP width in CA2/3 stratum oriens interneurons following chronic infusion of picrotoxin into the basolateral amygdala of rats (Gisabella et al., 2009), and depolarized RMP and increased excitability of CA1-PNs following systemic administration of the NMDA receptor antagonist MK-801 in mice (Kehrer et al., 2007).
To date, no thorough investigations of intrinsic and synaptic properties of CA1-PNs in DISC1 transgenic mouse models have appeared. To address this issue we performed the study detailed here, based on the DISC1tr mouse model developed by Shen et al. (2008). These mice express two copies of truncated mouse Disc1 closely mimicking the situation in the Scottish family with the Disc1 translocation. These mice are reported to display a variety of schizophrenia-related abnormalities including enlarged lateral ventricles, reduced cerebral cortex volume, and reduced counts of parvalbumin-positive interneurons in the hippocampus and medial prefrontal cortex. Behaviourally, an impairment in conditioning of latent inhibition and ‘depressive-like’ behaviours were described (Shen et al., 2008). Our results presented here represent the first description of the neurophysiological consequences of transgenic expression of DISC1tr in this mouse model.
Materials and methods
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- Materials and methods
As described by Shen et al. (2008), DISC1tr transgenic mice were originally generated with a bacterial artificial chromosome containing a truncated form of Disc1 which encodes the first eight exons. The original line was in a mixed genetic background of CBA/CaCrl and C57BL/6JCrl. Progenies, which were confirmed free of the Nnt and Snca mutations associated with C57BL/6JCrl and C57BL/6J Harlan substrains, respectively, were backcrossed with mutation-free mice of C57BL/6JRccHsd for nine generations, resulting in DISC1tr hemi mice prior to project initiation. C57BL/6JRccHsd mice were continuously used in all subsequent breeding to generate experimental DISC1tr hemi mice and wild-type (WT) littermate controls.
Male DISC1tr hemi mice and WT littermates were bred and aged at the University of Strathclyde, and subsequently shipped to the University of Bristol by road and housed singly on a 12 : 12-h light/dark cycle with ad libitum access to food and water. All procedures on experimental animals were approved by local ethical approval at the University of Bristol. Furthermore, all such work was carried out in compliance with UK Home Office regulations as set out in the Animals (Scientific Procedures) Act 1986 and consequently were also in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Two cohorts of DISC1tr and WT littermates were employed during this study. The first cohort was used for studies of intrinsic and synaptic properties employing patch-clamp methods and consisted of mice aged ~3.8 months of age (WT mean age 3.8 ± 0.1 months, range 2.7–4.6 months, n = 14 mice; DISC1tr mean age 3.7 ± 0.1 months, range 2.7–4.2 months, n = 15 mice). The second cohort was used for studies of synaptic plasticity with extracellular field potential recording and involved mice aged ~8.7 months (WT mean age 8.7 ± 0.1 months, range 8.2–9.1 months, n = 11 mice; DISC1tr mean age 8.7 ± 0.1 months, range 8.2–9.0 months, n = 10 mice).
Horizontal hippocampal slices were prepared, and patch-clamp and extracellular field recordings were made at ~34 °C from area CA1 as described previously (Clement et al., 2009; Brown et al., 2011; Randall et al., 2012) and summarized below.
Preparation of brain slices
Animals were killed by cervical dislocation and brains were quickly removed and placed into ice-cold sucrose-based artificial slicing solution comprising (in mm): sucrose, 189; d-glucose, 10; NaHCO3, 26; KCl, 3; MgSO4, 5; CaCl2, 0.1; NaH2PO4, 1.25. Horizontal slices of ventral hippocampus of thickness 300 μm were prepared using a Leica VT1200 vibratome (Leica Microsystems, Milton Keynes, UK). Following their preparation slices were transferred to normal artificial cerebrospinal fluid (aCSF) and incubated at 35–37 °C for 30 min, following which they were kept at room temperature until use. The composition of the standard aCSF was (mm): NaCl, 124; KCl, 3; NaHCO3, 26; CaCl2, 2; NaH2PO4, 1.25; MgSO4, 1; d-glucose, 10; the solution was equilibrated with 95% O2 and 5% CO2.
Single cell patch-clamp recording
For recording, slices were transferred to a submersion-type chamber mounted on an Olympus BX51 WI upright microscope (Scientifica, Uckfield, UK) equipped with infrared differential interference contrast optics to enable visual identification of neurons. Here slices were continuously perfused (~2.5 mL/min) with normal aCSF and maintained at ~33 °C. Pharmacological agents were applied via the perfusion system.
Recordings were made using 3–5 MΩ fire-polished glass microelectrodes filled with one of two internal solutions: a K-gluconate-based solution for current-clamp experiments and a CsCl-based solution for voltage-clamp recordings. The pairing of aCSF and pipette solutions produced liquid junction potential errors which were corrected for arithmetically. Signals were amplified using a MultiClamp 700B amplifier (Molecular Devices, Union City, CA, USA), digitized using an Axon Digidata 1440a data acquisition board (Molecular Devices) and stored on a personal computer using pClamp10.2 software (Molecular Devices).
Whole-cell current-clamp recordings were made using a K-gluconate-based internal solution containing (in mm): K-gluconate, 140; NaCl, 10; HEPES, 10; EGTA, 0.2; Na-GTP, 0.3; Mg-ATP, 4; pH 7.3. Current-clamp recordings were made using the bridge circuit of the amplifier to allow faithful voltage following. Recordings of intrinsic and AP properties were lowpass filtered at 10 kHz and digitized at 100 kHz. Sinusoidal current (ZAP current) recordings of intrinsic resonance were lowpass filtered at 0.5 kHz and digitized at 1 kHz. Recordings of synaptic responses in current-clamp were lowpass filtered at 4 kHz and digitized at 20 kHz.
To measure subthreshold, AP and excitability properties, a series of 500-ms square-wave current injection steps (−100 to +300 pA in 50-pA increments) were applied to cells. A sinusoidal current (ZAP current) with constant amplitude and linearly increasing frequency (1–20 Hz over 30 s) was used to characterize resonance properties. This was performed at three different pre-stimulus membrane potentials as these properties are inherently voltage-dependent (Hu et al., 2002). To examine fast after-spike potentials, single AP(s) were elicited with a strong but brief (2 nA, 2 ms) current injection.
To measure properties of synaptic responses in CA1-PNs, postsynaptic potentials were evoked by delivering brief (0.1 ms) electrical stimulation to tungsten bipolar stimulating electrodes (FHC Inc., Bowdoin, ME, USA) connected to constant-current isolated stimulator boxes (Digitimer, Welwyn Garden City, UK). Stimulating electrodes were placed in stratum radiatum to stimulate the SC pathway, and in stratum lacunosum moleculare to stimulate the temporoammonic (TA) pathway. Measurements of excitatory and inhibitory postsynaptic potential (EPSP and IPSP) summation were made by delivering trains of six stimuli at different frequencies (5–100 Hz). Stimulus intensity was adjusted to elicit a first EPSP of approximately 4–5 mV (SC) or 0.5–1 mV (TA) in amplitude and was kept constant throughout the stimulus trains.
Whole-cell voltage-clamp recordings were used to specifically measure AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA currents in the SC pathway using a CsCl-based internal solution containing (in mm): CsCl, 130; NaCl, 5; HEPES, 10; EGTA, 0.2; Na-GTP, 0.3; Mg-ATP, 4; QX314-Cl, 5; pH 7.3. As for current-clamp recordings of synaptic potentials, SC EPSCs were evoked with a stimulating electrode placed in stratum radiatum. To block inhibitory postsynaptic currents (IPSCs), which could contaminate evoked excitatory responses, the γ-aminobutyric acid (GABA)A receptor antagonist gabazine (5 μm) was included in the normal aCSF. Area CA3 was removed from the slice to prevent epileptiform discharges that can occur in hippocampal slices in the absence of GABA receptor-mediated inhibition.
Input–output curves were constructed at a holding potential of −80 mV by incrementally increasing stimulus intensity and recording the evoked response (5–40 μA). For subsequent experiments, stimulus intensity was adjusted to elicit an EPSC of approximately 250 pA (at a holding potential of −80 mV) and remained constant throughout. Paired-pulse profiles were constructed at a holding potential of −80 mV by delivering paired stimuli over a range of inter-stimulus intervals. To examine NMDA/AMPA ratios, single stimuli were delivered at holding potentials of −80, +40 and 0 mV. Holding potentials were adjusted to correct for the liquid junction potential error. Voltage-clamp data were lowpass filtered at 4 kHz and digitized at 20 kHz. Series resistance (range 10–20 MΩ) was monitored but not compensated for, and only cells with stable series resistance (<20% change throughout experiment) were included for analysis.
Area CA3 was removed from the slice before transfer to a submerged chamber (Scientifica) continuously perfused (~2.5 mL/min) with normal aCSF and maintained at ~33 °C. Pharmacological agents were added to the aCSF at the concentrations stated and applied to the slice via the perfusion system.
Recordings were made using 2–3 MΩ glass microelectrodes filled with normal aCSF which were fabricated from borosilicate capillary glass (Harvard Apparatus, Edenbridge, UK) using a P-97 Flaming Brown micropipette puller (Sutter Instrument Co., Novato, CA, USA). Stimulating electrodes and recording electrodes were placed in stratum radiatum and stratum lacunosum moleculare. Field excitatory postsynaptic potentials (fEPSPs) were evoked by delivering brief (0.1 ms) electrical stimulation to tungsten bipolar stimulating electrodes (FHC Inc.) connected to a constant-voltage isolated stimulator box (Digitimer). Signals were amplified using a MultiClamp 700 amplifier (Molecular Devices), digitized using an Axon Digidata 1322a data acquisition board (Molecular Devices) and stored on a personal computer using pClamp10.2 software (Molecular Devices). Recordings were lowpass filtered at 10 kHz and digitized at 50 kHz.
The SC and TA pathways were stimulated alternately every 15 s for input–output curves and LTP experiments. Input–output curves were constructed by incrementally increasing stimulus intensity and recording the evoked response (0–12 V in 1-V steps for the SC pathway, and 0–60 V in 5-V steps for the TA pathway). Paired-pulse profiles were constructed by delivering paired stimuli over a range of inter-stimulus intervals at a stimulus intensity that elicited an approximately half-maximal response.
Long-term potentiation was induced using a theta burst stimulation (TBS) protocol. A baseline period of at least 20 min stable fEPSPs was recorded at a stimulation intensity that elicited an approximately half-maximal response. Stimulation intensity remained constant throughout the experiment, including during the TBS protocol. Following the baseline period, the TBS protocol was delivered to one pathway and consisted of five bursts (10 stimuli at 100 Hz) at 5 Hz (theta frequency), repeated four times at an interval of 20 s. fEPSPs were then followed for 1 h before TBS stimulation was delivered to the other pathway and fEPSPs were followed for another hour. The order in which the pathways were stimulated with the TBS protocol was alternated between experiments.
Data analyses were carried out using pClamp10.2, Excel and custom-written routines in MatLab. A detailed description of the algorithms used for analysis of CA1-PNs can be found in Kerrigan et al. (2013). spss was used to carry out statistical analyses.