extfieldsofstudy: Biology Medicine Psychology

s2fieldsofstudy: Biology Psychology

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GluN2B antagonism affects interneurons and leads to immediate and persistent changes in synaptic plasticity, oscillations, and behavior.

Although antagonists to GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) have been widely considered to be neuroprotective under certain pathological conditions, their immediate and lasting impacts on synaptic, circuit, and cognitive functions are poorly understood. In hippocampal slices, we found that the GluN2B-selective antagonist Ro25-6981 (Ro25) reduced synaptic NMDAR responses and consequently neuronal output in a subpopulation of GABAergic interneurons, but not pyramidal neurons. Consistent with these effects, Ro25 reduced GABAergic responses in pyramidal neurons and hence could affect circuit functions by altering the excitation/inhibition balance in the brain. In slices from Ts65Dn mice, a Down syndrome model with excess inhibition and cognitive impairment, acutely applied Ro25-rescued long-term potentiation (LTP) and gamma oscillation deficits, whereas prolonged dosing induced persistent rescue of LTP. In contrast, Ro25 did not impact LTP in wild-type (wt) mice but reduced gamma oscillations both acutely and following prolonged treatment. Although acute Ro25 treatment impaired memory performance in wt mice, memory deficits in Ts65Dn mice were unchanged. Thus, GluN2B-NMDARs contribute to the excitation/inhibition balance via impacts on interneurons, and blocking GluN2B-NMDARs can alter functions that depend on this balance, including synaptic plasticity, gamma oscillations, and memory. That prolonged GluN2B antagonism leads to persistent changes in synaptic and circuit functions, and that the influence of GluN2B antagonism differs between wt and disease model mice, provide critical insight into the therapeutic potential and possible liabilities of GluN2B antagonists.

INTRODUCTION
N-methyl-D-aspartate receptors (NMDARs) are heteromeric receptors consisting of two GluN1 subunits and two GluN2A-D subunits, which have essential functions in brain development, synaptic signaling, and neuroplasticity. In the forebrain, GluN2A and GluN2B are the predominantly expressed NR2 subunits (Watanabe et al, 1993). Selective antagonists of GluN2B-containing NMDARs have been pursued for treating a variety of neurological conditions (Chazot, 2004;Mony et al, 2009), including ischemic stroke (Kaufman et al, 2012;Liu et al, 2007), Huntington's disease (Milnerwood et al, 2010;Okamoto et al, 2009), and Parkinson's disease (Calon et al, 2003;Wessell et al, 2004). A rationale for this approach is that  NMDARs are thought to be excessively activated in these indications and could thus be selectively inhibited to prevent the synapse loss and/or neuronal cell death observed in these indications. Moreover, selective inhibition of GluN2B-containing NMDARs could potentially avoid the known side effects of non-selective NMDAR antagonists (Chazot, 2004;Mony et al, 2009) because of their preferential presence at extrasynaptic but not synaptic regions on excitatory pyramidal neurons in adult brain (Harris and Pettit, 2007;Papouin et al, 2012;Tovar and Westbrook, 1999). However, aside from pathological activation, evidence from both genetic deletion of GluN2B and acute systemic administration of GluN2B antagonists suggest a physiological role for GluN2B receptors in the adult brain during memory function (Mathur et al, 2009;von Engelhardt et al, 2008). Furthermore, a significant portion of the excitatory drive to inhibitory interneurons is mediated by NMDARs (Lei and McBain, 2002;Wang and Gao, 2009), and GluN2B subunits are expressed in GABAergic interneurons (Lei and McBain, 2002;Porter et al, 1998). Therefore, it needs to be tested whether GluN2B antagonists can significantly alter circuit functions by affecting NMDARs on interneurons. This is an important question not only for providing a deeper understanding of the contribution of NMDAR subunits to circuitry functions, but also for critically evaluating the therapeutic potential of GluN2B antagonists. Any possible benefits of blocking pathologically activated extrasynaptic GluN2B receptors would need to be carefully weighed against any alterations to the physiological functions of neural circuitry.
To address this, we studied the effects of the GluN2Bselective antagonist Ro25-6981 (Ro25;Fischer et al, 1997) on interneuron excitation, neural circuitry functions, and cognitive behavior, after either acute treatment or prolonged dosing, in both wild-type (wt) mice and the Ts65Dn mouse model of Down syndrome that features excess inhibition and cognitive impairment (Braudeau et al, 2011;Chakrabarti et al, 2010;Costa and Grybko, 2005;Fernandez et al, 2007;Kleschevnikov et al, 2012Kleschevnikov et al, , 2004Perez-Cremades et al, 2010;Rueda et al, 2008). We found that Ro25 disinhibited hippocampal circuitry by reducing excitatory drive to a subpopulation of hippocampal interneurons. In vitro application of Ro25 rescued deficits in long-term potentiation (LTP) and gamma oscillations in Ts65Dn Down syndrome model mice. Although this in vitro rescue did not translate into improvement of memory function in the Down syndrome mice, circuit function and memory in wt mice were disrupted. These results demonstrate novel physiological roles for GluN2B-NMDARs in interneurons and neural circuitry, and have broad implications for considering GluN2B antagonists as therapeutics.

Animals
Three-to six-month-old male segmental trisomy 16 (Ts65Dn) mice and wt littermate controls were obtained from Jackson Labs (B6EiC3Sn a/A-Ts(1716)65Dn number 001924) or bred at Genentech. Some experiments, including the behavioral studies, used a related strain virtually identical to the original, but bred to not carry the recessive retinal degeneration mutation, Pde6b rd1 , (B6EiC3Sn.BLiA-Ts(1716)65Dn/DnJ number 005252; Costa et al, 2010). GABAergic interneurons were selectively targeted using 3-month-old GAD67-GFP knockin mice (Tamamaki et al, 2003). All animal studies were approved by the Genentech IUCAC, and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Field Recordings
EPSPs and population spikes (PSs) were measured from the stratum radiatum (s. radiatum) and stratum pyramidale (s. pyramidale) regions of CA1, respectively, in response to stimulation of Schaffer collateral inputs. NMDAR EPSPs were measured with the Mg 2 þ concentration in the ACSF reduced to 0.5 mM and in the presence of PTX and NBQX. LTP was induced under conditions with intact inhibition using 1, 2, or 3 bouts of theta burst stimulation (TBS) separated by 20 s, each consisting of 5 pulses at 100 Hz repeated 10 times at 200-ms intervals. Tetanus-induced oscillations were recorded in the s. pyramidale following 200 ms of 100 Hz stimulation to the nearby region of s. radiatum proximal to the recording site. Gamma oscillations were analyzed after bandpass filtering at 30-200 Hz (8 pole Bessel). Spectrograms were generated with Matlab, and total power was calculated as the sum of power at all frequencies and all time points during the 1 s following stimulation. Ro25 was applied to brain slices for 20 min before assay of LTP or gamma oscillations. Oscillation power and EPSP magnitude during maximal stimulation were analyzed by two-factor ANOVAs, with genotype and drug treatment as the factors. The steady-state LTP responses following 1, 2, or 3 bouts of TBS were analyzed by three-factor ANOVAs with genotype, drug treatment, and number of TBS bouts as the factors. Pre-planned comparisons were performed using a Student's t-test to test specific hypotheses of deficits in Ts65Dn mice, Ro25 rescue of Ts65Dn deficits, and Ro25 alteration of wt function.

Whole-Cell Recordings
For current clamp recordings, internal solution consisted of (in mM) 120 K-gluconate, 20 KCl, 0.5 EGTA, 10 HEPES, 2.5 MgCl2, 4 Na2ATP, 0.3 NA3GTP, and 10 phosphocreatine. For voltage-clamp recordings, K-gluconate and KCl were replaced with Cs-methanesulfonate and CsCl, respectively, and 5 mM QX-314 bromide was added. Evoked EPSPs were measured at resting potentials that were subthreshold to action potential generation in response to single stimulations, and evoked EPSCs were measured at a holding potential of À 70 mV. NMDAR EPSCs were measured at À 70 mV in 0.5 mM Mg 2 þ , NBQX, and PTX. Significance of differences between the means was assessed using a Student's t-test.

Y-Maze
Individual mice were placed into a Y-maze and were allowed to explore the maze for 6 min. The number of arm entries and the sequence of arms entered was recorded by an observer unaware of either genotype or drug treatment. Non-performers with less than 10 arm entries throughout the entire 6 min observation period were excluded from the analysis. A correct alternation was scored when a sequence of three consecutive arm entries (a triplet) included entries into all three arms of the maze. Percent alternations were calculated as 100 Â (number of correct alternations)/(total number of arm entires À 2). Percent alternations and number of arm entries were analyzed by two-factor ANOVAs, with genotype and drug treatment as the between-subjects factors. ANOVAs were followed by Student's t-test when only two groups were compared.
Barnes Maze. The Barnes maze is a spatial memory test that utilizes the animals' tendency to escape from a brightly lit open environment into a quiet, dark escape tunnel. Mice were first familiarized with the maze, which consists of a circular table, 91 cm in diameter with 20 holes (5 cm diameter) equally spaced around the perimeter of the maze. Training consisted of allowing the mouse to freely explore the maze for up to 3 min to locate the target hole (in a new location from pre-training). During these 3 min, the number of errors (wrong holes explored), distance traveled, and latency to enter target hole were recorded using Cleversys software (TopScan). Immediately after the mouse entered the escape box, the lights were dimmed and the mouse was allowed to stay in the tunnel for 10-20 s. Animals were run with an intertrial interval of 15 min. Training occurred over 3 consecutive days. Latency to enter target hole, distance traveled, velocity (m/s), and errors (total number of nose pokes into incorrect holes) were analyzed using a twofactorial MANOVA with repeated measures with genotype and drug treatment as between-subjects and training days as within-subject repeated measure.

Ro25 Selectively Reduces Synaptic NMDAR Responses in S. Radiatum Interneurons, Decreases Interneuron Spiking, and Reduces Inhibition of Pyramidal Neurons
We first examined the function of GluN2B-NMDARs on pyramidal neurons in the hippocampal CA1 region using field NMDA-EPSP recordings. Consistent with previous results (Harris and Pettit, 2007;Papouin et al, 2012), we found that bath application of the selective GluN2B antagonist Ro25 (3 mM) partially reduced isolated synaptic NMDAR responses in CA1 pyramidal neurons in slices from young (2 week) but not more mature (4 week) animals ( Figure 1a). We were able to block extrasynaptic NMDAR responses in pyramidal neurons from mature animals using Ro25 (Supplementary Figure S1A), indicating a selective exclusion of Ro25-sensitive NMDARs from synapses on pyramidal neurons.
Next we compared the effects of Ro25 on isolated synaptic NMDAR currents in interneurons located in s. radiatum or s. pyramidale of CA1, and found that NMDAR EPSCs in s. radiatum, but not s. pyramidale interneurons, were reduced by Ro25 in slices from mature animals ( Figure 1b). On the other hand, adding the GluN2A-prefering antagonist NVP-AAM077 (50 nM) in addition to Ro25 caused a substantial reduction in s. pyramidale interneuron EPSCs, but only a modest change in the blockade in s. radiatum interneurons compared with Ro25 alone (Figure 1b). Thus, in contrast to pyramidal neurons or pyramidale interneurons, significant functional GluN2B-NMDARs are present at synapses on s. radiatum interneurons in mature brain slices. These radiatum interneurons exhibited regular firing characteristics in response to injection of depolarizing currents, in contrast to the fast spiking exhibited by the pyramidale interneurons (Supplementary Figure S2), and likely have a distinct role in circuit function. Using current clamp recordings, we found that Ro25 application reduced evoked EPSP area and spiking elicited by suprathreshold synaptic stimulation in radiatum, but not pyramidale interneurons (Figure 1c). To test if reduced spiking in interneurons could result in reduced inhibition of pyramidal neurons, we measured IPSCs in pyramidal neurons before and after Ro25 application. In these experiments, we used repetitive stimulation to resemble the activity patterns seen under physiologically relevant conditions, such as induction of synaptic plasticity and network oscillations. We observed a significant reduction of IPSCs amplitude during the first stimulus, and the profile of response amplitudes during repetitive stimulation was changed from facilitating to depressing in the presence of Ro25 (Figure 1d).
The above results raised the possibility that GluN2B antagonists could be useful to reduce excessive interneuron function observed in certain disease models. One such mouse model is the Ts65Dn model of Down syndrome, which exhibits increased interneuron production and inhibitory function (Braudeau et al, 2011;Chakrabarti et al, 2010;Costa and Grybko, 2005;Fernandez et al, 2007;Kleschevnikov et al, 2012Kleschevnikov et al, , 2004Perez-Cremades et al, 2010;Rueda et al, 2008). To test if inhibition is excessive in CA1 of Ts65Dn mice, we measured the ratio of inhibition to excitation, using stimulation that evoked similar amplitude EPSCs in both Ts65Dn and wt mice. A significant increase in the inhibition/excitation ratio was seen in Ts65Dn mice compared with wt mice (Figure 1e). To test if Ro25 could reduce the excitatory inputs to interneurons in Ts65Dn mice, we repeated the measurements of evoked EPSPs and synaptically driven interneuron spiking in Ts65Dn mice in the absence and presence of Ro25 (Figure 1f), and observed a reduction in EPSP area and synaptically driven spiking activity in s. radiatum but not s. pyramidale interneurons ( Figure 1g). The magnitude of reduction was similar between Ts65Dn and wt mice. In addition, we observed that Ro25 could also effectively reduce evoked IPSCs of pyramidal neurons during repetitive stimulation in Ts65Dn mice (Figure 1h). At the same time, synaptic NMDAR responses in pyramidal neurons from adult mice were insensitive to Ro25 in both Ts65Dn and wt mice (Supplementary Figure S1B), indicating that the main target of Ro25 is likely to be glutamatergic synapses onto interneurons, but not pyramidal neurons in both wt and Ts65Dn mice. possible that GluN2B antagonist could counteract the excessive inhibition in Ts65Dn mice and restore LTP. We measured LTP following 1, 2, and 3 bouts of TBS in hippocampal slices from adult (3 month) wt and Ts65Dn mice. This protocol resulted in robust LTP in wt slices, but much reduced LTP in slices from Ts65Dn mice (Figure 2c and e). Acute bath application of Ro25 did not significantly affect LTP in wt slices, but restored LTP in Ts65Dn mice to a level similar to wt slices (Figure 2c and e). In addition to LTP of EPSPs, a similar rescue of deficits in population spike (PS) LTP was also seen in Ts65Dn mice in the presence of Ro25 (Figure 2d and f). Although this rescue of LTP is consistent with disinhibition by Ro25 in Ts65dn mice, it is also possible that Ro25 could cause an interneuron-independent impact on LTP. Therefore, we repeated the LTP experiments in the presence of the GABA A R antagonist, PTX, to test if Ro25 could have an effect on LTP in the absence of fast inhibitory synaptic transmission. Under these conditions, Ro25 had no impact on LTP ( Figure 3). As these results suggest reduced inhibition has a critical role in the rescue of LTP deficit in the Ts65Dn mice, we would expect that the impact of Ro25 occurs during LTP induction. To examine this, we analyzed spiking activity during the TBS used to induce LTP. Although spiking (as measured by PS area) was relatively sustained during TBS in wt mice, the ability to sustain spiking during TBS was significantly diminished in Ts65Dn mice, but rescued in the presence of Ro25 (Supplementary Figure S3). Consistent with this excitation deficit and rescue in Ts65dn mice during LTP induction being mediated by inhibition, the deficit disappeared, and the benefit of Ro25 was occluded in the presence of PTX (Supplementary Figure  S3). Interestingly, although total spiking (PS area) was sustained in wt mice regardless of Ro25 application, the apparent synchrony of spiking across the population of postsynaptic neurons (inferred from PS amplitude) was reduced by Ro25 application, an effect that was also occluded when inhibition was blocked (Supplementary  Figure S3). These results suggest that it is the total level of inhibition rather than the specific patterns of inhibition that influence LTP induction with this protocol.

Acute Application of Ro25 Rescues In Vitro Gamma Oscillation Deficits in Ts65Dn Mice but Impairs Gamma Oscillations in wt Mice
In addition to LTP, the balance between excitation and inhibition can also affect other network functions, such as the generation of synchronous oscillations. We induced oscillations in CA1 of hippocampal slices using tetanic stimulation, a phenomenon that depends on the activation of both excitatory and inhibitory synapses (Bracci et al, 1999;Vreugdenhil et al, 2005). Gamma oscillation power in slices from Ts65Dn mice was significantly reduced compared with wt mice (Figure 4). Acute Ro25 application restored oscillation power in slices from Ts65Dn mice, but significantly reduced oscillation power in slices from wt mice ( Figure 4). Interestingly, the frequency profile and time course of oscillations were not affected by GluN2B antagonism in either genotype (Supplementary Figure S4). These results demonstrate that the acute circuit impacts of GluN2B antagonism extend beyond plasticity induction.

Prolonged Dosing with Ro25 Persistently Restores Synaptic Plasticity in Ts65Dn Mice
Previous work demonstrated that prolonged treatment (2 weeks) with GABA A R antagonists, which reduce inhibition, rescued deficits in cognitive function and LTP in Ts65Dn mice (Fernandez et al, 2007). Surprisingly, these improvements persisted for weeks even after dosing was discontinued, indicating lasting modification of circuit function. Therefore, we tested whether prolonged dosing with Ro25 could result in a lasting improvement of LTP in Ts65Dn mice, which could be detected after drug washout.
In these experiments, mice were injected daily for 2 weeks with 50 mg/kg Ro25, a dose that achieves relevant brain exposure, with peak unbound brain levels reaching 0.958 ± 0.126 mM (n ¼ 3 mice) immediately after injection and remaining above the IC 50 of recombinant GluN2B-NMDARs (Fischer et al, 1997) for hours (0.005±0.003 mM at 6 h post injection). After 2 weeks of dosing, mice were allowed to recover for 2-4.5 weeks with no drug treatment before LTP was assessed (Figure 5a). Although this treatment regimen did not alter LTP in wt mice, it significantly improved LTP in Ts65Dn mice such that LTP was indistinguishable from wt mice (Figure 5b and d).
Similarly, although PS LTP was unaltered in treated wt mice, deficits in PS LTP were significantly improved in treated Ts65Dn mice, resulting in partial restoration to wt levels ( Figure 5c and e).

Prolonged Dosing with Ro25 Persistently Impairs In Vitro Gamma Oscillations and Increases Excitatory Synaptic Transmission in wt Mice
The persistent rescue of LTP in Ts65Dn mice indicates remodeling of circuit function following prolonged Ro25 Figure 1 Ro25-6981 (Ro25) selectively reduces synaptic N-methyl-D-aspartate receptor (NMDAR) responses and excitation-driven spiking in CA1 stratum radiatum (s. radiatum) interneurons and reduces inhibition of pyramidal neurons. (a) Representative pyramidal neuron field NMDAR-EPSP traces recorded before and after application of 3 mM Ro25 to slices from 2-and 4-week-old animals (scale bars: 0.25 mV/20 ms). Bar graph represents the mean±SEM of the NMDA-EPSP area blocked by either 3 mM Ro25 or 50 mM 2-amino-5-phosphonopentanoic acid (APV; to verify the NMDARdependence of the EPSPs). Ro25 blockade was 54.4±8.8% in 2-week-old and 7.8±4.8% in 4-week-old animals (n ¼ 4/age group, po0.01). APV blockade was 93.2±5.2% in 2-week-old and 93.7±2.2% in 4-week-old animals (n ¼ 4/age group). (b) GAD67-GFP mice were used to examine synaptic NMDAR currents in stratum pyramidale (s. pyramidale) and s. radiatum GABAergic (GAD-67-positive) interneurons from mature mice. Example traces are shown for each type of interneuron before and after addition of Ro25 alone, or after addition of Ro25 and the GluN2A-preferring antagonist NVP-AAM077 tetrasodium hydrate (NVP; scale bars: s. pyramidale 40 pA/50 ms, s. radiatum 200 pA/100 ms). Bar graph shows the % blockade of the baseline EPSC area (mean ± SEM). NMDA EPSCs in s. pyramidale interneurons were not blocked by Ro25 alone (0.0 ± 4.2%, n ¼ 9), but were significantly blocked when NVP was added in addition to Ro25 (46.0 ± 13.5%, n ¼ 9), whereas NMDA EPSCs in s. radiatum interneurons were significantly blocked by Ro25 alone (48.5 ± 7.0%, n ¼ 7). (c) Ro25 reduces EPSPs and synaptically driven spiking in s. radiatum, but not s. pyramidale interneurons. S. radiatum EPSP area in response to a single synaptic stimulation (arrow) was decreased following Ro25 application (scale bars: 10 mV/100 ms). When triple synaptic stimulations (100 Hz, arrows) were used to drive s. radiatum interneurons to fire multiple spikes, Ro25 application resulted in a reduction of spike number ( Figure S5). Although the reduction in gamma oscillation power in wt mice resembles the effect of acute in vitro application of Ro25, the lack of impact on oscillations in Ts65Dn mice contrasts with the increased power with acute application, suggesting functional circuit changes following prolonged treatment do not simply mimic the acute impacts of Ro25. Although conceivably the functional circuit alterations following prolonged dosing could simply be caused by changes in the NMDAR function, several observations argue against this possibility: (1) a similar reduction in gamma oscillation power was seen after complete blocking of NMDARs by the non-selective NMDAR antagonist 2-amino-5-phosphonopentanoic acid (APV) in slices from both vehicle-treated and Ro25-treated wt and Ts65Dn mice (Supplementary Figure S6A); (2) The extent of Ro25induced inhibition of isolated synaptic NMDAR responses in radiatum interneurons was not altered in either genotype (Supplementary Figure S6B); (3) No significant changes in GluN1, GluN2A, or GluN2B protein levels were detected in hippocampi from either genotype (Supplementary Figure  S6C). Together, these data suggest that the impairment of gamma oscillations in wt mice following prolonged dosing is unlikely to be caused by gross changes to the NMDAR expression or function.
Although the direct impacts of Ro25 are likely limited to synapses onto interneurons, persistent alterations to circuit function following prolonged Ro25 dosing could involve other parts of the circuitry. Therefore, we examined excitatory drive to CA1 pyramidal neurons following prolonged Ro25 dosing and washout, and observed , and the lower graph shows LTP in Ts65Dn mice (n ¼ 6, 7), with wt vehicle data replotted in gray for comparison. (b) Population spike (PS) LTP from the same experiments as in panel a is shown. (c) Binned EPSP LTP data from the last 10 min following 1, 2, or 3 Â TBS in the presence of PTX is shown. There were no significant effects of genotype or treatment, and planned comparisons did not show any significant deficits in EPSP LTP in Ts65Dn mice or any effects of Ro25. (d) Binned PS LTP data in the presence of PTX is shown. There were no significant effects of genotype or treatment, and planned comparisons did not show any significant deficits in PS LTP in Ts65Dn mice or any effects of Ro25.
GluN2B roles in interneurons and circuit function J Hanson et al enhanced basal synaptic transmission and output spiking in wt mice, but not in Ts65Dn mice (Figure 6c-e). This selective increase in excitatory synaptic function was also present when synaptic inhibition was blocked with PTX (Supplementary Figure S7), indicating that the enhanced synaptic excitation is not secondary to alterations to synaptic inhibition. Thus, prolonged dosing with Ro25 can lead to lasting perturbation of the basal excitatory synaptic properties of neural networks in wt mice, which could potentially underlie the observed deficits in in vitro gamma oscillations.

Ro25 Dosing Differentially Affects Cognitive Behavior in Ts65Dn and wt Mice
To test whether the Ro25-induced alterations observed in vitro lead to changes in cognitive functions, we examined Y-maze performance in wt and Ts65Dn mice. Vehicletreated Ts65Dn mice made significantly fewer alternations than wt mice in the Y-maze (Figure 7a). Although other processes such as novelty-seeking could impact this measure, reduced alternations in this assay are consistent with working memory impairment. Acute Ro25 injections (50 mg/kg) 1 h before the Y-maze assay resulted in a marked genotype-dependent effect on the alternations: Ts65Dn mice performed significantly better than wt mice (Figure 7a). This difference was caused by a significant impairment in the wt mice rather than an improvement in the Ts65Dn mice with Ro25. Furthermore, this effect was not due to altered activity levels by Ro25. Although Ts65Dn mice exhibited hyperactivity in general, there was no significant change in arm entries following Ro25 injection in wt or Ts65Dn mice (Figure 7a). This genotype-dependent effect on the Y-maze performance with Ro25 was replicated in a second study by an independent laboratory (SFBNL) using 10 or 50 mg/kg Ro25 (Supplementary Figure S8). These Y-maze results demonstrate a differential impact of acute Ro25 on wt vs Ts65Dn mice in vivo. On the other hand, prolonged Ro25 dosing did not affect the Y-maze performance of either genotype (Figure 7b). Prolonged treatment did affect cognitive behavior, however, as assessed with the Barnes maze, a distinct test of spatial memory in which Ts65Dn mice also show deficits (Figure 7c). In the Barnes maze, prolonged Ro25 treatment reduced performance in Ts65Dn mice but not in wt mice (Figure 7c), again emphasizing the genotype-and treatment regimen-dependent effects of Ro25. Overall, although cognitive impairment in the Ts65Dn mice was not rescued by GluN2B antagonism, these behavioral experiments provide in vivo confirmation of several of the major insights of the electrophysiological analysis as follows: (1) Ro25 has acute impacts; (2) prolonged Ro25 dosing induces lasting changes; and (3) both of these phenomena are genotype-dependent.

DISCUSSION
Alhough much previous work on GluN2B antagonists has focused on their neuroprotective roles in pathological situations, we have described novel impacts of GluN2B antagonism under normal physiological conditions via contribution to excitatory drive of interneurons. Antagonizing GluN2B-NMDARs led to differential changes in LTP and gamma oscillations in wt vs Ts65Dn Down syndrome model mice. Acute GluN2B antagonism also led to impaired memory performance in wt mice without altering memory deficits in Ts65Dn mice. The importance of GluN2B-NMDARs to normal circuit function is further emphasized by the lasting changes to neural circuit function following prolonged antagonist dosing.

Cell-Type Specific Impacts of GluN2B Antagonism
We found that GluN2B antagonists inhibited synaptic NMDARs on s. radiatum interneurons but not excitatory and Ts65Dn slices (purple, scale bars: 0.1 mV/100 ms). The total gamma power in wt mice during the 1-s epoch following tetanization is shown as mean ± SEM, *po0.05. There was a significant interaction between genotype and treatment (p ¼ 0.01), and planned comparisons showed that Ro25-treated wt slices had significantly less power than vehicle-treated wt slices (po0.05), whereas Ro25-treated Ts65Dn slices had significantly more power than vehicle-treated Ts65Dn slices (po0.05). (b) Average spectrograms from wt and Ts65Dn slices treated with Ro25 or vehicle (n ¼ 10 wt veh, 13 wt Ro25, 8 Ts65Dn veh, 11 Ts65Dn Ro25) show the differing levels of total power.  Prolonged Ro25-6981 (Ro25) dosing persistently rescues long-term potentiation (LTP) in Ts65Dn mice. (a) 2.5-3-month-old mice were injected with 50 mg/kg Ro25 or vehicle daily for 2 weeks, followed by at least a 2-week washout period before LTP was measured as in Figure 2 (over a period of 2.5 weeks). (b) EPSP LTP in slices from mice treated with vehicle or Ro25 in response to sequential bouts of 1, 2, or 3 Â theta burst stimulation (TBS) is shown. The upper graph shows LTP in wild-type (wt) mice (n ¼ 16, 12), and the lower graph shows LTP in Ts65Dn mice (n ¼ 11, 14), with wt vehicle data replotted in gray for comparison. (c) Population spike (PS) LTP from the same experiments as in panel c is shown. (d) Binned EPSP LTP data from the last 10 min following 1, 2 or 3 Â TBS is shown. There was a significant interaction between genotype and treatment (po0.01). Planned comparisons showed that LTP was significantly reduced in slices from vehicle-treated Ts65Dn mice compared with vehicle-treated wt mice (1 Â , po0.05), and that Ro25 treatment significantly enhanced LTP in Ts65Dn mice compared with vehicle (1 Â , po0.01; 2 Â , po0.05; 3 Â , po0.05). Wt vehicle data from panel b is in gray. All data in this figure is shown as mean±SEM. (e) Binned PS LTP data is shown. There was a significant effect of genotype on PS LTP. Planned comparisons showed that PS LTP was significantly reduced in vehicle-treated Ts65Dn mice compared with wt mice (1 Â , po0.01; 2 Â , po0.01; 3 Â , po0.01) and that Ro25 significantly rescued PS LTP in Ts65Dn mice treated with Ro25 compared with vehicle (1 Â , po0.05). All data is shown as mean±SEM, *po0.05, **po0.01.

GluN2B roles in interneurons and circuit function J Hanson et al
That GluN2B-NMDARs function is selective to s. radiatum vs s. pyramidale interneurons is consistent with the much longer time course of EPSPs in s. radiatum interneurons. These interneurons provide feed-forward inhibition of pyramidal neurons (Maccaferri and Dingledine, 2002) and have axonal projections that are associated with Schaffer collateral inputs to pyramidal neurons (Vida et al, 1998), and are thus positioned to regulate depolarization of pyramidal neurons during synaptic activation. Therefore, reduction of excitatory inputs to s. radiatum interneurons by GluN2B antagonism could affect the overall level of inhibition of pyramidal neurons, and thereby gate functions like synaptic plasticity. At the same time s. radiatum interneurons also provide inhibition to other interneuron types within CA1 (Chamberland and Topolnik, 2012), and could cause additional indirect impacts to circuit function when their activation is reduced by GluN2B antagonism.

GluN2B Antagonism Reduces Inhibition and Alters Circuit Function
We found that Ro25 reduced synaptic inhibition of pyramidal neurons and rescued LTP deficits in Ts65Dn mice that have excess inhibition. The rescue of LTP by Ro25 appears to be mediated via effects on inhibition, as Ro25's effects in Ts65Dn mice are occluded in the presence of the GABA A R blockers. We speculate that the gamma oscillation deficits that we report here for the first time could also be caused by excess inhibition in Ts65Dn mice, as they were also rescued by Ro25. Although we cannot exclude a role of extrasynaptic GluN2B-containing NMDARs in the circuit Although there wasn't a significant interaction between genotype and treatment (p ¼ 0.096), planned comparisons showed that oscillation power in slices from Ro25-treated wt mice was significantly lower than that from vehicle-treated wt mice (po0.05), whereas Ro25 treatment did not alter oscillation power in Ts65Dn mice. (b) Average spectrograms from recordings in wt and Ts65Dn mice treated with Ro25 (n ¼ 11 wt veh, 8 wt Ro25, 12 Ts65Dn veh, 14 Ts65Dn Ro25), show greatly reduced power in Ro25-treated wt mice. (c) Measurement of excitatory synaptic strength. Stimulus intensity (logarithmically spaced) vs EPSP magnitude relationships are shown for wt mice treated with vehicle or Ro25 (left). Example EPSP recordings are inset (scale bars: 2 mV/50 ms). The right panel shows simultaneously measured population spike (PS) data (n ¼ 13 vehicle, 14 Ro25; po0.05). (d) Stimulus intensity vs EPSP (left) and PS magnitude (right) relationships are shown for Ts65Dn mice treated with vehicle or Ro25 (n ¼ 13 vehicle, 15 Ro25). Data from vehicle-treated wt mice from panel a is shown in gray for comparison. (e) Maximal EPSP and PS responses are plotted for all genotype and treatment groups under baseline conditions. There was a significant interaction between genotype and treatment for both EPSP and PS responses (po0.05), and planned comparisons showed that both EPSP and PS magnitudes were significantly greater in slices from Ro25-treated wt mice compared with vehicle-treated wt mice (po0.05). Slices from vehicle-treated Ts65Dn mice did not significantly differ from vehicle-treated wt slices, and Ro25 treatment did not significantly alter maximal EPSP or PS magnitude in slices from Ts65Dn mice compared with vehicle treatment. Data is shown as mean ± SEM, *po0.05.

GluN2B roles in interneurons and circuit function
J Hanson et al level phenomena, the disinhibition achieved by the action of Ro25 at synaptic NMDARs on interneurons alone would be sufficient to explain the rescue of LTP and gamma oscillation in Ts65Dn mice. In wt slices, disinhibition likely leads to suboptimal function by shifting the balance between excitation and inhibition towards excessive excitation, and could underlie the reduction of wt gamma oscillation power with Ro25. Although we did not observe an impact of Ro25 on fast spiking interneurons, which directly control gamma oscillations (Sohal et al, 2009), our finding of altered oscillations with Ro25 is consistent with recent findings also demonstrating a contribution of GluN2B receptors to oscillations (Kocsis, 2012a;McNally et al, 2011), and suggests that s. radiatum interneurons could mediate this modulation.
In addition to acute impacts, we observed lasting impacts of prolonged dosing, which did not simply mimic the effects of acute application/dosing. Although acute GluN2B antagonism decreases inhibition, prolonged antagonism results in enhanced excitation in wt mice. This impact of prolonged dosing should cause a similar imbalance to circuit function and could account for the similar deficits in gamma oscillations in wt mice as seen with acute Ro25 application. We cannot exclude the possibility that the reported offtarget effects of Ro25, such as impacts on monoamine neurotransmission (Keiser et al, 2009), could contribute to some of the phenotypes seen with chronic treatment. However, that the rescue of LTP in chronically treated Ts65Dn mice resembles the effects of a similar treatment regimen using GABA A R antagonists (Fernandez et al, 2007) and is consistent with prolonged disinhibition by Ro25 being responsible for inducing lasting functional alterations.

Implications for Therapeutic use of GluN2B Antagonists
Our results highlight that the effects of GluN2B antagonism vary depending on disease state, as differential effects on circuit function and behavior were consistently observed in Ts65Dn vs wt mice with both acute and prolonged Ro25 treatment. That the specific consequences of GluN2B antagonist treatment are determined by the state of brain circuitry is in agreement with an in vivo study showing sleep-state dependent effects of Ro25 on gamma activity (Kocsis, 2012b), and the differential impacts of Ro25 on gamma oscillations in wt brain slices induced by a tetanus (our study) vs kainate application (McNally et al, 2011). The Ro25-6981 (Ro25) dosing differentially affects cognitive behaviors in wild-type (wt) and Ts65Dn mice, depending on dosing regimen (acute vs prolonged). (a) Y-maze alternations and entries are shown following acute Ro25 treatment. There was a significant genotype Â treatment effect on the Y-maze alternations following acute Ro25 treatment (po0.005; n ¼ 14/12 Ts65Dn and 14/13 wt vehicle/Ro25). Vehicle-treated Ts65Dn mice were impaired in percent alternations compared with wt mice (po0.05), indicating a deficit in Ts65Dn mice. However, Ro25-treated wt mice exhibited significantly impaired alternations compared with Ro25-treated Ts65Dn mice (po0.05). This was due to a significant impairment of alternations in Ro25-treated wt mice compared with vehicle-treated wt mice (po0.05), and no significant effect of Ro25 treatment on Ts65Dn mice. Although there was an effect of genotype on arm entries (po0.0001) reflecting increased activity of Ts65Dn mice, there were no genotype Â treatment or treatment effects on arm entries. (b) Y-maze alternations and entries are shown following prolonged Ro25 dosing. There was an effect of genotype (po0.01; n ¼ 20/ 19 Ts65Dn and 20/18 wt vehicle/ Ro25), with the Ts65Dn mice showing impaired alternations compared with wt mice, but there were no treatment or genotype Â treatment effects on alternations. There was also an effect of genotype on arm entries (po0.0001), with more arm entries in Ts65Dn mice than in wt mice, but there were no treatment or genotype Â treatment effects on entries. (c) Barnes maze performance across 3 days of training is shown following prolonged Ro25 dosing. All groups learned the task with improved latency, distance, and error levels across days of training (po0.0001; n ¼ 10/9 Ts65Dn and 9/8 wt vehicle/Ro25), but Ts65Dn mice showed significant impairments in both distance (po0.01) and errors (po0.0001) compared with wt mice. Significant genotype Â treatment effects were observed for latency (po0.05) and distance (po0.05). This reflected a significantly increased latency (po0.05) and distance (po0.05) in Ro25-treated Ts65Dn mice compared with vehicle-treated Ts65Dn mice, whereas Ro25 treatment did not significantly affect wt mice. All data is shown as mean±SEM.
GluN2B roles in interneurons and circuit function J Hanson et al results of our study suggest that a key aspect of the brain state that determines the effects of GluN2B antagonism could be the balance between excitation and inhibition.
In the context of Down syndrome, our in vitro results indicate that GluN2B antagonists compensate for excessive inhibition, and could potentially be useful for acute and persistent rebalancing of synaptic function. However, despite rescuing several electrophysiological measures in hippocampal slices, we did not observe acute or lasting improvement of cognitive deficits after Ro25 treatment in Ts65Dn mice with the doses/treatment regimens used. Thus, disinhibition of a subpopulation of interneurons would appear less powerful in rescuing cognitive functions than blocking all GABA A Rs, which has been shown to rescue Ts65Dn mouse memory deficits (Fernandez et al, 2007;Rueda et al, 2008). Nonetheless, although other approaches to manipulating inhibition may be more likely of benefit in Down syndrome, potential benefits of the circuit impacts of GluN2B antagonists bear further analysis in the context of other neurological diseases, including Alzheimer's disease that could involve altered inhibition (see Supplementary Discussion SI). In addition, in considering therapeutic application of GluN2B antagonists, impacts on circuit function must be considered alongside possible neuroprotective benefits.
In general, future studies will need to explore functions in not only excitatory neurons, but also in inhibitory interneurons and at the level of circuit function, to understand the diverse subunit-dependent contributions of NMDARs to normal physiological functions, as well as the potential benefits of their selective modulation in treating specific neurological diseases.


The Mechanisms of Epigenetic Modifications During DNA Replication

The term “epigenetics” was first used to describe “the causal interactions between genes and their products, which bring the phenotype into being” [1], and this definition initially refer‐ red to the role of the epigenetics in embryonic development, in which cells develop distinct identities despite having the same genetic information. However, today epigenetics refers to “the study of heritable changes in gene expression that occur independent of changes in the primary DNA sequence” [2]. This definition is now associated with in a wide variety of bio‐ logical processes, such as genomic imprinting [3,4], inactivation of the X chromosome [5], embryogenesis [6], tissue differentiation [7], and carcinogenesis [8].

Introduction
At the DNA replication step during cell division, not only fundamental information (i.e. nucleotide sequence) but also superficial information (i.e. "epigenetic" modifications) is faithfully reproduced on the newly synthesized DNA sequence. The faithful maintenance of the epigenetic pattern, which determines the gene-expression pattern of the cell, safeguards the maintenance of cell identity.
The term "epigenetics" was first used to describe "the causal interactions between genes and their products, which bring the phenotype into being" [1], and this definition initially referred to the role of the epigenetics in embryonic development, in which cells develop distinct identities despite having the same genetic information. However, today epigenetics refers to "the study of heritable changes in gene expression that occur independent of changes in the primary DNA sequence" [2]. This definition is now associated with in a wide variety of biological processes, such as genomic imprinting [3,4], inactivation of the X chromosome [5], embryogenesis [6], tissue differentiation [7], and carcinogenesis [8].
Epigenetic chemical modifications, such as DNA methylation and histone modifications, are known to be faithfully duplicated in each cell cycle and subsequently the chromatin structures are propagated through DNA replication [9]; however, little is known about how the chromatin structure is maintained during or reformed after DNA replication. Furthermore, several lines of recent evidence suggested that the superficial information on the DNA strand is more susceptible to change by environmental stress than the DNA strand itself. Therefore, for a better understanding of the DNA replication process, it is highly important and desirable, for biologists in general and molecular biology in particular, to learn about the epigenetic mechanisms.
In this chapter, we introduce the current understanding of the DNA methylation mechanism, 5-hydroxycytosine (the sixth base), histone modifications, and their significance in congenital and acquired diseases, and also discuss to which direction this field ought to proceed in the future.

DNA methylation during DNA replication
Not all genes are necessarily expressed in every cell of the organism. Most of these genes and genetic regions are programmed to remain repressed, which defines the identity of each cell. Epigenetic modifications are molecular mechanisms that can preserve the inactive state by regenerating a repressive chromatin structure on the "unnecessary genes and genomic regions" following each round of DNA replication in the cell. DNA methylation is one of the fundamental mechanisms known to be involved in this maintenance process [10].
Maintenance of such methylation pattern in DNA during replication is mediated by DNA nucleotide methyltransferase 1 (DNMT1) [11], which methylates newly synthesized CpG sequences, depending on the methylation status of the template strand ( Fig. 1). A bridging protein, known as UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), that interacts with DNMT1 and hemimethylated CpG is required to maintain the hemimethylated CpG dinucleotides pattern at the DNA fork [12,13]. Maintenance of DNA methylation pattern with 5-hydroxymethylcytosine (upper) and 5-methylcytosine (lower) during DNA replication and cell division. 5-hydroxymethylcytosine is relatively abundantly found in embryonic stem (ES) cells and its level decreases during development due to the declining levels of TET expression. Cytosine, 5-hydroxymethylcytosine and 5-methylcytosine are shown in white, blue and red circles, respectively [95].
The chromatin structure, modified by DNA methylation, is not stable, but it undergoes a wave of disruption and reassembly during DNA replication. These changes in the chromatin structures influence the dynamics of DNA replication by regulating the selection of replication origin sites and their initiation timings. Interestingly, active gene promoters are often found at these active replication origin sites. Thus, the coordination of replication and transcription is an important mechanism for the establishment and inheritance of differential gene expression patterns during cellular differentiation [2].
DNA methylation status is also involved in determining the chromosomal replication timing. Hypomethylation is associated with late-replication and late-replicating genomic regions are gradually demethylated with cell divisions, whereas DNA methylation of earlyreplicating regions is maintained during DNA replication [14]. Moreover, DNA replication in early S phase gets automatically repackaged with acetylated histones, whereas the regions that replicate late in S phase assemble nucleosomes containing deacetylated histones [15].
So far several DNA nucleotide methyltransferases (DNMTs), which includes DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L, have been found in mammals, all of which contain a methyltransferase catalytic domain. Of these, DNMT1 is the most abundant DNMT in differentiated cells; it has a preference for hemi-methylated DNA, and acts as a 'maintenance methylase', which allows it to efficiently methylate the hemi-methylated sites that are generated during DNA replication. Thus, the CpG methylation pattern is maintained in the genome after DNA replication [16]. Until recently, the biochemical and functional properties of DNMT2 remained unknown. However, the DNMT2 is now known to act as an RNA methylatransferase and the DNMT2-mediated methylation protects tRNAs against ribonuclease cleavage in drosophila [17].
DNMT3A and DNMT3B are expressed at high levels in mouse embryonic stem (ES) cells and at lower levels in differentiated cells. They act as 'de novo methylases', which catalyze the transfer of methyl groups to naked DNA, and are responsible for establishing the pattern of methylation during embryonic development [18]. Recent evidence suggests that besides playing the role as 'de novo methylases' DNMT3A and DNMT3B may also act as 'methylation completer' and 'methylation error corrector' -by completing the methylation process and correcting errors, respectively, left by DNMT1 -at least at highly methylated DNA regions, such as imprinted regions and repetitive elements [19].  Once a certain site is methylated, it could then act as a candidate region where the silent chromatin is established. For this purpose, the methylated site first recruits methyl-CpG binding domain (MBD) proteins; the MBD proteins subsequently recruit histone deacetylases, histone modification proteins. In other words, MBDs, which form bridges between the methylation site and other associated proteins, are the key proteins in epigenetic regulation (Fig. 2).
So far five MBD proteins, each containing a methyl-CpG binding domain, have been reported. Among these MBD proteins, MBD1 is unique because it is capable of repressing transcription from both methylated and unmethylated promoters [20].
MBD1 associates with chromatin modifiers such as the Suv39h1-HP1 complex, and enhances DNA methylation-mediated transcriptional repression [21]. MBD1 also associates with the H3K9 methyltransferase SETDB1 [22]. During S phase, the chromatin assembly factor CAF1 recruits the MBD1-SETDB1 complex to chromatin to establish new H3K9 methylation.
On the other hand, the removal of DNA methylation disrupts the formation of MBD1-SETDB1-CAF1 complex, which results in the loss of H3K9 methylation at the formerly methylated site [23].
MBD2 protein shares extensive sequence homology with MBD3. MBD2 binds to methylated CpGs and confer DNA methylation-mediated transcriptional silencing through its association with HDAC1 and HDAC2 in the NuRD chromatin remodeling complex [23]. Although Mbd2-null mice develops normally and remains viable and fertile [24], lack of Mbd2 affects immunological systems by inducing ectopic IL-4 expression in undifferentiated helper T cells [25]. Lack of Mbd2 also influences X-chromosome inactivation by inducing ectopic Xist expression in the active X chromosome [26].
MBD3, like MBD2, is an essential subunit of the NuRD complex. It has been suggested that MBD2 and MBD3 associate with the NuRD in a mutually exclusive way, thereby forming two distinct complexes [23]. Although there is a great sequence similarity between MBD2 and MBD3, the two proteins do not perform redundant functions during early development.
In contrast to Mbd2-mull mice which displayed a mild phenotype, MBD3-null embryos die on day 8.5, by failing to shut down the expression of undifferentiated cell markers such as Oct4 and Nanog [27].
MBD4 is a thymine glycosylase, which acts as a DNA repair protein and targets the sites of cytosine deamination. Spontaneous hydrolytic deamination of 5mC leads to 5mCpG-TpG transitions, whereas that of non-methylated CpG leads it to UpG, and MBD4 is able to excise and repair both 'mutated' nucleotides [28]. Consistent with this observation, Mbd4-null mice exhibit a two to three times higher number of 5mCpG-TpG transitions, indicating that Mbd4 indeed acts to reduce the 5mCpG-TpG mutation rate [29]. More importantly, when crossed with mice carrying a germline mutation in the Apc (adenomatous polyposis coli) gene, Mbd4-null mice show accelerated tumor formation [29]. In fact, mutations in MBD4 have been reported in various human carcinomas [30].
repressor like other MBDs, MeCP2 also acts as a splice regulator, by interacting with YB-1, a component of messenger ribonucleoprotein particles, in brain nuclear extracts [34]. Indeed, microarray splicing analysis of cerebral cortex mRNA isolated from Mecp2-mutant mice showed a number of aberrantly spliced genes [23]. Furthermore, MeCP2 deficiency activates L1 retrotransposition in neurons, which is possibly associated with the genomic diversity of brains [35]. Therefore, it is interesting that there exist several links between MBD-mediated repression, RNA processing and DNA-sequence diversity. It is also intriguing to find a link between epigenetic modification and its suppressive power on genetic diversity since, in addition to MeCP2, a histone modification enzyme (H3K9 methyltransferase, ESET) also contributes to silence retrovirus-like elements in the mammalian genome [36].

5-hydroxymethylcytosine -the sixth base in mammalian DNA
The 5-methylcytosine (5mC) has been recognized as "the fifth base". However, early work suggested the existence of a sixth base, 5-hydroxymethylcytosine (5hmC) (Fig. 1). 5hmC was first reported in T-even bacteriophages [37] and later in mammalian cells [38]. However, the reported finding, which claimed that this modified base accounted for ~15% of total cytosines in DNA extracted from the brains of adult rats, mice and frogs, could not be reproduced [39]. The topic received only little attention for the next 30 years until 2009, when work from two research teams brought it back to life [40,41]. Actually, it was found that 5hmC accounts for 0.6%, 0.2%, 0.03% of total nucleotides in Purkinje cells, granule cells, and mouse ES cells, respectively [40,41].
The presence of 5hmC in the mammalian genome depends on pre-existing 5mC, because 5hmC is converted from 5mC with the help of TET proteins, which utilize molecular oxygen to incorporate a hydroxyl group to 5mC. TET is named after Ten-Eleven Translocation (translocation between chromosomes 10 and 11) because it is initially found as a fusion protein partner of mixed-lineage leukemia gene (MLL) in acute myeloid leukemia (AML) patients carrying a t(10;11)(q22;q23) translocation [42,43]. The findings that ectopic expression of TET1 in HEK293 cells lacking TET1 led to reduced levels of 5mC and increased levels of 5hmC, and that the levels of 5hmC decreased upon RNAi-mediated depletion of TET1 in ES cells indicate that TET1 is able to catalyze the conversion of 5mC and 5hmC in cultured cells [41]. Also, it has been demonstrated that TET1 is capable of acting not only on fully-methylated DNA strands but also on hemi-methylated DNA strands [41]. Furthermore, not only TET1 but also other TET proteins (TET2 and TET3) is capable of converting 5mC to 5hmC [44].
In terms of gene regulation, the significance of this 5hmC modification is similar to that of non-methylated cytosine. In other words, the 5hmC modification is associated with transcriptional activity, which is different from the 5mC modification that is associated with transcriptional repression [16]. It has recently demonstrated that TET1-binding to the promoter region (presumably 5hmC modification at this site) induces the expression of Nanog in ES cells and that downregulation of Nanog via TET1 knockdown induces DNA methylation in the promoter region [44]. These findings indicate that the TET1 driven 5hmC modifi-cation contribute to maintenance of the nature of un-differentiation and pluripotency of ES cells, and support a working model by which TET1 and DNMTs coordinately regulate Nanog expression.
In ES cells, high levels of TET1 block the access of DNMTs for maintained Nanog expression. On the other hand, when TET1 is downregulated in ES cells by in vitro differentiation, DNMTs methylate the Nanog promoter, which leads to the downregulation of Nanog expression and loss of ES cell identity (Fig. 1) [44]. This hypothesis is supported by a recent finding in which the chromosomes containing 5hmC are gradually reduced during the development of preimplantation embryos [45]. However, another study showed that the 5hmC level in the mouse cerebellum during development increases from 0.1% of total nucleotides at postnatal day 7 to 0.4% of total nucleotides in the adult mouse [46].
As described above, TET1 was initially identified through a rare translocation case with leukemia [42,43]. Later studies have demonstrated that deletion and mutations in TET1, TET2 and TET3 are associated with myeloid malignancies [47]. In fact, mutations found in TET2 in myeloid cancers have been shown to impair hydroxylation of 5mC [48].
While our knowledge about 5hmC is rapidly growing, currently there is no reliable methodology available that would provide information on 5hmC at single-base-pair resolution. Although a 5hmC antibody is available for chromatin Immunoprecipitation, this method only provides some coarse information (i.e. detects presence of 5hmC but not that of 5mC in chromatin). A more sensitive method has been developed for 5hmC by capillary eletrophoresis, but this is not the one at sinlge-base-pair resolution [49]. Another method (namely, bisulfite sequencing) has proven to be a powerful tool for providing information on the methylation status at single-base-pair resolution. However, it too fails to discriminate between 5mC and 5hmC. Thus, if the bisulfite-treated DNA is used as a template for PCR analysis, cytosine will be read as thymine, whereas both 5mC and 5hmC will be read as cytosine [16]. Therefore, it is important to develop a methodology that can distinguish between 5mC and 5hmC at single-base-pair resolution in order to achieve complete understanding of the active demethylation mechanism, because TET protein-mediated 5mC oxidation may contribute to dynamic changes in global or locus-specific 5mC levels by promoting active DNA demethylation [50].

Histone modifications and DNA methylation during replication
DNA methylation and histone modifications not only occur separately, but they also work hand-in-hand at multiple levels to determine expression status, chromatin organization and cellular identity, and they are co-ordinately maintained through mitotic cell division, allowing for the transmission of parental DNA and for the histone modifications to be copied to newly replicated chromatin [51,52].
Lande-Diner et al. recently developed a DNMT1knockout cell line and demonstrated that an unmethylated state, caused by the lack of DNMT1, induced deacetylation of histones H3 The Mechanisms of DNA Replication and H4, resulting in transcriptional activation in many genes [10]. This observation clearly indicates that DNA methylation is associated with histone deacetylation. However, this group also demonstrated that in several other genes the unmethylated state, caused by lack of DNMT, did not induce histone H3 and H4 deacetylation, resulting in transcriptional repression. In addition, late replication in S phase was observed at these loci, suggesting that the replication timing may be independent of DNA methylation [10]. Rather, histone acetylation is associated in controlling the replication timing [53].
DNA methylation is not only correlated to histone 'acetylation', but also associated with histone 'lysine methylation'. Genome-wide DNA methylation profiles suggest that DNA methylation is associated with the absence H3K4 methylation and the presence of H3K9 and H3K27 methylation [54].
In fact, DNA methylation induces histone H3K9 methylation through an MBD, thereby establishing a repressive chromatin state [55]. SETDB1, a H3K9 trimethylation (H3K9me3) methyltransferase, contains a putative MBD domain with two conserved DNA-interacting arginine residues, which are also present in the MBD domains of MBD1 and MeCP2 and are known to make direct contact with the DNA in the structures of MBD1-DNA and MeCP2-DNA complexes [56,57]. This result suggests that SETDB1 acts as an H3K9me3 'writer' in corporation with DNA methylation 'reader'. Likewise, SUV39H1/2, another H3K9me3 'writer', interacts with HP1, the H3K9me3 'reader' to create a repressed status in their recruited genomic region [58]. These are the mechanisms for propagating and maintaining repressive chromatin marks on both DNA and histones during DNA replication.
A histone methyltransferase, in turn, can direct DNA methylation to specific genomic targets by recruiting DNMTs to stably silence genes [59]; accordingly, disruption of the histone lysine methyltransferase gene with specificity for H3K4 (MLL) in mice not only induces the loss of H3K4 methylation but also induces de novo DNA methylation at several gene promoters [60,61]. In another study, it was shown that the lack of histone H3K9 methyltransferase induced demethylation at the imprinting center in SNPRN locus on the maternal chromosome, whereas the lack of DNMT1 failed to induce demethylation of histone H3K9, indicating that the modification order at this locus is histone modification followed by DNA methylation [62]. Taken together, histone methylation marks play important roles in predicting the methylation status of the genome [63].
Whereas DNMT1 is stabilized by a histone demethylase (HDM) to maintain DNA methylation [64], DNMTs can direct the local status of histone methylation patterns, recruiting MBDs and HDACs to achieve gene silencing and chromatin condensation [65,66]. Recently, DNMT3L has been shown to act as a sensor for H3K4 methylation. Thus, when methylation is absent, DNMT3L induces de novo DNA methylation by docking DNMT3A to the nucleosome, which is one of mechanisms by which methylated regions are newly created during the replication step [67].
The interplay of these modifications creates an epigenetic landscape that regulates the way the mammalian genome expresses itself in different cell types, developmental stages and disease states. The distinct patterns of these epigenetic modifications present in different cel-lular states serve as a guardian of cellular identity [2]. Whereas it is well accepted that DNA methylation patterns are replicated in a semi-conservative fashion during cell division via the mechanisms discussed earlier, how histone modification patterns are similarly replicated remains to be elucidated.

Abnormalities in epigenetic mechanism and their possible inheritance
Thanks to identification of molecules that contribute to epigenetic gene regulation, we now know how that abnormalities in these molecules cause a number of congenital diseases.
The first group of diseases with abnormal epigenetic mechanism is genomic imprinting diseases [3]. Genomic imprinting is a mechanism in which only one of the two parental alleles is expressed in a gene. For example, in the case of SNRPN gene, the paternal allele of the SNRPN gene is expressed, whereas the maternal allele is suppressed by DNA methylation in normal individuals, and abnormal suppression of the normally expressing paternal allele causes a congenital obesity disease, known as Prader-Willi syndrome [4]. In the case of UBE3A gene, which locates adjacent to the SNRPN gene, the maternal allele is expressed, whereas the paternal allele is suppressed in neurons [68]; abnormal suppression of the expressing maternal allele causes a congenital epileptic disease, known as Angelman syndrome [69].
X-chromosome inactivation is another epigenetic mechanism in which only one of the two X chromosomes is activated and the other X chromosome is inactivated in females [5]. Females with aberrant X-inactivation (i.e. both two X chromosomes are activated) are thought to be embryonic lethal, since somatic clones with aberrant X-inactivation are aborted [70].
Abnormal functioning of the proteins related to epigenetic regulation also causes diseases. For example, mutations in the DNMT3B gene, which lead to hypomethylation at the paracentromeric chromosomal regions, cause the immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, which is characterized by immunodeficiency, centromere instability, facial abnormalities, and mild mental retardation (Fig. 3A) [71][72][73]. On the other hand, over-expression of DNMTs is associated with hypermethylation found in colorectal, breast, and hepatocellular carcinomas (Fig. 3C) [74][75][76]. Another example is Rett syndrome caused by MECP2 mutations, which is characterized by seizures, ataxic gait, language dysfunction and autistic behavior [77,78]. In this disease, MECP2 mutations induce abnormal regulation of a subset of neuronal genes [79,80] (Fig. 3B).
Besides these "DNA methylation diseases" caused by mutations in DNA methylation-related enzymes and proteins, "histone modification diseases" caused by mutations in histone modification-related enzymes have recently been reported. For example, Say-Barber-Bieseker-Young-Simpson syndrome is caused by mutations in the histone acetyltransferase gene, KAT6B, which is a multiple anomaly syndrome characterized by, an immobile mask-like face, abnormal narrowing of palpebral fissures (short eyelid), anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, agenesis of the corpus callosum, and severe intellec-tual disability [81]. Another example is Kleefstra syndrome caused by deletion or mutation in the histone H3K9 methyltransferase gene, EHMT1, which is characterized by childhood hypotonia, distinctive facial features, and intellectual disability with severe expressive speech delay [82].
Recently, it has been shown that short-term environmental stress could also cause aberrant epigenetic status associated with various diseases. Thus, aberrant epigenetic mechanism can not only cause congenital diseases, but can also cause acquired diseases. For example, shortterm mental stress after birth, in which the mother is separated from the offspring, causes DNA hypermethylation in the promoter of the glucocorticoid receptor (GR) gene in the rat brain, resulting in persistent abnormal behavior [83]. Malnutrition in the fatal period is also known to induce DNA hypomethylation in the promoter of the peroxisome proliferator-activated receptor alpha (PPARα) gene, a so-called "thrifty gene", in the liver, which may be associated with the developmental basis of adult diseases (i.e. obesity and diabetes mellitus) [84,85] (Fig. 3D). This hypomethylation event has later been confirmed in human individuals who suffered prenatal malnutrition during the period of famine [86,87]. Several lines of evidence suggested that acquired DNA methylation changes described above are transmitted to the next generation. Epigenetic marks allow the transmission of gene activity states from one cell to its daughter cells. Initially, it was assumed that epigenetic marks were completely erased and re-established in each generation. However, recent studies using several model organisms indicate that the erasing process is incomplete at some loci and so the epigenetic changes acquired in one generation are inherited by the next generation.
For example, it has been shown in mice that the mental stress caused due to maternal separation in offspring not only changes the DNA methylation status in the first generation but also in the next generation through changes in the sperms of the first generation in mice [88]. Moreover, the observed changes in the DNA methylation status altered the expression level of corticotrophin releasing actor receptor 2 (Crfr2) in the brains of next generation mice, which could be associated with their abnormal behavior [88].
This phenomenon is termed "transgenerational epigenetic inheritance", which is expected to provide a biological proof for the apparent heritability of acquired characteristics [89][90][91].

Concluding remarks
One of the major differences between DNA sequence and epigenetic modifications is tissue specificity. Epigenetic modifications vary according to the tissue type, which consequently allows generating tissue-specific expression patterns. However, how determines the epigenetic modification (epigenomic) pattern in each tissue type is not fully understood.
Thus, it is essential to categorize epigenomic patterns in each human tissue at the nucleotide resolution [92,93]. In fact, the NIH Roadmap Epigenomics Program under the US National Center for Biotechnology Information (NCBI) and the International Human Epigenome Consortium (IHEC) have initiated the large-scale epigenomic mapping studies in order to generate epigenome maps for each human cell type for this purpose [94].
Understanding the human epigenome will be fundamental to the study of congenital and acquired diseases, and will also be invaluable for analyzing the linkage between birth defects and environmental factors. However, biological studies to understand the epigenome are in their initial phase. Further studies are necessary to elucidate the molecular mechanism by which the epigenome pattern in each cell type differs, epigenomic patterns are altered by environmental factors, and process of inheriting the epigenomic pattern from the previous generation could be avoided. The authors expect that these molecular mechanisms would hopefully be discovered by the "next generation" of researchers.


s2fieldsofstudy: Biology Medicine

text:
Impaired Baroreflex Function in Mice Overexpressing Alpha-Synuclein

Cardiovascular autonomic dysfunction, such as orthostatic hypotension consequent to baroreflex failure and cardiac sympathetic denervation, is frequently observed in the synucleinopathy Parkinson’s disease (PD). In the present study, the baroreceptor reflex was assessed in mice overexpressing human wildtype alpha-synuclein (Thy1-aSyn), a genetic mouse model of synucleinopathy. The beat-to-beat change in heart rate (HR), computed from R–R interval, in relation to blood pressure was measured in anesthetized and conscious mice equipped with arterial blood pressure telemetry transducers during transient bouts of hypertension and hypotension. Compared to wildtype, tachycardia following nitroprusside-induced hypotension was significantly reduced in Thy1-aSyn mice. Thy1-aSyn mice also showed an abnormal cardiovascular response (i.e., diminished tachycardia) to muscarinic blockade with atropine. We conclude that Thy1-aSyn mice have impaired basal and dynamic range of sympathetic and parasympathetic-mediated changes in HR and will be a useful model for long-term study of cardiovascular autonomic dysfunction associated with PD.

INTRODUCTION
Baroreflex failure is documented in patients with Parkinson's disease (PD) and is often considered as the root cause for observed orthostatic hypotension (1,2). Recently, orthostatic hypotension, baroreflex failure, and sympathetic denervation have been reported to occur early in PD, before the onset of overt motor symptoms, making cardiovascular autonomic dysfunction a potential PD biomarker for the development of diseasemodifying treatments (3)(4)(5)(6)(7). To date few studies have reported cardiovascular autonomic dysfunction in PD mouse models and the pathologic mechanisms underlying PD-related cardiovascular autonomic dysfunction remain to be elucidated (8-11).
In PD, Lewy bodies containing alpha-synuclein have been observed in peripheral sympathetic areas including stellate ganglia and the cardiac plexus, as well as in central brainstem areas important for regulation of blood pressure and peripheral vascular resistance and compliance (12)(13)(14). Patients with a form of familial PD associated with alpha-synuclein locus triplication show impaired baroreflex-cardiovagal gain and reduced cardiac sympathetic innervation suggesting a potentially important role for alpha-synuclein in the pathogenesis of autonomic dysfunction in PD (15). Therefore, in this study we sought to characterize baroreceptor function in transgenic mice overexpressing alpha-synuclein under the Thy1 promoter [Thy1-aSyn; (16)]. These mice have increased alpha-synuclein levels in central and peripheral neurons and develop proteinase K-resistant alpha-synuclein aggregates in multiple brain regions, including the substantia nigra, locus coeruleus, and olfactory bulb (16-18). Thy1-aSyn mice display both motor and non-motor impairments including gastrointestinal and olfactory dysfunction reminiscent to that observed in PD (18)(19)(20)(21)(22). In addition, by 14 months of age, these mice have a significant decrease in striatal dopamine and l-DOPA responsive behavioral deficits (23). In the present study, we assessed morphometry parameters, baroreceptor reflex, and left ventricular contractility in Thy1-aSyn and wildtype (WT) mice.

ANIMALS
Animal care was conducted in accordance with the United States Public Health Service Guide for the Care and Use of Laboratory Animals, and procedures were pre-approved by the Institutional Animal Care and Use Committee at the University of California Los Angeles. Transgenic mice overexpressing human alphasynuclein under the Thy1 promoter (Thy1-aSyn) were crossed into a hybrid C57BL/6-DBA/2 background (16). Animals were maintained on the hybrid C57BL/6-DBA/2 background by breeding mutant females with male mice on the hybrid background (18, 24,25). Littermates were never inbred. The genotype of all Thy1-aSyn and WT mice was confirmed with polymerase chain reaction (PCR) amplification analysis of DNA from tail tips at birth and www.frontiersin.org confirmed at the end of the experiment. All mice were individually housed with free access to water and standard rodent chow.
Male mice from a total of 21 litters were used in this study. Litter sizes ranged from 2 to 11 mice. Separate cohorts of mice were used in the hemodynamic-baroreflex protocol (Thy1-aSyn = 9, WT = 9) and for the heart rate (HR) and contractility dobutamine challenge protocol (Thy1-aSyn = 7; WT = 7) at 9-12 months of age. An additional separate cohort of mice (Thy1-aSyn = 3-4, WT = 3-5) was implanted with arterial transducers and tested at 3-5 months of age. A younger cohort in the arterial transducer experiment was included because the survival rate in older animals was too low.

MORPHOMETRY
Following hemodynamic assessment surgeries Thy1-aSyn and WT mice were euthanized and heart, lung, and liver weights were measured and tibia length determined.

HEMODYNAMIC ASSESSMENT
Hemodynamic assessment was performed as previously described (26)(27)(28)(29). Briefly, in Thy1-aSyn and WT mice anesthetized with ketamine, xylazine, and buprenorphine, both femoral arteries were catheterized with flame-stretched PE-50 tubing (30). One catheter was interfaced to a pressure transducer connected to the PC computer data acquisition system. The other catheter was used for drug injections. The functionality of the sympathetic and parasympathetic branches of the autonomic nervous system were independently evaluated by intravascular injections of the vasoconstricting α-1 adrenergic receptor agonist phenylephrine (5 and 25 µg) and the vasodilating nitric oxide donor sodium nitroprusside (5 and 15 µg). This was followed by autonomic nervous system blockers glycopyrrolate (a peripheral acting quaternary amine muscarinic receptor antagonist, 20 µg) and propranolol (β-adrenergic receptor antagonist, 50 µg) and reinfusions of phenylephrine and sodium nitroprusside to confirm abolition of the baroreceptor reflex. All drug injections were administered intrarterially in a volume of 100 µl of 0.9% aqueous saline heparinized at 7 U/ml and injected over 7 s. HR was determined from the beat-to-beat systolic pressure peak intervals.

DOBUTAMINE CHALLENGE
We used dobutamine to probe the integrity of the cardiac β-1 adrenergic receptor as reflected in HR and rate of left ventricular pressure change during systole (inotropic response) and diastole (lusitropic response). Thy1-aSyn and WT mice were anesthetized with ketamine, xylazine, and buprenorphine and placed on a warming pad. HR and temperature were continuously monitored during surgery. Once anesthetized, the right carotid artery was catheterized and a 1.4 Fr catheter (Millar Instruments, Houston, TX, USA) was advanced into the left ventricle to obtain a dynamic pressure signal that was mathematically differentiated into in vivo indices of cardiac muscle inotropy (+dP/dT ) and lusitropy (−dP/dT ) (26). A femoral artery was also catheterized as above for injection of dobutamine, a β-1 adrenergic receptor agonist. Dobutamine was injected in serial doses of 3, 6, 15, and 30 ng/g with at least a 5-min recovery period between injections as an adrenergic post-synaptic receptor challenge test. Measurements were acquired with Hem Software (Notocord Systems V4.2, Croissy sur Seine, France). HR and left ventricular pressure were recorded 150 s after injections and averaged over a 10-s period. Maximum and minimum dP/dT were calculated during this 10-s period.

TELEMETRIC ASSESSMENT
Arterial pressure and cage activity was measured simultaneously in awake, freely moving Thy1-aSyn and WT mice using radio telemetry transducers (TA11PA-C10; Data Sciences International Inc., St. Paul, MN, USA). Transmitter units were implanted subcutaneously along the mouse's ventral side under the ketamine, xylazine, and buprenorphine anesthesia. The transmitter blood pressure catheter was fed over the shoulder and inserted into the left carotid artery under sterile surgical conditions (30). Data recording from the mice began immediately following implantation via an antenna receiver under the cage connected to a computer system. Pressure waveforms and activity data were collected, analyzed, and displayed with the telemetry software. For all studies, 20 s of data were collected every 10 min for the duration of the study. Data waveforms and parameters were analyzed with the DSI analysis program (ART 4.0) to determine differences in systolic, diastolic, and mean arterial pressures plus cage activity and HRs between Thy1-aSyn and WT mice during the entire light-dark diurnal cycle. Data for the hours surrounding the weekly scheduled cage changing were excluded from analysis. The data were averaged at the same time of day to create "foldagrams" to reveal any variances (dysfunction) and compare changes between groups under baseline conditions (30).
We tested tonic vagal release of acetylcholine at the muscarinic receptor of cardiac pacemaker cells with the muscarinic antagonist atropine [1 mg/kg, ip; (31)] administered to both Thy1-aSyn and WT mice. We documented blood pressure and baroreceptor reflex induced HR changes after phenylephrine [3 mg/kg, ip; (32)] and sodium nitroprusside [1 mg/kg, sc; (33)]. Each drug was administered at the same time of day at least 2 days apart to allow for complete drug clearance from the ip injection. Saline was used as the vehicle and administered before each drug injection. Blood pressure and HR were recorded continuously in the home cage, following saline, and following drug administration.

DATA EXPRESSION AND STATISTICS
The hemodynamic experiment plots (Figures 1A-F) depict the baseline and acute post-drug pressure and HR data pairs as means and standard errors, computed as a function of condition, based on the nine mice in each experimental cell. The slope and R 2 statistics were derived with standard bivariate linear regression analyses, determined from the raw systolic and beats per minute data pairs, as a function of the genotype × drug conditions, but collapsed across the three doses of each drug (i.e., n = 27 for each genotype × drug cell). These and the chronic telemetric data are also expressed as the ratio of the change in HR in BPM to the change in systolic blood pressure in mmHg, or ∆HR/∆SBP. For the dobutamine challenge test and cardiac contractility experiments data are expressed as left ventricular +dP/dT max and −dP/dT min . A mixed design ANOVA was used to compare genotypes and dose of drug in the hemodynamic and contractility experiments. Student's t -test or Mann-Whitney U were applied Frontiers in Neurology | Movement Disorders FIGURE 1 | Systolic pressure and heart rate at baseline (squares) and in response to phenylephrine (A) and sodium nitroprusside (B) in anesthetized WT (gray; n = 9) Thy1-aSyn (Black; n = 9) mice at 9-12 months of age. Mice received 5 µg (circles) and 25 µg (diamonds) of intravenous phenylephrine and 5 µg (circles) and 15 µg (diamonds) of intravenous sodium nitroprusside. The slope and R 2 values were derived from four separate regression equations, based on all available pairs of systolic blood pressure (mm/Hg) and heart rate (beats per minute) scores for each condition and drug, rather than the condition means and standard errors that are depicted in the plot. The baroreceptor sensitivity index (∆HR/∆SBP) following serial doses of phenylephrine alone and phenylephrine (25 µg/kg) following autonomic blockade with glycopyrrolate and propanolol (C). ∆HR/∆SBP following serial doses of sodium nitroprusside alone and sodium nitroprusside (15 µg/kg) following autonomic blockade with glycopyrrolate and propanolol (D). *, **Represents p < 0.05, 0.01, respectively compared to WT at the same dose. Heart rate measurements at baseline (0.0 µg/kg) and following serial doses of phenylephrine [5 and 25 µg/kg; (E)]. Heart rate measurements at baseline (0.0 µg/kg) and following serial doses of sodium nitroprusside [5 and 15 µg/kg; (F)]. *Represents p < 0.01 compared to WT at same dose, DD represents p < 0.01 compared to baseline (0.0) within same genotype (Student's t -test and mixed design ANOVA followed by Fisher's LSD).
www.frontiersin.org to analyze morphometry parameters and arterial telemetry data between Thy1-aSyn and WT mice. Fisher's Least Significant Difference was used for post hoc analyses. All statistics were calculated with GB-Stat software (Dynamic Microsystems, Inc., Silver Spring, MD, USA, 2000). The level of significance was set at p < 0.05.

MORPHOMETRY PARAMETERS
Morphometry measures included measurements of body, heart, liver, and lung weights, and tibia length in Thy1-aSyn and WT mice ( Table 1). Comparisons between WT and Thy1-aSyn mice using Student's t -test showed that Thy1-aSyn mice weighed significantly less than WT mice, a difference we reported previously in this mutant line (19). While there were no significant differences in any of the other measures, heart and liver weights tended to be smaller in Thy1-aSyn mice but the difference did not reach significance (p = 0.08 and p = 0.06, respectively).

BASELINE HEART RATE AND SYSTOLIC BLOOD PRESSURE
Baseline systolic blood pressure measurements did not differ between conscious or anesthetized WT and Thy1-aSyn mice (p > 0.05). However, baseline HR measures were significantly different between genotypes under anesthesia during the acute hemodynamic studies ( Table 2); Thy1-aSyn mice had a higher HR compared to WT mice (p < 0.05). In contrast, conscious WT and Thy1-aSyn mice equipped with arterial telemetry had comparable baseline HRs during both the light and dark cycles ( Table 2).

HEMODYNAMIC ASSESSMENT
The baroreflex response to the injection of phenylephrine and sodium nitroprusside in anesthetized Thy1-aSyn and WT mice are plotted in Figures 1A,B. Though the plots show averaged pressure and heart values for each condition for clarity, the individual baseline systolic pressures were measured over a wide range of values from 48 to 159 mmHg in WT mice and from 41 to 157 mmHg in Thy1-aSyn mice. Linear regression analysis from the raw data pairs (n = 27 pairs per study) are drawn through the averaged data points under each condition in Figures 1A,B. These data are also plotted as the ∆HR/∆SBP score (baroreflex sensitivity index) in Figures 1C,D. These data clearly show that phenylephrine produced a similar increase in systolic blood pressure and reflex decrease in HR in WT and Thy1-aSyn mice resulting in ∆HR/∆SBP scores that did not differ between genotypes over this range of baseline pressures ( Figure 1C). As expected with combined β-adrenergic and muscarinic receptor blockade, the ∆HR/∆SBP response to phenylephrine following β-adrenergic and muscarinic blockade also did not differ between genotypes ( Figure 1C). In contrast, the response to sodium nitroprusside in WT mice showed the characteristic decrease in systolic blood pressure and increased HR, while in Thy1-aSyn mice systolic blood pressure decreased but HR remained close to their initial baseline values regardless of dose (Figures 1B,D). Here, Thy1-aSyn regression slope was very shallow and ∆HR/∆SBP scores were significantly lower compared to WT mice (p < 0.05). The ∆HR/∆SBP response to sodium nitroprusside following β-adrenergic and muscarinic blockade did not differ between genotypes ( Figure 1D).  Analysis of HR in WT and Thy1-aSyn mice showed phenylephrine caused similar HR decreases in both genotypes following administration of phenylephrine [ Figure 1E; F (1,16) = 4.33, p > 0.05 for genotype and F (2,32) = 39.47, p < 0.01 for drug dose]. However, analysis of HR following sodium nitroprusside revealed that WT but not Thy1-aSyn showed significant increases in HR after each dose [ Figure 1F; F (1,16) = 1.69, p > 0.05 for genotype and F (2,32) = 25.65, p < 0.01 for drug dose, and F (2,32) = 7.34, p < 0.05 for genotype × drug dose interaction].

TELEMETRIC ASSESSMENT
Because Thy1-aSyn mice had a higher HR at baseline under anesthesia we wanted to determine if the differences we observed were due to an abnormal response to the anesthesia or to impaired baroreflex. Therefore, a separate cohort of Thy1-aSyn and WT mice were implanted with arterial telemetry transducers. Due to poor post surgery survival at the 9-to 12-month age in Thy1-aSyn mice the separate cohort of mice for telemetry was younger in age, 3-5 months. Similar to the hemodynamic experiment Frontiers in Neurology | Movement Disorders under anesthesia, conscious mice were challenged pharmacologically with intraperitoneal phenylephrine and nitroprusside (ip). In addition the response to muscarinic blockade was measured using atropine. At 3-5 months of age baseline HR and systolic blood pressure did not differ between WT and Thy1-aSyn mice. Similar to the anesthetized experiment WT and Thy1-aSyn mice displayed comparable responses to phenylephrine and nitroprusside over a similar range of systolic pressures. There was no difference between the genotypes in ∆HR/∆SBP or HR scores following phenylephrine injections (p > 0.05; Figures 3A,D). However, in response to sodium nitroprusside Thy1-aSyn mice once again showed a blunted HR response while WT showed characteristic tachycardia. Thy1-aSyn ∆HR/∆SBP scores were significantly decreased compared to WT (p < 0.05; Figure 3B). Analysis of HR alone showed that HR significantly increased in WT but not in Thy1-aSyn mice following nitroprusside (p < 0.05; Figure 3E). In response to muscarinic blockade ∆HR/∆SBP scores did not significantly differ between genotypes however, analysis of HR alone showed that HR significantly increased in WT but not in Thy1-aSyn mice following atropine (Figures 3C,F). The activity response did not significantly differ between genotypes in any of the drug conditions (Student's t -test, p > 0.05).

DISCUSSION
Baroreflex failure has been reported in both sporadic and alphasynuclein associated familial forms of PD (4,15). While cardiovascular autonomic dysfunction is well established in PD, little is known about the underlying cause of the dysfunction. Until recently, there has been a lack of basic research on the nonmotor impairments associated with, and often present very early in humans with PD. However, with the potential early occurrence of many of the non-motor symptoms, a keen interest in these symptoms as possible early disease biomarkers has developed. For example, impaired HR variability observed in REM sleep behavior disorder may be an early indicator of PD (7). In the present study we show that overexpression of human alpha-synuclein in mice leads to impaired baroreflex control of HR prior to changes in striatal dopamine content and reminiscent of the baroreflex dysfunction observed in PD (1,2). These findings are important because they provide a foundation for future studies aimed at identifying brain structures and pathological mechanisms associated with cardiovascular autonomic dysfunction in PD. In addition, these results underscore the usefulness of Thy1-aSyn mice in the study of early non-motor symptoms associated with PD.
Overall, with the exception of body weight, morphometry measures did not statistically differ between genotypes. We have previously reported decreased body weight in Thy1-aSyn compared to WT mice at 12 months of age without differences in overall food intake (19). We did however note a trend for heart and liver weights to be decreased in Thy1-aSyn mice.
The most profound functional differences were observed in response to administration of the vasodilator sodium nitroprusside. Here, anesthetized WT mice displayed the characteristic tachycardic response to the sudden drop in blood pressure produced by sodium nitroprusside, over a wide range of initial baseline values. But the Thy1-aSyn mice showed no significant change in HR during an equivalent transient decrease in systolic blood pressure suggesting impairments in autonomic modulation of cardiac function in Thy1-aSyn mice at an early age, before reductions in striatal dopamine content are observed (23). During these experiments it was noted that Thy1-aSyn mice appeared more sensitive than WT to the anesthetic combination of ketamine, xylazine, and buprenorphine. This is consistent with a recent study in the same Thy1-aSyn mouse line where the authors report increased sensitivity to the common rodent anesthetic xylazine (22). Due to this increased sensitivity it is possible that the HR differences observed in the Thy1-aSyn mice could be due in part to the anesthesia and not impaired baroreflex function. Therefore, we performed an additional set of experiments where WT and Thy1-aSyn mice were equipped with arterial telemetric transducers to allow HR and SBP measurements in conscious/awake mice. In this experiment we found that baseline HR and SBP were not different between genotypes suggesting the difference seen in anesthetized mice may have been a consequence of abnormal response to the anesthesia, or only develop at a later age. However, HR and SBP responses to phenylephrine and sodium nitroprusside were similar to what we observed in the previous experiments under anesthesia. In Thy1-aSyn mice sodium nitroprusside administration resulted in a significant decrease in SBP and a blunted HR response. Typically, during a drop in blood pressure efferent sympathetic outflow www.frontiersin.org is increased (34,35). Thus, in the present study the blunted HR response to sodium nitroprusside suggests the sympathetic component of the baroreflex is impaired in Thy1-aSyn mice over a wide range of ages. In PD patients with or without orthostatic hypotension, a similar blunted HR increase has been reported in response to the feet down tilt maneuver and in the Valsalva maneuver (5,36,37). It has been proposed that the combination of baroreflex failure and widespread noradrenergic denervation contributes to the development of orthostatic and post-prandial hypotension and supine hypertension commonly seen in PD (38).
Adrenergic supersensitivity in response to sympathetic denervation has been reported in patients with PD (39). Therefore, similar to Nakamura et al. (39) we tested left ventricular contractility of the heart in response to administration of the β-adrenergic agonist dobutamine in WT and Thy1-aSyn mice. There were no significant differences in inotropy or lusitropy between WT and Thy1-aSyn mice indicating no evidence of receptor supersensitivity. This is consistent with a recent finding showing no differences in contractility in patients in the early stages of PD (40).
Acetylcholine released from parasympathetic nerves reduces HR by binding primarily to M2 subtype muscarinic receptors on sinoatrial nodal cells in the heart. Blockade of M2 muscarinic receptors results in an increase in HR (31). In the present study WT but not Thy1-aSyn mice showed a significant increase in HR in response to the systemic muscarinic antagonist atropine. In addition, although not statistically significant (p = 0.08), WT mice showed a mean increase in HR in response to glycopyrrolate while Thy1-aSyn mice showed on average a decrease in HR in response to the peripheral muscarinic antagonist. The abnormal response to atropine in Thy1-aSyn mice is similar to a recent study measuring HR variability parameters in mice that express A53T mutant human alpha-synuclein under the Thy1 promoter. Here, the authors show increased baseline HR and reduced change in HR in response to systemic atropine in A53T compared to WT mice suggesting parasympathetic dysfunction in the mutant mice (11). However in contrast, mice with A53T or A30P mutations in alphasynuclein generated using PAC transgenesis showed no alterations in HR variability parameters at 12 months of age, perhaps due to the low level of expression of the transgenic protein (10). The abnormal cardiovascular response to atropine taken together with the impaired HR response to sodium nitroprusside and previous work showing gastrointestinal dysfunction in Thy1-aSyn mice suggest impairment of both sympathetic and parasympathetic systems in Thy1-aSyn mice (19, 20). Thus, the Thy1-aSyn mice show a potentially broader range of autonomic anomalies than previously reported in other mouse models of synucleinopathy (10, 11).
The pathological mechanisms underlying cardiovascular autonomic dysfunction in PD remain unclear. In PD sympathetic denervation is observed in patients with and without orthostatic hypotension and has been reported to develop before and after the diagnosis of PD (4,38,41). In addition to sympathetic denervation alpha-synuclein containing Lewy bodies can be observed in cardiac and brain regions important for sympathetic regulation of blood pressure and HR in humans suggesting that alpha-synuclein pathology may contribute to cardiovascular autonomic anomalies commonly observed in PD (12-14). In the brain Frontiers in Neurology | Movement Disorders of Thy1-aSyn mice human alpha-synuclein mRNA expression is high throughout the brainstem and proteinase K-resistant alphasynuclein inclusions develop in the locus coeruleus and substantia nigra (16, 17). In a recent study in the same line of Thy1-aSyn mice human alpha-synuclein protein was observed in the ventricular and atrial walls of the heart localized within noradrenergic fibers (22). Thus, it is unclear at this point whether the functional impairments are associated with either peripheral or central pathology or a combination of both. Functional deficits in central noradrenergic and dopaminergic systems have been observed in this mouse line (23,24,42, Maidment and Chesselet, unpublished observations) and dysregulation of central noradrenergic and dopaminergic systems has been shown to alter baroreceptor function in rodents suggesting central catecholamine system dysfunction could be a contributing factor (8, 27). Sodium nitroprusside, in addition to its peripheral vasodilator actions, is also known to activate central noradrenergic neurons in the locus coeruleus (43). Interestingly, the most profound impairment in Thy1-aSyn mice was a lack of nitroprusside-induced tachycardia, which was shown in both anesthetized and awake conditions. However, further research is needed to determine the exact contribution of peripheral and central noradrenergic and dopaminergic dysfunction in the autonomic regulation of blood pressure and HR in Thy1-aSyn mice.

CONCLUSION
The present study shows that Thy1-aSyn mice, a model of synucleinopathy, have impaired baroreflex control of HR analogous to what is observed in patients with the synucleinopathy PD (5,36,37). Furthermore, the cardiovascular autonomic dysfunction detected in the Thy1-aSyn mice occurs prior to dopaminergic alterations in the striatum. Similarly in PD cardiovascular autonomic dysfunction is also reported to occur early, before the onset of overt motor symptoms. While the mechanisms contributing to cardiovascular anomalies in PD remain to be established the Thy1-aSyn mice provide a novel genetic model in which to study the contribution of alpha-synuclein pathology to autonomic dysfunction in PD.