Low-frequency sound localization depends on the neural computation of interaural time differences (ITD) and relies on neurons in the auditory brain stem that integrate synaptic inputs delivered by the ipsi- and contralateral auditory pathways that start at the two ears. The first auditory neurons that respond selectively to ITD are found in the medial superior olivary nucleus (MSO). We identified a new mechanism for ITD coding using a brain slice preparation that preserves the binaural inputs to the MSO. There was an internal latency difference for the two excitatory pathways that would, if left uncompensated, position the ITD response function too far outside the physiological range to be useful for estimating ITD. We demonstrate, and support using a biophysically based computational model, that a bilateral asymmetry in excitatory post-synaptic potential (EPSP) slopes provides a robust compensatory delay mechanism due to differential activation of low threshold potassium conductance on these inputs and permits MSO neurons to encode physiological ITDs. We suggest, more generally, that the dependence of spike probability on rate of depolarization, as in these auditory neurons, provides a mechanism for temporal order discrimination between EPSPs.
Animals can locate the source of a sound by detecting microsecond differences in the arrival time of sound at the two ears. Neurons encoding these interaural time differences (ITDs) receive an excitatory synaptic input from each ear. They can perform a microsecond computation with excitatory synapses that have millisecond time scale because they are extremely sensitive to the input's "rise time," the time taken to reach the peak of the synaptic input. Current theories assume that the biophysical properties of the two inputs are identical. We challenge this assumption by showing that the rise times of excitatory synaptic potentials driven by the ipsilateral ear are faster than those driven by the contralateral ear. Further, we present a computational model demonstrating that this disparity in rise times, together with the neurons' sensitivity to excitation's rise time, can endow ITD-encoding with microsecond resolution in the biologically relevant range. Our analysis also resolves a timing mismatch. The difference between contralateral and ipsilateral latencies is substantially larger than the relevant ITD range. We show how the rise time disparity compensates for this mismatch. Generalizing, we suggest that phasic-firing neurons—those that respond to rapidly, but not to slowly, changing stimuli—are selective to the temporal ordering of brief inputs. In a coincidence-detection computation the neuron will respond more robustly when a faster input leads a slower one, even if the inputs are brief and have similar amplitudes.
Copyright: © 2010 Jercog et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by National Institutes of Health (NIH) grants DC008543 and MH62595 to PEJ, GS, and JR and also NIH grant DC006864 to DHS and VCK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In order to localize acoustic objects along the horizontal plane, the nervous system is able to distinguish microsecond differences in the arrival time of sound at the two ears, referred to as interaural time differences (ITDs). Low sound frequencies are the most useful signals for detecting ITDs, and animals with good sensitivity below 1,500 Hz tend to perform best at this perception . In mammals this computation is first performed by medial superior olivary neurons (MSO) in the auditory brain stem. Each MSO neuron receives two sets of excitatory inputs on its bipolar dendrites, with each set activated by one ear. When both excitatory pathways are activated within a narrow time window, the MSO neuron detects the coincident excitatory synaptic inputs and fires action potentials. When the pathways are activated asynchronously, the MSO neurons do not respond. Thus, an ITD response function is the representation of the variation of MSO discharge rate with the relative delay of the two inputs and, therefore, the position of a sound along the horizontal plane .
One influential theory holds that ITD encoding is based on an arrangement of axonal delay lines . In this model, the differences in the sound's time of arrival at the two ears is transformed into a spatial map of ITD detecting neurons, sometimes referred to as a "place" code. Thus, an MSO neuron would discharge maximally when a specific ITD is exactly compensated by an internal delay that arises as a consequence of differences in the length of axons that are driven by the two ears. In fact, evidence for this mechanism has been found in birds and mammals –. However, since the discharge rate of many MSO neurons increases over the physiological range of ITDs –, this information could also be used to encode the azimuthal position, sometimes referred to as the "slope" code . Additionally, there is evidence to suggest that inhibitory inputs to MSO play a role in tuning the response function within the physiological range of ITDs ,.
In previous models of ITD processing, the propagation time between the ipsi- and contralateral ears to the MSO neurons is implicitly assumed to be equal (excluding Jeffress's internal delay lines). However, MSO neurons are positioned to one side of the brainstem, and the ipsilateral pathway is expected to be shorter than the contralateral. For example, one study has shown in vivo that many superior olivary neurons display longer latencies for the contralateral pathway . Thus, any mechanism that relies on temporal precision must take this into account. We have tested this premise using a novel in vitro preparation that preserves each pathway. Our results support a new mechanistic explanation for the compensation of a longer contralateral response latency, and the positioning of the ITD response function in the physiological relevant range. The mechanism takes advantage of a difference in the dynamics of ipsi- and contralateral excitatory synaptic inputs. Using a computational model, we demonstrate that these asymmetric excitatory synaptic dynamics can significantly alter the ITD responses of MSO neurons.
Asymmetries in circuit architecture can have a significant effect on ITD processing. Specifically, the contralateral projections from ventral cochlear nucleus (VCN) to MSO are longer than those from the ipsilateral side (Figure 1A, difference in afferent lengths between ipsilateral VCN to MSO and contralateral VCN to MSO ≈2.45 mm; Paul Nakamura and Karina Cramer, personal communication). To measure this difference functionally we used a thick brain slice preparation from gerbils that preserves the afferent pathways to the superior olivary complex (Figure 1A; see Methods). Whole cell recordings were obtained from MSO neurons while activating each pathway at the same anatomical position on each side; the pathway between the stimulation point and the cochlea, which is eliminated in this preparation, is assumed to be identical for each side (Figure 1A). We first found that the response latency did, in fact, differ between the two pathways. An analysis of evoked postsynaptic potentials (PSPs) and currents (PSCs) showed that the latencies to peak of contralateral responses were on average about 500 µs longer than those of ipsilateral responses on the same recorded neuron (Figure 1B,C; average differences in latency to peak for PSPs: 573±62 µs, n = 54; for PSCs: 589±81 µs, n = 37, see Methods section). This difference was apparent on a cell-by-cell basis because the difference of latencies (contralateral - ipsilateral) was significantly different than zero (see gray bars in Figure 1B).
Figure 1. Time difference processing in gerbil MSO in vitro.
(A) Schematic of thick slice preparation (500 µm) through the ventral auditory brain stem. Afferent projections from the ipsi- and contralateral VCN are segregated on MSO dendrites. MNTB inhibitory afferents provide contralaterally evoked inhibition to MSO neurons. It is important to recognize that the stimulating electrodes are placed at a position on the auditory pathway that has the same axonal length to the ears, respectively, on each side. (B) PSPs- and PSCs-latency to peak responses (different population sets: voltage clamp data were recorded with intracellular cesium and QX-314). Population's average and standard deviation to show the range of MSO delays. Statistical intervals of confidence were expressed using t test with respect to zero for the difference between ipsilateral and contralateral responses on the same neuron (right column: Cont-Ipsi). (C) Average and standard deviation of PSPs and PSCs (average of 50 trials per neuron) for two different sample neurons. The superimposed traces show that the contralateral response occurs at a longer latency. (D) ITD response function in vitro. Bilateral stimulation of VCN afferents elicits action potentials in MSO neurons through coincidence detection of the bilateral PSPs. Gerbil's physiological relevant range (gray bar). Spikes were counted at different stimulation delays to mimic physiological ITD response functions (Measured). Based on the average delay between ipsilateral and contralateral PSPs latency to peak, the ITD response function should be maximal at 580 µs on the contra-leading side ("Predicted" curve is hypothetical, based on bilateral PSP-peak coincidence). The ITD response function peak is close to zero-delay when the bilateral PSPs are summated by the neuron, creating a paradox between the predicted and measured responses. (E) Average and standard deviation of PSCs (average of 50 trials for a sample neuron). The superimposed traces show that the ipsilateral rising slope is steeper. (F) Population data for PSC slopes. Statistical intervals of confidence were expressed using t test versus zero for the difference between ipsilateral and contralateral responses on the same neuron (right column: Cont-Ipsi). PSCs in (E) are marked with colored circles. Difference in PSCs is our explanation for the paradox stated in D.
In presenting the following experiments, we refer to the in vitro inter-stimulus time difference as ITD. Thus, if threshold were to depend solely on PSP amplitude, then the measured disparity in PSP latencies would predict that the peak ITD response would occur when the contralateral PSP leads by approximately 500 µs (Figure 1D; predicted, thin curve). This ITD value is sufficiently large that the response function would fall largely outside of the physiological range for gerbils, which is ±130 µs . In contrast, we found ITD response functions in which MSO firing rate was maximal when bilateral stimuli were delivered with smaller delays of ≈100 µs (Figure 1D; measured, thick curve). This finding suggests that an intrinsic integration mechanism must compensate for the longer contralateral path.
MSO neurons are exquisitely sensitive to the rate of depolarization. Therefore, in order to understand the integration of subthreshold bilateral inputs that lead to a spike, we examined the dynamics of synaptic inputs. Our starting assumption had been that synaptic properties are identical for each of the two excitatory inputs to MSO. We examined this assumption by measuring the rising PSP slopes because their time scale is within the same range as the coincidence detection window as manifested by the width of the ITD response function (i.e., 0–250 µs). Ipsilaterally evoked PSCs had significantly steeper rising slopes than contralateral PSCs (Figure 1E,F) (ipsilateral: 1.04±0.15 nA/ms, contralateral: 0.62±0.06 nA/ms; p = 0.01, n = 35). This difference was apparent on a cell-by-cell basis because the difference of PSC slopes (contralateral - ipsilateral) was significantly different than zero (see gray bar in Figure 1F). This result was independent of stimulus amplitude in all tested neurons (see Figure S1). The differences in the slopes of the PSCs could compensate, in part, for the disparity in delay between the two pathways. Our computational model (below) showed that even a modest asymmetry in rising slopes could shift the ITD response function from its hypothetical position (based on latencies to peak) to the observed location in the in vitro experiment (Figure 1D).
To determine how this asymmetry in excitatory synapse kinetics might compensate for the differences in path length, it was first necessary to determine the contribution of synaptic inhibition. To address this issue, we obtained ITD response functions under current clamp (CC), before and after application of a glycine receptor antagonist, strychnine (SN). As shown in Figure 2A and 2B, when synaptic inhibition was present (control), the maximal firing occurred for contralateral leading stimulation, consistent with in vivo recordings –. When synaptic inhibition was blocked (Figure 2A and 2B, SN) the maximal firing rate was close to zero ITD, also consistent with an in vivo study . We calculated the ITD at which peak firing probability occurred ("best ITD") for the population of recorded neurons (Figure 2C) and found that under control conditions the peak was at 105±35 µs (contra-leading), while under SN conditions it was at −62±38 µs (ipsi-leading). Therefore, the effect of synaptic inhibition was to shift ITD tuning towards contralateral leading stimuli. Since this shift is in the wrong direction to compensate for the longer contralateral path, we next considered the role of asymmetric excitatory responses.
Figure 2. Effect of asymmetric PSPs and EPSPs in setting best ITD position.
(A) Voltage time courses of somatic depolarization under bilateral stimulation at different delays (example cell, P21, T = 32°C). Each column shows three representative trials for a specific ITD value. The dashed lines show the spike identification-threshold for our experiments, and these were used to plot the ITD functions shown in (B). When glycinergic inhibition was blocked (SN), firing rate increased for ipsilateral leading ITD stimulations (compare column 1 (1 spike in 3 trials) with column 3 (3 spikes in 3 trials)). (B) ITD response function for control case and when blocking synaptic inhibition (SN) for 10 stimulation trials. Shaded area is the physiologically relevant range (±130 µs). (C) Best ITD response for different cells under control and with SN. Predicted best ITDs for control should be around 580 µs on average. Instead our data show that best ITD responses are closer to a delay of zero. When inhibition is blocked the best ITDs shift towards ipsi-leading responses (N = 12, postnatal days 17–25; disconnected points are from experiments that were performed either in control or under SN only). (D) Increase in slope when inhibition is present. Slopes for normalized EPSP (SN, no-inhibition) are: ipsi 2.61±0.11 ms−1(square), contra 2.21±0.14 ms−1 (triangle); p = 0.031, n = 17. When inhibition is present (Control) PSP slopes are: ipsi 2.71±0.12 ms−1, contra 2.49±0.10 ms−1; p = 0.064, n = 17. Stronger effect of the inhibition on contralateral responses (contra-inputs difference 0.28 ms−1, p = 0.05, n = 17; ipsi-inputs difference 0.10 ms−1, p = 0.05, n = 17). Bilateral EPSPs are even more asymmetric than the PSPs, suggesting a possible explanation for why best ITDs are more shifted to the ipsi-leading side under SN (see below model results, Figure 5).
In the presence of inhibition (control), the ipsilaterally evoked normalized PSP slope was 2.71±0.12 ms−1 and the contralateral slope was 2.49±0.10 ms−1 (Figure 2D). When inhibition was blocked (SN), evoked EPSP slopes were significantly different between ipsi- and contralateral responses (ipsilateral: 2.61±0.11 ms−1; contralateral: 2.21±0.14 ms−1, see Figure 2D and also Figure S2). Blockade of glycinergic inhibition increases the differences in the PSP slopes. More specifically, inhibition always increases the slope (Figure 2D, from squares to triangles), but more so for the contralateral responses (Figure 2D, right column). Such steepening occurs for either fast or slow inhibitory synaptic conductance transients (see Figure S3 for theoretical support). In the fast case (Figure S3, left), the decaying brief IPSC coincides with rising EPSC and the summed current therefore rises faster than the EPSC alone. The effect is stronger on contralateral inputs because the IPSC will more fully decay during the EPSC rise. In the slow case, the IPSC transiently reduces the effective time constant, accelerating the rise although less dramatically than does a fast IPSC (Figure S3, right). The effect is stronger for contralateral inputs partly because integration of slower inputs is affected more by time constant changes (leakage matters in addition to capacitive integration). Another major contributing factor related to active currents is explained below with our model. Thus, we confirmed that synaptic inhibition reduced the effect of shifting the ITD response function towards zero ITD, and leads us to suggest that the compensation arises from the excitatory asymmetry described above (Figure 1).
How can such a small asymmetry in EPSP slope influence ITD sensitivity in MSO neurons? We addressed this question by using a computational MSO neuron model that was driven by bilateral trains of excitatory and inhibitory inputs temporally modulated with a periodic function representing VCN responses to pure tone stimuli. Each cycle's composite input was generated from many small excitatory postsynaptic conductances (EPSGs) with statistics that depended on VCN afferent activity that varied with sound frequency and amplitude (see Methods; ,). Figure 3 shows a simplified version of the simulated MSO inputs to illustrate the variability of the composite EPSGs and integrated EPSPs due only to the jitter on the mini-EPSGs time release. Here, we exclude firing rate modulation throughout the sinusoidal input's cycles, although it is employed in the detailed model used for the simulated ITD functions. Using only differences in vector strength of the simulated inputs from the VCN arriving to each dendrite of the MSO neuron model we modeled differences in rising slope of the bilateral EPSPs (Notice: without delaying the composite EPSP peak, see triangles in Figure 3 for EPSG peaks). These differences led to shifts in the ITD response function that are large enough to compensate for the longer contralateral input pathway. For a given EPSG input, the evoked EPSPs and spike threshold will be determined by the active currents. In MSO and other auditory processing centers, a low threshold potassium current (IKLT) exerts control on spike threshold –. This fast IKLT imposes a filtering effect on the synaptic inputs allowing only steep EPSG slopes to evoke an action potential ,. Therefore, steeper EPSGs are more likely to trigger spikes, even when shallower EPSGs may have greater amplitude, as is shown in our simulations.
Figure 3. Example of composite EPSGs for an input train (500 Hz) using an idealized auditory nerve fiber model (sinusoidally modulated Poisson rate for mini-EPSG times).
It is less realistic than Carney's model (1993) that we used for Figures 4 and 5. (A) Raster plots with event times for ipsilateral excitatory inputs (red) and contralateral excitatory inputs (blue). Superposition of composite EPSGs for the two cases. Mini-EPSGs are alpha functions with time constant of 0.1 ms (see Methods). The difference in vector strength (degree of synchrony) between the events dictates the shape of the composite EPSGs. (B) Average over 25 cycles of composite EPSGs for the two different input cases. In the two cases a function (dashed) was fitted to the average PSG. Alpha function is the simplest description of PSGs (long dashes); it fits the rising phase but not the falling phase. For our summated EPSGs a more complicated function (see below) matches better the envelope or composite EPSG (in the case of excitation). EPSGs integrated through our MSO neuron model give EPSPs rising slopes that are steeper when vector strength is larger (normalized ipsi-EPSP's slope 2.78 ms−1, contra-EPSP's slope 2.12 ms−1). Composite EPSGs in (B) are fitted by functional form that is proportional to (1−(exp(−(t−t0)/τrise))).3.(exp(− (t−t0)/τdecay)). Ipsilateral inputs (red): τrise = 2.5, τdecay = 0.14. Contralateral inputs (blue): τrise = 2.8, τdecay = 0.18. For the alpha functions fits, for ipsilateral inputs (red): τ = 0.48; for contralateral inputs (blue): τ = 0.69. All τ values in ms. Note: the position of the peak of the summated EPSP does not depend on vector strength (see dark triangles in B).
When bilateral subthreshold inputs arrive at an MSO neuron, there is a higher probability of eliciting a spike when the steeper EPSG arrives first. Figure 4A shows how a pair of EPSGs, one fast and one slow, can produce a very different outcome, depending on their order of arrival. When a faster input arrives first this will enable spike generation (Figure 4A and 4B, left side). When a slower input arrives earlier it leads to a slower rising EPSP that recruits more IKLT conductance, which hinders spike generation even though a faster EPSG arrives subsequently (Figure 4A and 4B, right side).
Figure 4. The differential dependence of spike probability on EPSP's slope disappears when IKLT is disabled.
(A) Pairings of EPSGs with different time constants and different temporal order. Left: steep EPSG (red curve) arrives earlier than shallow EPSG (blue curve). The summated EPSG pair (steeper then shallower; ipsi then contra) leads to a spike. Right: same EPSGs with reverse order but same time difference does not generate a spike. Inputs' delays different than zero in which steeper EPSP is arriving earlier will generate more spikes than the inputs' delays with the opposite order of arrival time. (B) Voltage time courses (black curves) for the case of spike and non-spike illustrated in (A). GKLT (orange and light blue curves) corresponding to the same two temporal ordering cases. Amount of GKLT recruited (arrows) for the same voltage amplitude in the two time order cases. The deflection in voltage prior to spike generation is indicated by a dashed line. Notice: the amount of GKLT is higher in the case of the shallower EPSP leading; this yields the same proportional increase for IKLT since driving force is the same in both cases. For a given pair of EPSPs, the steeper EPSP arriving first recruits less GKLT and therefore decreases spike threshold level. (C) ITD function shifting due to asymmetric EPSPs. Simulation for ITD detection with asymmetric EPSPs using Carney's (1993) synaptic input model (bilateral EPSP trains at 500 Hz). Colored time courses: schematics of the combination of EPSP-pairs (without spike; vertical lines are to identify relative time delays), three cases: ipsi-leading (steeper then shallower, A), perfect coincidence ITD = 0 (B), and contra-leading (C). Pairs in (A), (B), and (C) cases have different rising slope. Maximum firing rate is generated for the steepest rising EPSP-pair (steep ipsi-EPSP leading) as explained in (A). (D) Differential dependence on EPSP's slope disappears when IKLT is disabled. Thin black curve: ITD function for bilaterally symmetric inputs (vector strength, R = 0.9). Thick black curve: ITD function for asymmetric inputs (RIPSI = 0.95, RCONTRA = 0.63), same curve as in (C). Thick brown curve: GKLT is frozen at its rest value preserving the model's passive properties (IKLT behaves as a passive current). For comparison, EPSGs are adjusted to give the same spike probability as in the previous case.
To show the essence of the ITD response function shift due to the asymmetry in the kinetics of the excitatory inputs we delivered inputs to the model with different vector strength (Figure 3) and calculated their probability to evoke spikes for different input delays (ITD response function, Figure 4C,D). If the contralateral composite EPSP was slower-rising, the bilateral combined EPSP had different rising dynamics when the ipsilateral inputs led than when the contralateral inputs led (Figure 4C, EPSPs schematics). Consistent with previous findings ,–, the shallower-leading combined EPSP was associated with a lower probability of firing. Therefore, the ITD function shifted towards the ipsilateral leading side (Figure 4C). The asymmetry in firing rate probability caused by an asymmetry in inputs' rising slopes is due to the voltage-dependence of IKLT conductance. We explain this (Figure 4D) by showing that with the same set of bilateral asymmetric EPSPs that generate a shift of ∼400 µs (Figure 4D, thick black curve), the shift of the ITD's response function disappears (Figure 4D, brown curve) if we fix the IKLT conductance at its resting value, in order to maintain the neuron model's time constant and input resistance intact.
We next asked whether the asymmetry in the excitatory inputs could compensate for an intrinsic input delay of ≈500 µs as measured in our in vitro preparation. The simulations showed that the integration of hypothetical symmetric EPSPs led to an ITD response function that was shifted to the contralateral leading side due to the intrinsic contralateral axonal delay (Figure 5, thin black curve). When asymmetric EPSGs were introduced in the model to generate EPSP slopes similar to those found in our experiments, the ITD function shifted towards the ipsilateral-leading direction due to the favorable response when a steep EPSP occurs first (Figure 5, thick black curve).
Figure 5. Model prediction of ITD response function using experimental data.
R is the vector strength of the monolateral presynaptic input. Bilateral input trains of 500 Hz. Thin black curve: simulated ITD function if EPSGs are symmetric. Longer contralateral delay of 500 µs, as our experimental data show. If the pre-synaptic afferents are symmetric, each with R = 0.90, the rising phase (only) of the summated EPSGs was fit by an alpha-function with τexc = 0.14 ms (EPSP-slope = 2.75 ms−1). Thick black curve: same contralateral delay as the previous case but with asymmetric excitatory inputs. Contra- is shallower than ipsi-, slope-ipsi-EPSP = 3.1 ms−1 (R = 0.93, mini-EPSG-τexc = 0.1 ms), slope-contra-EPSP = 1.6 ms−1 (R = 0.60, EPSG-τexc = 0.1 ms); for EPSP shapes see also Figure S4. Violet curve: adding contralateral inhibition preceding excitation by 0.2 ms shifts ITD function towards contralateral leading inputs; slow IPSPs (IPSP-slope = 0.6 ms−1 (R = 0.5, IPSG-τinh = 0.4 ms)). Orange curve: faster IPSP than previous case creates larger shift (IPSP-slope = 0.95 ms−1 (R = 0.7, IPSG-τinh = 0.4 ms)).
Our experimental data were consistent with this theoretical explanation: most of the neurons displayed this asymmetry in excitatory inputs. Thus, when we subtracted contralateral slope from ipsilateral slope for each individual neuron, the average difference was 0.69±0.18 nA/ms for EPSCs and 0.40±0.12 ms−1 for normalized EPSPs. Inclusion of synaptic inhibition made the simulated EPSPs less asymmetric. The hyperpolarization from inhibition transiently reduced IKLT. The reduction of this conductance would no longer favor spike generation when fast EPSPs are followed by slow EPSPs. The ITD response function was reduced on the ipsilateral-leading side, giving the appearance of a shift towards the contralateral-leading side (Figure 5, orange and violet curves), as observed experimentally in vitro (Figure 2B) and as reported previously in vivo ,.
Our experimental and computational findings identified key biophysical factors that, together, position the ITD response function in the biologically relevant range. We first confirmed the presence of an internal delay of the longer contralateral pathway (Figure 1B). In itself, this would cause MSO neurons to fire mostly to ITDs with stimuli having large contralateral leading stimuli that are outside the physiological range. Our experimental and computational results suggest a novel excitatory synaptic mechanism that could compensate for the disparity in path length. An asymmetry in the slopes of EPSPs (Figure 2D) can bias the ITD coding in favor of the ipsilateral-leading inputs (Figures 4 and 5), and this repositions the ITD function within the physiological range, as found in vivo –.
The presence of a fixed internal latency difference will affect all models of ITD processing. Jeffress  assumed tacitly that the two paths were equal in length except for the small differences along one spatial axis of the encoding nucleus. Others have suggested that the shorter path length from the ipsilateral ear is compensated by an additional span of axon (e.g., see schematic in ), or a difference in myelination between the two pathways . If the difference in path length to MSO for the gerbil is ≈2.45 mm (Paul Nakamura and Karina Cramer, personal communication), then our electrophysiological measurements of response latency difference of 500 µs would yield a propagation speed of 4.9 m/s. Thus, it appears that there is an internal latency difference to gerbil MSO that is not compensated for by an axonal property. It is this functional characteristic that must be addressed if MSO neurons are to encode ITDs in the physiological range (±130 µs; ).
Our electrophysiological measurements indicate that the rising PSP slope is larger for the ipsilateral input to MSO neurons on a cell-by-cell basis (Figures 1 and 2). The functional implications for this finding are illustrated in a computational model which demonstrates that this property can compensate for the aforementioned difference in path length (Figure 5). The general principle, which is that the rising slope of an EPSP determines the probability of firing, is consistent with findings from other systems ,,,. Here, we have adapted this principle to resolve the general problem of compensating for different input latencies due to path length.
How might the EPSP asymmetry arise? In the model we allowed for more jitter in the arrival times of identically shaped unitary (minimal) EPSPs on the contralateral side, which slowed the rise of the composite EPSPs. This idealization, for demonstrating plausibility in the context of our point neuron model, could be elaborated and explored in a neuron model that has bilateral dendrites with cable properties . Many alternative mechanisms are also possible. Bilateral differences in dendritic morphology or the dendritic positioning of excitatory terminals could also lead to an asymmetry in the rising slope of composite EPSPs ,. Although longer electrical distances would promote broadening of composite EPSPs in a passive dendrite, IKLT in the dendrites can reduce the effect by shortening the tail of EPSPs as they propagate toward the soma in MSO neurons and cable models . Alternatively, the distribution of active currents could modulate the dendritic integration of synaptic inputs. For example, dendritic sodium channels are able to selectively boost EPSPs on one dendrite, and this would modify their rising slope (cortex: ).
It is important to consider the in vivo time scale of inhibition and excitation because it will determine the temporal integration window and the extent to which ITD curves will be affected by the mechanisms described above. It is possible that the time scales in vivo are faster than in the brain slice because a cell is in a high conductance state (e.g., many more active inputs as compared to brain slice). In addition, the degree of afferent synchrony could have been unnaturally high in our preparation because the stimulus simultaneously recruits all VCN afferents to MSO. However, the model demonstrated that the effect of slope is robust when implemented with vector strength values that have been reported in vivo (Figure 3; using model from ). Since we also showed that synaptic inhibition somewhat counteracts the shifting effect of the asymmetric excitation, it is important to consider its kinetics. The time scale for inhibition has only been studied in vitro, and even the fastest IPSPs have either been recorded from animals between 12 to 25 postnatal days , or at room temperature . Interestingly, we found that while the magnitude of the inhibitory effect depends on IPSP time scale, it is likely to play an important role in ITD coding no matter what the actual time scale value turns out to be (Figure 5; Figure S3).
The faster rising EPSPs that were elicited by ipsilateral afferents could overcome the penalizing effect of a rapidly activating outward current like IKLT (Figure 4B). Many previous reports have demonstrated a robust effect of IKLT on the integration time of EPSPs ,,. In this study, we applied this property to anatomically independent bilateral inputs and demonstrated computationally that IKLT influenced the ITD function.
Together, our findings lead us to propose a general principle. Passive neuronal integration to a threshold would not distinguish the temporal ordering in inputs that may have different rising slopes. Subthreshold dynamic negative feedback such as IKLT (comparably as fast as integration) will bias the integration. Firing will be favored when the steeper-rising input occurs first. Inhibition, by deactivating the negative feedback, can reduce the bias. The competition between these two effects in the MSO, leads to a positioning of the ITD response function with its slope in the physiological range, as seen in vivo . Thus, the synaptic property compensates for the intrinsic latency disparity. Time-difference encoding could exploit these mechanisms in this extremely short window of integration time (130 µs) or, more generally, in other windows where the biophysical components and time scales are appropriately matched. Generalizing, we propose a novel neuronal mechanism for temporal order selectivity. Subthreshold dynamic negative feedback can increase a neuron's firing probability to segregated subthreshold inputs when faster ones precede slower ones, even if the slower one is of similar or larger amplitude.
All protocols were reviewed and approved by New York University Institutional Animal Care and Use Committee. Postnatal day (P) 17–25 gerbils (Charles River) were used to generate thick (450–500 µm) horizontal slices (N = 91) from the ventral auditory brainstem. Each slice contained the MSO nucleus, the medial nucleus of the trapezoid body (MNTB), and the lateral nucleus of the trapezoid body (LNTB). Animals were deeply anesthetized (chloral hydrate, 400 mg/kg), perfused intracardially with artificial cerebrospinal fluid (ACSF: 123 mM NaCl, 4 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 24 mM NaHCO3, 15 mM glucose, 2.4 mM CaCl2, 0.2 mM ascorbic acid; pH = 7.35 after bubbling with 95% 02/5% CO2) at 32°C. The brain was then dissected free in 32°C oxygenated ACSF, and one horizontal slice was obtained with a Leica vibratome. The slice was incubated at 36°C for 20 min, and at 22°C for 1 h before being transferred to the recording chamber where oxygenated ACSF was perfused at a rate of 2 ml/min at 32°C; temperature was regulated by L&N temperature controller.
The afferents arising from both VCNs were visualized as compact bundles. Thus, ipsilateral and contralateral bundles were stimulated at the site of their origins with bipolar tungsten electrode and stimulation was delivered by two stimulus isolation units (Dagan). The distance between the MSO and the two stimulation sites was approximately 0.5 mm for the ipsilateral pathway and 1.5 mm for contralateral pathway. Whole cell current-clamp recordings were obtained mostly from medial and dorsal MSO neurons (Axoclamp2A). The recordings and stimulation were computer driven (Windows XP) through Labview software (National Instruments). The neurons were visually identified using infra-red differential interference contrast (IR-DIC) microscopy (Olympus). The internal patch solution contained (in mM) 127.5 potassium gluconate, 0.6 EGTA, 10 HEPES, 2 MgCl2, 5 KCl, 2 ATP, 10 phosphocreatinine (Tris salt), and 0.3 GTP (pH 7.2) in the case of CC protocol and (in mM) 127.5 cesium gluconate, 0.6 EGTA, 10 HEPES, 2 MgCl2, 5 KCl, 2 ATP, 5 QX-314,10 phosphocreatinine (Tris salt), and 0.3 GTP (pH 7.2) in the case of voltage clamp (VC) protocol. In order to block synaptic inhibitory inputs, we used SN in CC experiments and SN and bicuculine to block glycinergic/gabaergic inputs in VC experiments.
EPSPs in CC and EPSCs in VC were recorded when single square pulses repeatedly (20 Hz) of 25–50 µs were delivered via the stimulating electrodes to initially evoke minimum amplitude responses, maximum amplitude subthreshold responses, and subthreshold-unilateral/suprathreshold-bilateral responses. High stimulus currents (0.5 to 10.0 mA) and short pulse durations (25–50 µs) were used to avoid the overlap of stimulus artifact with evoked responses. The data were analyzed following these basic criteria: slopes of the rising phase (20% to 80%) of the responses, for unilateral stimulations. For all the parameters that were measured for bilateral stimulations responses (i.e., peak-delay, slope), the intervals of confidence (p values) were computed using t test over the difference between ipsi- and contralateral responses on the same neuron. All data variability is expressed in standard deviation. In addition, 100 to 500 Hz stimulus trains of 10 stimuli were applied (total number of spikes per train delay were counted) to generate ITD tuning response function. A minimum of four trials were run to get a smooth ITD response function. In the case of CC data the slopes of PSPs and EPSPs were computed when bilateral responses were similar in amplitude, to avoid differential effect of active currents, and were normalized to decrease population variability due to biophysical heterogeneity among neurons.
We used a computational model of MSO neurons based on the parameters described by Rothman and Manis (2003) for a point VCN neuron . We chose a membrane time constant of 0.3 ms, similar to the one reported for MSO neurons after P20 . Bilateral input trains with different delays were created by injecting currents (conductance based synaptic-like currents) such that the trains of EPSPs consisted of composite minimal EPSPs (32 or 64 minimal EPSPs were used to create a ∼8 mV composite EPSP; more EPSGs were used for higher input frequencies (1.1 KHz) to generate a smooth voltage time course). Minimal EPSGs had fixed form: alpha functions with time constant τsyn of 0.1 ms for excitation and 0.4 for inhibition, scaled to have specified area and peak proportional to 1/τsyn. Different minimal EPSG statistics led to different slopes and half-widths, which are summed in order to create the composite suprathreshold EPSGs (see Figure S4). These EPSPs have envelopes resembling alpha functions with time constants that ranged from 0.1 to 0.8 ms . This range of (in vivo based) EPSP time constants was slightly faster than those obtained from our experiments because our recordings were made at 32°C and the simulations were performed at 37°C. The same results were obtained using values of rising EPSP slopes from our experiments at 22°C as well as the kinetics of our computational model, to eliminate any temperature effect.
The asymmetry in simulated EPSP kinetics was modeled by varying the jitter of unitary events. The amount of jitter was based on the observed variability in EPSC amplitudes, slopes, and half-widths obtained in our brain slice recordings. ITD functions were created from bilateral EPSP or PSP trains (40 cycles) at frequencies ranging from 250 to 1,100 Hz. A minimum of 10 trials (per ITD) were run to get a smooth ITD response function.
The differential equations of the model were integrated numerically using fourth-order-Runge-Kutta scheme with a time step between 1 and 0.25 µs; refining the time step did not lead to noticeable differences in the computed solutions.
In all the simulations the contralateral inhibitory input leads the contralateral excitation by 0.2 ms. This time difference was imposed between the peak of the composite IPSPs and the composite EPSPs from the contralateral input side. The result in Figure 4 showing that inhibition shifts the ITD response function towards contralateral leading side holds even for bigger delays between contralateral inhibition and excitation (unpublished data).
PSP-rising-slope and -peak-delay versus PSP response amplitude for different VCN-afferents stimulation strength. (A) Examples of individual neurons showing that asymmetry of rising slopes between bilateral inputs remains, when the response amplitude changed (top: current clamp experiment, P21; bottom: voltage clamp experiment, P23). (B) The distribution of peak latencies for PSP responses is almost flat for the range of subthreshold PSPs and PSCs (top and bottom plots, respectively, same neurons in A). (C) Summarized data for 14 experiments in current clamp and 16 in voltage clamp configuration (see Methods). Left: rate of change for slope at different amplitude responses is similar between sides, supporting the result that when PSP slopes are asymmetric between sides they will remain asymmetric through the subthreshold range. Right: rate of change of latencies is close to zero for different response amplitudes. Even though the individual monolateral response has jitter, the ITD response function is the average latency of the PSP responses and this is statistically unchanged with the amplitude of the response.
Subthreshold evoked-PSPs stimulating the afferents of the VCN onto the MSO neurons on our thick slice preparation. (A) In most of the neurons recorded ipsilateral responses were bigger in amplitude than the controlateral; for this reason all the data in the paper are normalized by amplitude to avoid bias on the comparison between contralateral and ipsilateral rising-phase slopes. Cells had been recorded from both olives on the same slice, keeping the stimulating electrodes on the same location to avoid possible sources of asymmetries due to the stimulation artifacts. Time courses show that PSPs are a composition of many synaptic mini-PSPs released with a particular distribution of locations and/or timing. Dendrite morphology could play a role on the asymmetry in EPSPs' shapes. Superposition of ipsilateral and contralateral responses shows a consistent trial-to-trial difference on rising phase. (B) When inhibition is blocked voltage time courses show that EPSPs are a composition of many synaptic mini-EPSPs released with a particular time distribution. Decay phase is similar between the bilateral responses dictated by "effective" membrane time constant (combination of active currents) and integration properties of bipolar dendrites. (C) Top row: slopes versus halfwidth for PSPs evoked from contralateral and ipsilateral stimulations. Ipsilateral responses are steeper than contralateral, with similar halfwidth for both responses. Passive propagation of EPSP through asymmetric dendrites is not enough to explain the difference on asymmetric responses recorded at the soma compartment due to the similarity in halfwidths. Normalizing the halfwidths and slopes by the corresponding individual PSP amplitudes show consistent results supporting the fundamental observation that the bilateral asymmetry is intrinsic in the neurons independently from the trials and response amplitude. Bottom row: same as the top row but now glycinergic inhibition is blocked. Note: EPSP shapes are similar to the ones obtained from the model when we simulate the synaptic excitatory conductance as population of inputs with different jitter for each bilateral input (see also Figure 3 and Figure S4). In vivo EPSP slopes could be smaller than the ones recorded in our experiments due to the fact that in our preparation there is a high degree of synchronicity due to the simultaneous stimulation of the VCN bundle. Larger ipsilateral response will strongly support our results. For simplicity and taking a conservative position we will use for the modeling equal amount of conductance between bilateral inputs. Only for some schematics will we use the same amplitude for bilateral inputs.
Increase in slope due to fast and slow (extreme values) inhibitory conductance (from our parameter study this effect is seen if 0.2 (ms)<δ for τinh = 0.1 (ms) and 0.75 (ms) <δ for τinh = 1.0 (ms)). EPSPs with a shallower slope are more affected by synaptic inhibition for a large range of inhibitory synaptic input's time scale. Therefore, for the time scale of our recorded contralateral inputs, they will be more affected by inhibitory conductance than their ipsilateral counterparts. (A) Time courses for PSPs in the case of fast and slow synaptic inhibition (δ is the time that inhibition leads excitation). (B) Simulation (using the neuron model, see Methods) of PSP (EPSP + IPSP) generated with synaptic fast IPSGs having time scale of τinh = 0.1 ms and advanced with respect to the EPSGs by δ = 0.2 ms. Two different EPSGs examples: 0.15 ms (red, for ipsilateral input) or 0.25 ms (blue, for contralateral input). (C) Simulation with synaptic IPSGs with time scale of τinh = 1.0 ms and advanced with respect to the EPSGs by δ = 1.0 ms (point of full activation). Two cases: τexc = 0.15 ms (example for ipsilateral input, red), 0.25 ms (example for contralateral input, blue). For an equivalent change in synaptic inhibitory conductance (ΔGinh), the slope of the shallower EPSP displayed a greater change (arrow) than steeper EPSPs. If contralateral EPSPs have shallower rising slopes, then they are more affected by inhibition than ipsilateral EPSPs (Note: the range of total inhibitory conductance is the same in (B) and (C), since Ginh,MAX is proportional to τinh).
Composite EPSPs and IPSPs for an input train (500 Hz) using an idealized auditory nerve fiber model (sinusoidally modulated Poisson rate for mini-PSG times), less realistic than (Carney 1993). (A) Raster plots with event times for ipsilateral excitatory inputs (red), contralateral excitatory inputs (blue), and contralateral inhibitory inputs (green). Superposition of composite PSGs for these three cases. Mini-EPSGs are alpha functions with time constant of 0.1 ms, and mini-IPSGs are alpha functions of 0.4 ms time const. The difference in vector strength (degree of synchrony) between the events dictates the shape of the composite EPSGs and IPSGs. Similar composite IPSGs can be generated with mini-IPSGs made of alpha functions of 0.1 ms time constant and lower vector strength (R = 0.48). (B) Composite PSCs corresponding to the PSGs from the final cycle of the time series in (A). EPSCs obtained with higher vector strength have steeper rising slope and shorter halfwidth (red, ipsilateral inputs; blue, contralateral inputs). IPSCs look similar to inhibitory conductance time course, because the temporal summation reached a steady state dynamic. (C) PSPs for the last cycle in (A) for the three input cases. Superimposed with a thick line is the average time course for EPSPs and IPSPs. Rising slopes are steeper when vector strength is larger (ipsilateral EPSP has steeper slope). (D) Average over 25 cycles of composite PSGs for the three different inputs. In the three cases a function (dashed) was fitted to the average PSG. Alpha function is the simplest description of PSGs (long dashes); it fits the rising phase but not the falling phase. Composite EPSGs were fitted with a functional form that is proportional to (1−(exp(−(t−t0)/τ rise))).3.(exp(−(t−t0)/τ decay)). Ipsilateral inputs (red): τrise = 2.5, τdecay = 0.14. Contralateral inputs (blue): τrise = 2.8, τdecay = 0.18. For the alpha function fits, for ipsilateral inputs (red): τ = 0.48; for contralateral inputs (blue): τ = 0.69. In the case of inhibition the best fitting was obtained with a periodic function (a.sin((t−t0)/n)+b), due to the time constant of the individual components and the temporal summation generated at this frequency.
We thank Jaime de la Rocha, Max Schiff, Anne Takesian, Sach Sokol, and Alex Reyes for fruitful discussions and comments on an earlier version of this manuscript.
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: PEJ. Performed the experiments: PEJ. Analyzed the data: PEJ. Contributed reagents/materials/analysis tools and trained PEJ in doing the experiments: GS VCK. Wrote the paper: PEJ. Direct the research and manuscript preparation: DHS JR.
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Q: DocuSign Connect notifications in DocuSign demo account We are having issues with Connect notifications from our DocuSign demo account.We are not able to receive any updates from our Connect configuration. Though, when I republished the XML from Connect logs, I did receive a Connect update to a specified URL.
When I created new envelopes using API as well as from DocuSign account itself, I never got any notifications to the same URL.I do not even see a Connect log.
We do not have any issues with Connect notifications for our DocuSign live account.
We have never experienced this issue before with Connect updates from our DocuSign demo account.
Please advise.

A: Just a hunch - do you use http or https? connect can only work over port 443 with a valid SSL certificate. If you're on localhost - you may need to get a temp certificate or something like that but also using azure websites is good way to avoid this issue since they're pretty much just as good as working on your localhost

redpajama_set_name: RedPajamaStackExchange


Back in April I made a long road trip to Chihuahua from Zacatecas, Mexico its about 550 miles in distance from capital city to capital city of Chihuahua and Zacatecas. The in between route its all freeways (mostly 4 lanes), lots of views such corn fields, hamish towns, deserts (most of the time) and mountain ranges.
I spent one night in Chihuahua city, then the next day the trip continued to Basaseachi Falls, which the entire segment its all rain forest.
The first stop was Basaseachi falls, I went in April which the fall was in its lowest point, I mean there were drought so I couln't it splendid fall... but I was lucky to get down the fall, which usually its a closed place. Basaseachi its a national park program, so there are basic services such restrooms, caping sites, market trails, chairs, lookpoints etc..
The first picture It's what you see in the park, its as impressive as like Yosemite in California, huge mountains in sharp valleys.
The view at any time of the day are amazing!
This is the mexican skywalk, lol, you step over a metallic balcony and you feel dazzled if you look down, it was awesome... better than the grand canyon skywalk in Arizona... even this one is free!
Also a cable car! that takes you to the other side of the canyon.
rad! this is a section of the world I want to explore more. I've been to Chihuahua only once - crossed over at Palomas and headed west on highway 2 to Sonora.
Mexico overall doesn't sound like it's really all that dangerous (although some parts really aren't that safe), as long as you travel by day. In my lifetime I plan on driving a lot in the US, Canada, and Mexico. There really isn't that much that you can't see in the world that is in North America. Mexican Fed. Highway 16 looks like a pretty cool route to take on Google Maps. I really want to drive it one day. My problem is that I don't have a lot of money yet, so I can't afford to do everything I want to do.
I really wish that the US and Canada would accept Mexican insurance and that Mexico would accept US / Canadian insurance. The import permit / tourist card thing seems like a way to get money from foreigners. I mean you don't need a tourist card and an import permit for the border zone, so it's not like they couldn't just get rid of both all together. The military checkpoints don't seem like a bad idea and I would be fine with Mexico keeping those.
By the way how safe is it to travel MX 24 and MX 45 through that area? Maybe those roads would be a cheaper way to get to the new autopista from Durango - Mazatlan. The only reason I asked is because Chihuahua has a high homicide rate.
I really wish that the US and Canada would accept Mexican insurance and that Mexico would accept US / Canadian insurance.
Major differences between the US/Canada and Mexican legal systems might make it impractical for Mexican insurers to cover US/Canada and vice versa, even if such were allowed (and my impression is that Mexico doesn't allow foreign insurers to offer coverage in its territory). Even with some jurisdictions like Louisiana whose legal systems have French rather than English roots, the legal environments are much more consistent among the states/provinces/territories than between any of them and Mexico.
I took the train from Chihuahua to Los Mochis back in the late 1980s. It skirts the top of the canyon and you can get out and see the canyon from one point, but nothing to the extent you picture here. Great photos.
Beautiful country! Thanks for posting.


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They were very nice, and took care of our transmission purge and tune up. Marc was very nice and professional. I would recommend them.
As always the service was great. I trust them to always take good care of me, Mark and his staff are very friendly and I trust them to always be honest. They let me know when there are things I should take care of but never push unnecessary repairs.
I was 100% satisfied with the service I received here. They're very courteous, prompt and provide very good service. I've been there several times before, as I have 4 different cars I take there.
The owner, Mark is a really nice guy. Even when I didn't know what was wrong with my vehicle and it ended up being something small. I appreciated that Mark was very fair and very honest with me. I have no complaints at all. I absolutely recommend them.
I appreciate the good work they did. I had a couple of questions before I came in and I spoke to Mark that runs the place several times before I had the vehicle towed in.The service was courteous and they were polite and everything went well. I would definitely recommend them to a friend.
I was very happy with the service and everything is running fine. They are very polite and do a good job. They are very accommodating. I've already recommended them to several people.
Mark over there is always great and he always gets us in and works around our schedule. It's very convenient. He even helped arrange for a car rental for me when I broke down so I'd have a vehicle. We take all our cars there.
Once I went into the hospital 2 days after dropping my car off for repairs, Mark was so kind for working with my husband so he were able to pick up the car at a time when he was able to (over 2 weeks later) between me in the hospital and taking care of the kids. Great people there that really care.
Since my visit to this center, my vehicle is running perfectly and shifting very nicely now. What I liked best about this AAMCO was that they were prompt, courteous and the service was complete.
The car running great the service was great!
Knock on wood the car is running great, so far so good! The mechanics do a good job on my car every single time! I definitely would recommend AAMCO to my friends!
It's always a pleasure doing business with AAMCO! I have four cars in the family, we always go to AAMCO for our car needs. Their mechanics are great, and on top of that this place is truly honest. I know I'm leaving my cars in good hands with Mark the owner. I always tell my friends to go to AAMCO!
I've never had a problem here! The service is really great and the mechanics do a great job! I have nothing bad to say about this place which is exactly why I keep bringing my car back here and I also bring my daughter's cars here too! I would absolutely recommend AAMCO to all of my friends!
They took the car in and fixed all that needed to be fixed. Now my car is running great! I would absolutely recommend AAMCO to my friends!
From the manager to everyone who worked on my vehicle there great!
I am happy with AAMCO's work. They got the car running well and thats all it matters.
The AAMCO staff was honest, friendly and very professional. The work was done on time and well. Everything was perfect, I advocate them!
Easily one of the best customer service I have experienced from an auto shop. AAMCO did a fantastic job in fixing my vehicle because it has not given me any issues. I would highly recommend this shop to everyone.
I am extremely happy with the staff and owner of this AAMCO shop. They did an excellent job and because of that I have already recommended them to a bunch of my friends around the area. I am very pleased with their work and will certainly come again.
My 2009 Jeep Liberty is running fine and the service was great and the personal service manager was great to work with and I recommend my friends to AAMCO.
Unfortunately after I had my Hyundai fixed at AAMCO a week later my car was totaled, but my car was running beautifully and they did a wonderfully fine job my Hyundai Accent and I recommend my friends to AAMCO.
At AAMCO, they took care of everything that needed to be done. I brought my car in for a transmission rebuild and I brought it back for a double check and for them to finish the work, and so far so good! I was very satisfied with them.
So far my car is great, and so was the shop - when you're at AAMCO, you're well taken care of, and every detail was talked about so you know exactly what's going on!
The gentleman that I dealt with at AAMCO was great to work with - he was very up front and he was able to get everything done. My car is running great, and I would definitely recommend them!
My car is running great after working with Aamco. I was very impressed with their service, and even though I called them last minute to bring my car in, they still got me in and out very quickly! They did a great job, and I would absolutely recommend them to my friends!
My car is running great - I'm actually planning to trade it in because my lease is almost up but I was very impressed with the service at Aamco! I believe the man that I dealt with was the owner of the shop and he was very pleasant, and that was a nice surprise. The turnaround time was great - it was exactly what I expected, and I was very happy with the work that they did. I'd absolutely recommend this shop!
Everybody at the Aamco shop was very friendly and helpful. The manager was great to work with! They took my car in right away, and overall it was just a great experience. I would definitely recommend my friends to Aamco!
The work on my car was done to perfection and I'm 100% satisfied. I've been dealing with Mark at Aamco for about 20 years now and I won't take my car to any other place but him! I've already recommended a few people over there, and I have no complaints whatsoever!
The staff and the owner of this Aamco shop were very friendly and easy to work with, and also the turnaround for my car was only a few hours! I was very happy with the work that they did and I would definitely recommend my friends and family to them!
They do fantastic work here! The guys are all friendly and polite but above all, great mechanics. The prices are very reasonable and fair. I highly recommend them!
I am happy to say that since AAMCO had my vehicle, it has not given me any problems. The work was done very efficiently and fairly priced. I am certainly a return customer for the future. Thank you AAMCO!
Extremely quick and efficient work by AAMCO. The place was very clean and it looked very professional. I recommend everyone to go and check them out!
AAMCO went above and beyond for me. The people were extremely nice and accommodated me in everyday possible. After their amazing work the car has been running outstanding! Now everywhere I go, I recommend everyone to AAMCO.
I brought my car to Aamco for my air conditioning and they did a great job with it. The guys at the shop were honest and friendly, and all in all I had a good experience with them. I'd absolutely recommend Aamco!
The service at AAMCO was great and everybody there was very nice, and helpful. My car is running great, in fact I just brought it back to AAMCO for an oil change! I would absolutely recommend my friends to them.
I was impressed with the service at AAMCO and they did a fantastic job with my car... They were very honest and they were great to work with!
I brought my car to AAMCO to get my brakes changed and so far everything is great! It seemed like they really took the time to check my car and make sure there were no problems. I'd recommend them, I was very satisfied!
So far my car is running great since I got it back from AAMCO. The manager at the shop was very up-front and gave me all of the details with what my car needed, so there was no surprises. I was very satisfied with their service - they did an excellent job!
I was impressed with the speed and efficiency at AAMCO! I was able to drop my car off, and I came back in 2 hours and it was ready. I'd definitely recommend them, they did a great job.
The service at AAMCO was very professional and I was definitely satisfied! My car is running great after getting it back from them.
We took our daughter's car to AAMCO and so far it's running well. We haven't had any issues with it, and we're definitely satisfied with the service.
The car is running excellent. The guys were supreme. I highly recommend AAMCO. Outstanding work.
I'm not having any more issues with my brakes locking up since I got my car back from AAMCO! I was impressed by their promptness and their professionalism at the shop, and so far everything is good. So far, I'd say that I'd recommend them!
I brought my car to AAMCO for an inspection and I really liked how fast it was! I was in and out very quickly. I've already recommended someone to them!
I went to AAMCO for an oil change, and it was close to their closing time but they still took me in anyway. I was impressed how fast they got me in and out! I was very satisfied with their service, and I'll probably be back again.
AAMCO did a great job with my car! They got me off the side of the road and got me home, so I'm very happy with them.
My car is running great after getting it back from AAMCO, and they did a good job with it. If my friends had any trouble with their transmissions I'd send them over to this shop for sure!
When I brought my car to AAMCO, the service was quick and easy, and my car is running great now! They did an excellent job and I'm very satisfied.
The service at AAMCO was great! They had my truck in and out quickly , and they did a great job with it. If my friends needed to get work done on their cars I would absolutely send them to AAMCO!
AAMCO provided excellent service on my car and the guys at the shop were very helpful. Now, my car is running, and I'd definitely send my friends over there.
I was absolutely satisfied with the service at the AAMCO shop. They did a good job, and they were quick. I would recommend them.
My car is running PERFECTLY after getting it back from the shop. I'm 100% satisfied with AAMCO's service, and I liked that I was able to speak with the owner, and his assistant at the shop. They did an excellent job and I'd absolutely recommend them!
I was satisfied with the service at AAMCO, and they did everything I requested them to! My car's running well again, and I'm very happy with it.
My car is running great, and I was happy with the work that AAMCO did on my car! Everybody was very nice, and very helpful - and I'd absolutely send my family and friends to them.
I liked that the Aamco center went and got the vehicle for me. I would recommend Aamco to my friends.
I appreciated being able to get service on New Year's Eve.
I really appreciated how the manager worked with me on pricing and got the car fixed.
I was pleased with the cost of the service and would recommend AAMCO to my friends.
The service was very prompt and they got my car fixed quickly.
So far I liked everything about AAMCO from the quality work to the personnel.
They are right down the street from where I work. Justin is my sales rep and he always helps me out. I'd recommend AAMCO in a heartbeat and matter of fact I already have recommended AAMCO to everyone I know.
What I like most about AAMCO is that they are convenient. I recommend my friends to AAMCO.
I recommend my friends to AAMCO they explained to me very detailed about the work my car required and within four to five days I had my car back.
The shop I used to go to had me replacing a lot of parts for thousands of dollars and all you did was make an adjustment and my car is fixed. I am quite satisfied with AAMCO.
I brought my truck in for one thing. It turns out that there was another issue with it and while I waited they took care of both problems for me. I recommend my friends to AAMCO.
The day I picked my car up they gave me a lifetime warranty on my transmission. I recommend AAMCO to my friends.
The guys fixed my car and they put a lot of time and effort into working on my car. I recommend my friends to AAMCO.
Everything was good. The guys fixed my car fast and quickly delivered it to me. I recommend my friends to AAMCO.
The guys worked quickly and efficiently on my car to have it quickly returned to me. I brought my husband's car into the shop to have his transmission rebuilt. I recommend my friends to AAMCO.
The car is running good. Everything is good. You guys did a good job. Everything was done really quick I got my car back in a day or two I was really impressed by that. I recommend AAMCO to my friends and family.
I use AAMCO all the time for my personal and also business cars, so I trust them in anyway possible. This is a great shop and I would be glad to recommend them to everyone. The car is running great and I will be back in the future if I have to.
The service is excellent. I've been a customer of theirs for some time now. I dealt with the owner Mark. He is very amicable and on top of things. He explained everything thoroughly and from the first instant I had that feeling I could trust them and rely on them. I also like the fact they would not do more than necessary to inflate the bill.
Had some leaky valve covers causing my wires and spark plugs to be soaked in oil. They fixed it right up and car runs better than ever. Fair price and friendly staff. It should be noted I had a minor issue when they returned the car to me which seems like a negative. However, they were really understanding and made a quick adjustment the next day and problem was mitigated in 15-20 mins with no extra charge. As long as I live around here I will trustingly take my vehicle there.
These folks are friendly, courteous, helpful, and knowledgeable. I came in with a transmission problem technically out of warranty (okay by time, over by miles). They worked with me and the originating store to reach a fair arrangement. When I left, transmission was working better than I had ever seen it. Had some symptoms a day later (common - residual sludge from prior issue), and I was a couple hours away. They guessed at the correct diagnosis and worked with me by phone to resolve. Turnaround a day slower than initially promised, but given the intershop communications, not out of line. Thank you!!
We have been taking our cars to Mark for years. He and his guys know what they're doing! They explain things in a way that is very easy to understand, and I can always count on their work being thorough.


God help me to remain calm. Its like i know im loved by you and satan picks the exact things that would irratate me and cause me to lose it. Pleaswe heal my back. It hurts from sleeping on a couch and i pray doors open so we can get an intern house with my own bed.


Save the Dunes has been helping the Indiana dunes for over 65 years. Contributions from our members, donors, and sponsors enable us to protect the precious natural resources of the dunes region that are so important to Northwest Indiana. Your continued generosity will help us ensure our land and waters are healthy for generations to come.
Save the Dunes will provide you with a record of giving for your donations, which comes in the form of a thank you letter. Please hold onto this thank you letter for your taxes. For more information about charitable contributions, please see IRS Publication 526. To review a copy of the organization's 990, please click here.


text:
Q: How to import a GeoJSON file from GeoServer using OpenLayers I have been trying to import the layer that I created in GeoServer. I need to use OpenLayers. This is what I have so far.
<!-- language: js -->

<script type="text/javascript">
var serviceUrl = 'https://gis.arlingtonva.us:6443/arcgis/rest/services/public/Bike_Routes/MapServer/';
var layer = '0';
var otherURL = 'Specialty/ESRI_StateCityHighway_USA/MapServer';

var raster = new ol.layer.Tile({
      source: new ol.source.OSM()
    });

var map = new ol.Map({
  target: 'map',
  layers: [raster],
  view: new ol.View({
    center: ol.proj.fromLonLat([-77.0910, 38.8816]),
    zoom: 13.58
  })
});

var bikes = new OpenLayers.Layer.WMS(
 "Bike Routes of Arlington", "http://localhost:8080/geoserver/ArlingtonBikePath",
 {
         srs: 'EPSG:2283',
         layers: 'Bike_Route',
         styles: '',
         format:'image/png'
 }
);

map.addControl(new OpenLayers.Control.LayerSwitcher());
var myswitch = new OpenLayers.Control.LayerSwitcher();
map.addControl(myswitch);

</script>


Cosy Apartment for Sale in this prestigious development located close to all amenities. Accommodation has been finished to the latest modern standards consisting of an open plan kitchen/living leading to a nice double bedroom and a bathroom…From its balcony one can see a beautiful public garden. Property is an excellent rental investment.


text:
Q: PWA: safari web push notifications Does Safari 12.1 support service workers PWA push notifications?
I tried this demo on iOS but it still does not work for me.
Is there any chance to get them?
Thanks.

A: There is no communication about this feature at this moment... Apple doesn't support Push API.
Web Push notifications with iOS 11.3


Lusty Glaze Beach is a privately owned beach on the eastern end of the five main beaches that make up the coast of Newquay on the North Cornwall coast.
A funnel-shaped beach that grows dramatically at low water, Lusty Glaze faces northwest and is backed by 150 foot high cliffs. To the southwest lies Tolcarne Beach whilst over the headland in the northeast lies (St. Columb) Porth Beach and Trevelgue Head.
The beach itself consists of pale golden sand and stretches from the small headland and rocks at the 'Criggars', below Barrowfields in the southwest to the headland above Wine Cove in the northeast.
Access to the beach is down a flight of of 368 steps, making Lusty Glaze not viable for those with pushchairs or the less mobile. It is possible to walk around from Tolcarne Beach and in the other direction from Porth at low spring tides; but care must always be taken to know the exact state of the tide to avoid being cut off.
Lusty Glaze, a name which translates from the Cornish as 'Place to see the Blue Boats', is home to the Lifeguard National Training Centre. It is the premier venue for first aid and emergency rescue training in the South West providing a range of vocational courses as well as the National Beach Lifeguard Qualification. This qualification is an essential requirement for those working as a lifeguard on Britain's beaches and is also valid in a number of countries throughout the world.
Lifeguard cover at Lusty Glaze is usually from early March to early September.
Facilities include a cafe, restaurant, beach shop, bar, crèche, toilets and watersports equipment hire; beach huts are also available for hire. There is also a number of 'Cliff Activities' including abseiling and the 'Terror Tight Rope' - a walk from one cliff to another high above the beach.
Further developments and improvements have now made Lusty Glaze Beach 'the place to be seen' in Newquay.
Its sheltered position and range of facilities make Lusty Glaze very popular in high season - visitors may find the southwestern end of the beach less crowded.


text:
Coordinate may refer to:
 An element of a coordinate system in geometry and related domains
 Coordinate space in mathematics
 Cartesian coordinate system
 Coordinate (vector space)
 Geographic coordinate system
 Coordinate structure in linguistics
 Coordinate covalent bond in chemistry
 Coordinate descent, an algorithm

See also
 Coordination (disambiguation)
 Coordinator (disambiguation)

redpajama_set_name: RedPajamaWikipedia


TJ Boulting is delighted to present the third solo show with the gallery of British artist Boo Saville. This new series of drawings and paintings, whilst using her trademark biro pens and oil paint, is a departure to previous representational work, and consists of large format abstract colour fields. The first works were begun over a year and a half ago and since then have been repeatedly returned to, informing the consecutive works in a back and forth cycle of formal explorations. Each is also infused less visibly with the personal experience of the artist, through an ongoing proximity to the works over time.
The drawings are the essence of the whole series, the starting point where ideas feed the subsequent paintings, oscillating between the two mediums. Bold, imposing colour fields are created from layers and layers of different colour biros – the more usual red, blue and green, as well as a new palette of orange, pink, yellow and purple. Predominantly dark and various hues of black as a result of so many layers, she has pushed the medium to its extremes with regards to colour and form. The result is a vibrating solid mass that eludes subject matter whilst retaining an ominous presence.
The idea of the machine-made finish, removing the mark of the artist as much as possible, and the intense long process of production, mean the work is like a black hole of artistic input - they hold everything but seemingly show nothing. In the drawings, line disappears; colour and vibration take over, as if they are acting as paintings. At the same time, they are optical but without expression. They reflect the digital age in colour and form as would a screen made of millions of pixels, but in reality are entirely manual and created through hours of intense process. There is nothing instant in these works, they possess and silently exude thought and time.
The title of the show references the 1959 film of the same name, a science fiction where there are only three people left in the world. There is a visual reference also to the chapel designed by Mark Rothko in Houston, Texas, where his large black panels surround, somber but harmonious. As he stated, "I'm interested only in expressing basic human emotions: tragedy, ecstasy, doom." For Saville, the triumvirate is similarly present here in her world, flesh and devil.


The weekend took a turn for the better when a last minute invite to the reputable James Cochran EC3 landed on our desk. The restaurant has re-opened on Saturday nights, and a 6 course 'Last Supper' menu was served on Saturday to christen the event.
Arriving early, we have the choice of any table before the dedicated diners rush in. The restaurant soon reaches capacity.
The space is understated and elegant. Manager Pierre comes over to greet us; a charming man who is attentive throughout the evening and even invites us to the kitchen to meet James after our meal… more on that later.
The walls are a muted grey. Photographs adorn the walls and are an exhibition in their own right. Taken by James' best friend, they're a reflection of his life to date. James is half Caribbean and half Scottish, shown in the black and white shots are Caribbean beaches and Scottish woodlands. A fabulous image taken at Whitstable Bay is thrown in to acknowledge his time living by the seaside town.
Our dinner starts with a Whitstable Rock Oyster, served with a welcome glass of Moet. We contemplate the down-in-one method, but our oysters are sitting pretty in a Yoghurt and Champagne Foam requiring the assistance of a spoon.
After a full day of walking – from Camden to Hackney and Soho in between – we've worked up quite the appetite. Our next course can't come soon enough. Two life size 'Cigars' are presented to us filled with a Fois Gras parfait. The course is a rich introduction to the Supper menu.
Third in line is Roast Orkney Scallop; a gentle dish in comparison. An outburst of Mmm's and Ahhh's ensue as we tuck in to the beautifully warm course plated with Jerusalem Artichoke, Crispy Chicken Skin and Almonds.
Sipping on a mellow Italian white, we reflect on our busy day and laugh our way to the next course. Scottish Lobster is served with Whiskey Butter, Seaweed, Savoy Cabbage and Peanuts. It's a total melt-in-the-mouth dish.
Alas, we're on to the main event. It's as though James has put his heart and soul in to cooking the Galloway Beef. The Roast Fillet is served with Pressed Potato, Black Garlic, Cepes and Truffle. It's a winner of a dish and one that we won't forget for a long time.
An interval of Waterloo and Truffle Cheese is married with a juicy and vibrant Fig Jam. It's paired well with crispy Oat and Sourdough Crackers. Dessert follows; mini Dulce de Leche Doughnuts are freshly fried to order and arrive warm at the table.
Six courses complete, we prop ourselves up at the bar for an after dinner cocktail and listen to ambient music, Bonobo and Zero 7 to note.
We had the privilege of meeting James after our meal and learning of his passion for the restaurant. He tells us of his signature Fried Chicken created during his time cooking at the Harwood Arms, a gastro-pub in Fulham. He's also recently opened his second restaurant in Angel, James Cochran N1 notably. We jest he's a real man about town.
Before we leave, James brings us a takeaway box of his signature Fried Chicken. Its soaked in Buttermilk and coated in a Tapioca and Semolina Breadcrumb mix – Gluten Free cooking at it's finest!
We wish James all the luck with his restaurants and will certainly be returning soon for another delicious supper.


text:
Q: Django. How do I add a field to a query result? I have a Room model and I want to add the is_member boolean field to a queryset with rooms. How can  do this? I was thinking of using .annotate (), but that doesn't work for my task.
models.py
from django.db import models

class Room(models.Model):
    name = models.CharField(max_length=150)
    members = models.ManyToManyField(User, blank=True)

views.py
from rest_framework.views import APIView
from rest_framework.response import Response
from .serializers import RoomSerializer
from .models import Room

class RoomView(APIView):
    def get(self, request):
        rooms = Room.objects.all() # get all rooms
        user = request.user # get current user

        for room in rooms:
            members = room.members.all() # get members
            is_member = user in members # set boolean value
            room.is_member = is_member # set in room

        serializer = RoomSerializer(rooms, many=True)
        return Response(serializer.data)

serializers.py
from rest_framework import serializers
from .models import Room

class RoomSerializer(serializers.ModelSerializer)
    is_member = serializers.BooleanField(read_only=True)

    class Meta:
        model = Room
        fields = "__all__"

I solved this issue this way, but is there any other options to do it?
Help me please

A: When you are using an M2M like this, you could use your own through model rather than have django do it automatically.
That gives you the ability to add your own fields to the relationship, or properties or customise the manager/queryset.
Docs for this are here; https://docs.djangoproject.com/en/3.1/topics/db/models/#extra-fields-on-many-to-many-relationships
So by querying the through model, you could establish if a user was a member of a given room.
A very rough example of a custom through model approach;

class Room(models.Model):
    users = models.ManyToManyField("User", through=ThroughModel)

class User(models.Model):
    text = models.TextField()

class ThroughModel(models.Model):
    room_id = models.ForeignKey(Room)
    user_id = models.ForeignKey(User)
    is_member = models.BooleanField()

# this will return a list of ThroughModel objects
ThroughModel.objects.filter(user=instance_of_user, is_member=True)

# this will return a list of A objects based on an extra field on the through table
Room.objects.filter(users__ThroughModel__is_member=True)

# keep in mind that limiting by one of the foreign keys on the through model is easier
Room.objects.filter(users=instance_of_user)

A: Can you try this. This will not add is_member in a queryset but i think you are looking for something like this.
#  Serializer
class RoomSerializer(serializers.ModelSerializer):
    is_member = serializers.SerializerMethodField(read_only=True)

    class Meta:
        model = Room
        fields = "__all__"

    def get_is_member(self, obj):
        user = self.context["request"].user
        if user in obj.members.all():
            return True
        return False

#  View
class RoomView(APIView):
    def get(self, request):
        rooms = Room.objects.prefetch_related("members").all()  # get all rooms
        serializer = RoomSerializer(rooms, context={"request": request}, many=True)
        return Response(serializer.data)

And you should get the desired response
[
    {
        "id": 1,
        "is_member": true,
        "name": "first",
        "members": [
            1
        ]
    },
    {
        "id": 2,
        "is_member": true,
        "name": "second",
        "members": [
            1,
            2
        ]
    }
]


text:
International Tie-ups
Knowledge Partners
ABOUT MREI
MREI are accredited by respective Indian and globally acknowledged bodies. Like all leading names, it is reviewed for reaccreditation from time to time. The institutions meet the standards for accreditation established by an accrediting agency. These standards ensure that an institution has appropriate and clear goals, sufficient resources to achieve them, is fulfilling its objectives, and will continue to do so. The process provides MREI with an opportunity for reflection, honest assessment of strengths and weaknesses, along with a chance to develop strategies for continued improvement. Over a span MREI have achieved respectable rankings and academic accolades. This puts them in a different league altogether. Besides academics, they have received awards and recognition in sports and extracurricular activities. MREI are a major center for research and development covering various areas. Corporates in partnership, take full advantage of the expertise and resources that drive innovation. Here, an atmosphere of open innovation sparks new ideas, jump-starts discovery, accelerates progress and expands the frontiers of science.
ABOUT MRIIRS
MRIIRS ranked among top institutions by Ministry of Education's NIRF Ranking 2021. In Dental Studies; Faculty of Dental Sciences, Manav Rachna Dental College, MRIIRS has secured the 39th rank all-across India. In Engineering, jumping 56 ranks in a year; MRIIRS is ranked at No. 118 across India. MRIIRS has also moved ahead by one complete band (an approximate jump of 60 places) in the University category where it is placed in the Rank Band 101-150.
Accreditation Rankings of MRIIRS
ABOUT MRU
Manav Rachna University (MRU) is a State Private University (established by Haryana State Legislature Act No 26 of 2014 & under section 2(f) of UGC Act 1956) and has evolved from Manav Rachna College of Engineering (MRCE, established in 2004), a NAAC Accredited 'A' Grade Institution. It is among the Top 2 Emerging Engineering Institutions of India and is the No 1 Engineering Institution in India for Research Capability & Placements as per the Times Engineering Institutes Ranking 2021.
Manav Rachna University has been bestowed with QS I-GAUGE, Overall DIAMOND rating. It has received QS I-GAUGE DIAMOND RATING for Excellence in Teaching and Learning, Faculty Quality, Facilities, and Social Responsibility, and QS I-GAUGE PLATINUM RATING for Employability and Academic Development.
Manav Rachna University is the Founder Member of the prestigious "College Board's Indian Global Higher Education Alliance".
ABOUT MRDC
Manav Rachna Dental College under Faculty of Dental Sciences (FDS), Manav Rachna International Institute of Research and Studies (MRIIRS) is one of the flagship institutions of Manav Rachna Educational Institutions. Manav Rachna Educational Institutions (MREI) is one of the India's leading educational groups, producing some of India's most employable professionals. MREI is the brainchild of renowned educationist and philanthropist, Dr. O.P. Bhalla, Founder Visionary..
Manav Rachna Dental College (under FDS) was established in 2006, is recognized for BDS Degree and MDS Degree by Ministry of Health & Family Welfare, Govt. of India and Dental Council of India and is affiliated to Manav Rachna International Institute of Research and Studies Faridabad.

redpajama_set_name: RedPajamaCommonCrawl