All animal procedures were approved by the Ethics Committee of the Hebrew University (Ethic number MD-15-14274-1).

Neurons were isolated from lumbar dorsal ganglions of 6–9 week old Sprague Dawley male rat as previously described (Binshtok et al., 2008; Nita et al., 2016). In short: lumbar DRGs (L1-L6) were removed and placed into DMEM with 1% penicillin-streptomycin, then digested in 5 mg ml⁻¹ collagenase, 1 mg ml⁻¹ Dispase II (Roche) and 0.25% trypsin, followed by addition of 0.25% trypsin inhibitor. Cells were triturated in the presence of DNase I (250 U) and centrifuged through 15% BSA. The cell pellet was re-suspended in 1 ml Neurobasal media, containing B27 supplement (Invitrogen), penicillin and streptomycin, 10 mM AraC, 2.5S NGF (100 ng ml⁻¹, Promega) and GDNF (2 ng ml⁻¹). Cells were plated onto poly-D-lysine (100 μg ml⁻¹) and laminin (1 mg ml⁻¹) coated 35 mm tissue culture dishes (Corning) at ~10 K cells per well, at 37°C with 5% carbon dioxide. Unless otherwise stated the materials were purchased from Sigma.

Recordings were performed from small (~25 μm), capsaicin-sensitive (not shown) dissociated rat DRG neurons, up to 24 h after culturing. These neurons have been described in the literature to be nociceptive (Cardenas et al., 1995). Cell diameter was measured using Nikon Elements AI software (Nikon), from images acquired by a CCD camera (Q-Imaging). In experiments studying the role of I_M in nociceptor-like DRG neurons (Figures 1, 2, 3A,B,E,F, Supplementary Figure 1), whole-cell membrane currents and voltages were recorded using a nystatin-based perforated patch technique (Horn and Marty, 1988) in voltage clamp and fast current-clamp modes, respectively, using a Multiclamp 700B amplifier (Molecular Devices), at room temperature (24 ± 2°C). All other experiments (Figures 3C,D, Supplementary Figure 2) were performed using voltage and current clamp in whole cell configuration. In all experiments, only the cells that showed less than 10% change in access resistance during the entire recording period were analyzed. Data were sampled at 50 kHz and were low-pass filtered at 20 kHz (-3 dB, 8 pole Bessel filter). Patch pipettes (3–5 MΩ) were pulled from borosilicate glass capillaries (1.5 mm/1.1 mm OD/ID, Sutter Instrument Co., Novato, CA, USA) on a P-1000 puller (Sutter Instrument Co.) and fire-polished (LWScientific). Access resistance was in the range of 4–8 MΩ. For voltage-clamp recordings, capacitive currents were minimized and series resistance was compensated by about 80%. Command voltage and current protocols were generated with a Digidata 1,440 A A/D interface (Molecular Devices). Data were digitized using pCLAMP 10.3 (Molecular Devices). Data averaging and peak detection were performed using Clampfit 10.3 software. Data were fitted using Origin 8 (OriginLab).

Nystatin-based pipette solution (290 mOsm) for perforated patch recordings was freshly prepared in the dark every 2–3 h and contained (in mM): 140 KCl, 1.6 MgCl₂, 2 EGTA, 10 HEPES, 2.5 Mg-ATP, 0.5 Na-GTP (pH = 7.4).

Nystatin (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) to obtain a 50 mg ml⁻¹ stock solution, which, after 1 min ultra-sonication, was diluted in pipette solution to obtain a working concentration of 125 μg ml⁻¹.

The intracellular solution for measuring capsaicin-induced current contained (in mM): 145 KCl, 5 NaCl, 2.5 MgCl₂, 2 MgATP, and 0.1 EGTA (pH = 7.4).

The intracellular solution for measuring capsaicin-evoked firing contained (in mM): 140 K-Aspartate, 10 NaCl, 2 MgCl₂, 4 MgATP, 10 HEPES (pH = 7.4).

The extracellular solution contained (in mM): 145 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 D-Glucose (pH = 7.4).

Pipette potential was zeroed before seal formation and membrane potential was corrected for liquid junction potential of −4.5 mV.

Only cells that generated at least one action potential in response to current injections were then used for capsaicin application and analysis.

Only neurons that demonstrated a stable resting potential and stable action potential threshold during a 5 min application of vehicle (extracellular solution) were analyzed. In some experiments we applied vehicle solution for an additional 15 min to rule out possible time-dependent changes in neuronal excitability. No changes in the neuronal excitability were observed during the period of vehicle application (n = 9).

The action potential thresholds and neuronal firing properties were assessed by analyzing the neuronal responses to a series of depolarizing steps (1–2 nA in increments of 0.05 nA each) and 1.5 s depolarizing current ramps (250 pA/s). Action potential threshold was obtained by analyzing phase plots (dV/dt) of first spike evoked by 400 pA, 500 ms step when plotted versus time or versus membrane voltage. The voltage of the threshold was measured using “first local minimum” of the function before the peak. The “first local minimum” was determined as the first minimal value of dV/dt after the peak, followed by an additional increase in the dV/dt, while analyzing the function from its positive peak to time “0.” The time for the first local minimum was defined as the time of threshold, and its voltage was then detected from the original trace. For the verification of threshold values, the thresholds were also determined from the response to the depolarizing ramps, as the potential at the onset of a clear sharp deviation from passive response with subsequent action potential generation.

Measurements of changes in action potential threshold and evoked firing, following application of XE991 were made at the original (control) resting membrane potential (V_m), maintained constant by injecting an appropriate steady current.

To evoke I_M 500 ms hyperpolarizing voltage steps incrementing by −5 mV, were applied from a holding potential of −20 mV (Halliwell and Adams, 1982; Caspi et al., 2009; Tzour et al., 2016). These steps induced slow current “relaxations” after the instantaneous inward current drops (instantaneous current), representing the slow I_M deactivation. Current relaxations were fitted by bi-exponential curves (starting after the capacitance artifact) and were extrapolated back to the beginning of the hyperpolarizing command pulses. I_M amplitudes were assessed as the differences between the instantaneous peak currents at command onset and the steady-state currents just before command offset.

The I-V curves were calculated according to Adams et al. (Brown and Adams, 1980) and Wang and McKinnon (1995) as follows: the intersection of I-V curves of normalized instantaneous and steady-state currents—V_M, obtained from currents acquired as explained above, were measured. In our conditions V_M was −75 mV (not shown). Then, the I_M currents, were normalized to their maximal values and plotted vs. membrane voltages (Adams et al., 1982). The resulting I-V curve was upward shifted to align I(V_M) to zero. Leak, obtained by extrapolation of the linear portion of the I-V curve between −80 to −70 mV, was then subtracted according to Passmore et al. (2003).

The conductance of I_M (g_Kv7/M) was assessed according to Adams et al. (1982). Briefly, we averaged the currents at each command-voltage from all recorded neurons. The resulting I-V curve was upward shifted to align I(V_M) to zero. Leak, obtained by extrapolation of the linear portion of the I-V curve between −80 to −70 mV, was then subtracted according to Passmore et al. (2003). The current values were then divided by the driving force and normalized to the maximal value. The data were fitted by a Boltzmann curve:

where ν_1/2 is voltage at which the half-maximal activation of the channels occurs. The activation curve was best fitted with the following parameters: ν_1/2 = −42mV, k_l = 12, A₁ = 1.13462, A₂ = 1.2071.

To test if I_M prevents spontaneous activity of nociceptive DRG neurons we performed perforated-patch clamp recordings in current-clamp mode from acutely dissociated small (less than 25 μm in diameter), nociceptor-like DRG neurons. At resting potential (−58.15±0.9 mV, Figure 1, right “Before”), all neurons were quiescent (n = 26, Figure 1). Focal puff application of the I_M blocker XE991 (10 μM; applied ~2 min after the onset of recording) caused, within 3-5 min, a substantial depolarization (12.35 ± 1.8 mV; Figure 1A, right “XE991”) in all recorded neurons followed by repetitive firing in 83% of the neurons (5/6; Figure 1A, left). This excitatory effect persisted throughout drug application (1-5 min) and in most cases was also observed during washout (not shown).

Several recent reports demonstrated that 10 μM XE991 can also affect Kv1 and Kv2/Kv9 channels (Zhong et al., 2010) and delayed rectifier (I_K) and A-type (I_A) potassium currents (Xia et al., 2002). We therefore performed the abovementioned experiment using focal application of a lower concentration of XE991 (3 μM) and showed similar effects on nociceptor-like DRG neuronal excitability. Puff application of 3 μM XE991 lead, within 4–6 min, to 17.58 ± 2.4 mV membrane depolarization in all neurons and repetitive firing in 91% of the neurons (10/11; Figure 1B). To reassure that the observed spontaneous activity was due to XE911 mediated I_M inhibition and not due to mechanical interference caused by pressure during puff application, we recorded changes in nociceptor-like DRG neurons activity following puff application of vehicle (see Methods). Puff applied vehicle exerted no neuronal effects during sustained recordings (up to 15 min; n = 9; Figure 1C). In the latter condition the neurons displayed normal evoked firing behavior (n = 9; Figure 1C).

The abovementioned results, showing that selective inhibition of I_M leads to spontaneous activity in nociceptor-like DRG neurons, indicate that I_M provides sufficient outward current to prevent suprathreshold neuronal depolarization in the resting-to-threshold membrane potential range. To characterize the activation properties of the K_v7 channels underlying I_M in this membrane potential range, we first measured the action potential threshold of nociceptor-like DRG neurons using perforated-patch clamp recordings in the current clamp mode. We measured spike threshold by analyzing the phase plot (dV/dt) of single action potential evoked by 500 ms steps. We further verified the action potential threshold using 500 ms current ramps (see Methods). In our experimental conditions the threshold for action potential in nociceptor-like neurons was −31.1±2.2 mV (n = 14), which was similar to the one measured using ramps (−29.4±2.2, mV n = 9, see inset for Figure 2B).

Next we characterized the voltage dependence of I_M activation. We used a well-established voltage-clamp protocol in the perforated-patch whole cell configuration to isolate I_M and to generate I-V and conductance slope curves (Brown and Adams, 1980; Passmore et al., 2003; Wang and McKinnon, 1995; see Methods; Figure 2A, inset). In our conditions I_M was activated at −60 mV (n = 18, Figures 2B,C) similarly to what was previously shown (Passmore et al., 2003). Considering the reversal potential of I_M (Figure 2B), these data indicate that I_M is active and hyperpolarizatory at resting-to-threshold potentials in nociceptor-like DRG neurons.

Finally, we verified that I_M in nociceptor-like DRG neurons was attenuated by 3 μM XE991 (Supplementary Figure 1).

Our data show that XE991 abolishes I_M and leads to spontaneous firing. To link between these two effects of XE991 and to show that I_M is sufficient for averting nociceptive ectopic activity, we built a single-compartment computational model of a nociceptor-like DRG neuron using NEURON simulating environment (Hines and Carnevale, 1997). We used our experimental data to determine the kinetic properties of the I_M-mediated conductance and tuned the I-V relationship according to the steady-state I-V curve shown in Figure 2B. We adapted the I_M current model from Shah et al. (Shah et al., 2008) and adjusted its parameters to fit our findings. The properties of the resulting simulated current resembled the amplitude, kinetics and voltage dependence of I_M recorded from nociceptor-like DRG neurons (Figure 2C, inset). To simulate electrical properties of DRG neurons, we based our model on previously described models of nociceptor-like DRG neurons (Gudes et al., 2015; Herzog et al., 2001; Baker, 2005; see Methods). Under these conditions the resting potential of the simulated membrane was −57.85 mV and the threshold was −30.27 mV, which were similar to the experimental values (shown in Figure 2B, insets). Importantly, the simulated responses to current steps (Figure 3A) and depolarizing ramps (Figure 3B) replicated well the experimental findings. We then validated the response of the modeled soma to “natural” stimuli. To that end, we simulated a current induced by application of capsaicin, an activator of the noxious heat-sensitive TRPV1 channel, expressed by nociceptive neurons. We and others have previously demonstrated that application of capsaicin produces depolarization of nociceptive neurons, followed by a stereotypic pattern of action potential firing (Nita et al., 2016; Blair and Bean, 2003; see also Supplementary Figure 2, here). To mimic the capsaicin-induced current, we first introduced an inward current with the activation and inactivation kinetics resembling TRPV1-induced inward current evoked by short (5s) puff application of capsaicin (1 μM) into our single-compartment model (see Methods). The stimulation of the soma with a modeled 5 s application of capsaicin (capsaicin-like stimulation) resulted in an inward current and action potential firing, which resembled the experimental data (Figures 3C,D, compare to Supplementary Figure 2A,B respectively, see also Nita et al., 2016).

Next, we explored whether our model would reflect the hyperexcitable changes following inhibition of I_M. It is widely accepted that I_M inhibition apart from membrane depolarization (Figure 1) leads to decrease in spike threshold and increase in firing rate (Yue and Yaari, 2004; Gu et al., 2005; Linley et al., 2008; Liu et al., 2010; Tzour et al., 2016). Similarly, in our experimental conditions inhibition of I_M decreased spike threshold (−34.94±4.9 mV, n = 8) and increased action potential firing evoked either by depolarizing steps (compare Figure 3A, left to Figure 3E, left) or ramps (compare Figure 3B, left to Figure 3F, left). To examine if our model would reflect the hyperexcitability induced by I_M inhibition we omitted the I_M-like conductance (g_Kv7/M) from the model without altering any of the other parameters. The negative steady state current was applied to hyperpolarize the membrane to its initial value. In these conditions, the action potential threshold was decreased to −32.82 mV. Moreover, application of depolarizing steps or ramps accurately reflected the changes we observed in our experimental conditions when 3 μM XE991 was puff applied onto the nociceptor-like DRG neurons (Figures 3E,F). Thus, our model accurately predicts excitable properties of nociceptor-like DRG neurons, under the limitations and assumptions made (see Methods).

Finally, using this model, we show that gradual attenuation of g_Kv7/M (see Methods) lead to membrane depolarization followed by spontaneous action potential firing (Figure 3G). The action potential firing was induced only when at least 98% of somatic g_Kv7/M (gKv7/MSoma) was blocked (Figure 3G, bottom). These simulated results suggest that I_M is sufficient to prevent spontaneous activity of nociceptor-like DRG neurons.

Nociceptive DRG somata robustly express K_v7 channels which underlie I_M (Passmore et al., 2003). In addition, K_v7 channels are present at nociceptive peripheral terminals innervating the target organs (Passmore et al., 2012). Thus I_M, as an integral part of the terminal conductance may contribute to the terminal excitability state. Indeed, subcutaneous application of XE991 into the hind paw induced increased grooming and flinching, suggesting that inhibition of I_M at the terminals and distal axons leads to acute pain (Linley et al., 2008). It is therefore plausible that K_v7 channels, which are expressed at the nociceptive terminals prevent spontaneous activation of terminal and hence the ectopic activity of nociceptive neurons. To examine whether I_M is sufficient to prevent spontaneous activation of nociceptive terminals we built a realistic multi-compartment model of nociceptive peripheral terminal and peripheral and central axons connected to cylindrical soma via a T-junction and stem axon (Figure 4A). To be able to closely replicate the geometry of the nociceptive terminal branches, we first assessed the structure of the nociceptive terminal tree in-vivo using multiphoton imaging of nociceptive terminal branches innervating the skin. To that end, we expressed a fluorescent marker in the sensory neurons innervating the mouse hind paw by infecting mice sciatic nerve unmyelinated C-fibers (Iyer et al., 2014) with an AAV6 carrying the expression cassette for GFP. The intact axons and terminals innervating the mice hind paw were visualized in-vivo and reconstructed in 3D, using multiphoton microscopy, two weeks after sciatic injection, as exemplified in Figure 4A, inset right. The 2D render of this 3D reconstruction was then used as a base for building a geometrical modeled structure of the terminal tree (Figure 4A, inset left).

We based some of the electrical parameter values of the model (see Methods) on data available from the literature (Pristera et al., 2012; Vasylyev and Waxman, 2012; Feng et al., 2015; Sundt et al., 2015), while some were obtained by data extrapolation. The model incorporates a repertoire of voltage-gated Na⁺, K⁺ conductances homogeneously distributed along the axons and the terminals and tuned to resemble experimental values (see Methods). To model terminal g_Kv7/M (gKv7/MTerminal), we used the parameters of somatic g_Kv7/M (gKv7/MSoma) adjusted for the area of the cable-like terminal (see Methods). When the gKv7/MTerminal was intact (1×gKv7/MTerminal, see Methods) the simulated neuron was quiescent (Figure 4B, upper traces). In the latter condition, stimulation of one of the terminal branches (Figure 4A, inset right, red dot) by a 500 ms square pulse of 100 pA current, elicited activation of the distal axon which propagated along the soma and reached the central terminal (Figure 4B, lower traces). When gKv7/MTerminal was zeroed (0×gKv7/MTerminal) in the whole terminal tree (shown in Figure 4A, inset, right) a spontaneous firing with a rate of 13 Hz (the maximal firing rate in the model) was observed, which started at the peripheral axon and propagated throughout the neuron (Figure 4C). These data suggest that I_M acting at the nociceptive terminals is sufficient to prevent spontaneous activation and therefore ectopic activity of nociceptive neurons.

We next estimated, using our multi-compartment model, what is the value of gKv7/MTerminal which is necessary to prevent spontaneous activation of nociceptive neurons. To that end, we gradually reduced the gKv7/MTerminal and examined at which gKv7/MTerminal the simulated neuron starts to fire spontaneously (Figure 5). We found that even after inhibition of 63% of gKv7/MTerminal the remaining gKv7/MTerminal is sufficient to prevent spontaneous firing (Figure 5A). Lowering gKv7/MTerminal beyond 37% elicited spontaneous firing with a frequency which progressively increases with decrease of gKv7/MTerminal (Figures 5A,B).

K_v7/M channels are modulated by various signaling molecules. These channels require bound PIP₂ for opening, and GqPCRs such as the muscarinic acetylcholine receptor causes I_M inhibition by PLC-induced PIP₂ depletion (Suh and Hille, 2002; Zhang et al., 2003; Liu et al., 2010). Moreover, both IP₃-induced increases in intracellular Ca²⁺ concentration (Jones et al., 1995) and PKC activation (Marrion, 1994; Lee et al., 2010) have been implicated in I_M inhibition. These signaling molecules and downstream signaling cascades are activated during inflammation. It is therefore plausible that during inflammation even partial inhibition of gKv7/MTerminal could increase the sensitivity of nociceptive neurons to incoming stimuli, thus leading to inflammatory hyperalgesia (Liu et al., 2010). We examined this hypothesis using our multi-compartment model in which we “inhibited” half of the gKv7/MTerminal(0.5×gKv7/MTerminal). Interestingly, although in these conditions, simulated neurons remained silent (Figure 6A, upper traces), stimulation of the terminal branch (as shown in Figure 6A, right, red dot) by a 500 ms square pulse of 100 pA current produced robust activation of the distal axon which propagated along the soma and reached the central terminal (compare Figure 6A, lower traces with Figure 4B, lower traces).

Next, we examined if partial inhibition of I_M would enhance the responsiveness of the simulated neuron to “natural” stimuli. We introduced the modeled TRPV1/capsaicin current (see Methods) into the terminals of the multi-compartment model. We applied a capsaicin-like stimulation onto the same terminal branch of the simulated neuron, previously used for electrical stimulation (Figure 6B, left, red dot), while varying the gKv7/MTerminal. In “normal” conditions, i.e. with an intact gKv7/MTerminal, application of the capsaicin-like stimulus induced relatively weak activation of the terminal (Figure 6B, middle) which was transmitted toward the central axon (Figure 6C, lower inset). We next examined whether inhibition of gKv7/MTerminal, to levels which are not sufficient to induce spontaneous firing, would be sufficient to affect capsaicin-mediated responses of the multi-compartment model neuron. Inhibition of up to 30% of gKv7/MTerminal did not change the response of the terminal to capsaicin (not shown) and capsaicin-induced firing (Figure 6C). Further decrease of gKv7/MTerminal lead to a gradual increase in response to capsaicin-like stimulus (Figure 6C). Inhibition of more than 40% of gKv7/MTerminal induced robust, step-like increase in activation of the terminal (exemplified for 0.5 × gKv7/MTerminal in Figure 6B, right), translated into about 11 Hz firing which was fully propagated toward the central terminal of the simulated neuron (Figure 6C, exemplified for 0.5×gKv7/MTerminal in Figure 6C, upper inset).

Nociceptive membrane potential undergoes subthreshold fluctuating perturbations which are increased after nerve injury (Amir et al., 1999, 2002; Liu et al., 2000). To examine the contribution of I_M in impeding spontaneous firing during membrane fluctuations we have stimulated the terminal tree of the multi-compartment model by injecting an Onstein-Uhlenbeck-based current noise (see Methods), in which the current amplitude varies normally in each time-step of 0.25 ms (Olivares et al., 2015), while changing gKv7/MTerminal. We varied the standard deviation of the current amplitude's normal distribution (σ) thus changing the probability of reaching a higher injected currents and causing higher membrane fluctuation amplitude (Figures 7A–D, upper traces). In normal conditions, i.e. when gKv7/MTerminal was intact, the modeled neuron remained quiescent despite fast membrane fluctuations at the single terminal of up to 15 mV (up to σ = 0.007; exemplified for σ = 0.004 in Figure 7A; exemplified for σ = 0.006 in Supplementary Figure 3; Figure 7F, black). When gKv7/MTerminal was reduced to half (Figure 7B), which, as we showed above, was not sufficient to induce spontaneous firing of the neuron (Figures 5, 6) neuronal membrane fluctuations of even 6 mV (σ = 0.004), were sufficient to elicit repetitive firing at the terminal tree which propagated toward the central terminal (Figures 7B,E, red).

In suprathreshold fluctuations, in which σ was increased to evoke low frequency firing (exemplified for σ = 0.007 in Figure 7C) reduction of gKv7/MTerminal to half led to a substantial increase in firing frequency of nociceptive neurons (Figures 7D,E). These data predict that I_M reduces susceptibility of nociceptive neurons to membrane fluctuations. When gKv7/MTerminal is decreased the nociceptive neuron is activated by small perturbations of 6 mV, while when gKv7/MTerminal is intact, the neurons will still be quiescent even upon 15 mV fluctuations.

Subthreshold excitability of nociceptive neurons is controlled, among other factors, by depolarizing conductances, contributed largely by sodium channel isoform 1.9 (Na_(V)1.9) which underlie persistent TTX-resistant sodium current (Herzog et al., 2001; Dib-Hajj et al., 2002; Baker, 2005). To characterize the interaction between gKv7/MTerminal and terminal g_Na(v)1.9, we simulated, using our multi-compartmental model, the effect of changing these conductances on the excitability of a modeled nociceptive terminal and whole neuron. When gKv7/MTerminal was zeroed neurons remained silent if g_Na(v)1.9 was inhibited (Figure 8). In our conditions, at least 65% of terminal g_Na(v)1.9 were required for spontaneous firing when gKv7/MTerminal was zeroed. These data suggest that at subthreshold potentials I_M counteracts TTX-resistant persistent sodium current, thus precluding spontaneous firing.

The enhancement of g_Na(v)1.9 under pathological conditions has been shown to lead to activation of nociceptive neurons (Baker, 2005; Amaya et al., 2006; Binshtok et al., 2008; Maingret et al., 2008; Gudes et al., 2015). We examined the ability of gKv7/MTerminal to prevent spontaneous activity when g_Na(v)1.9 was enhanced. We show that when gKv7/MTerminal is intact the simulated neuron remains silent even when g_Na(v)1.9 is increased up to 145% (Figure 8). I_M was also able to prevent stimulus-induced hyper-responsiveness when g_Na(v)1.9 was increased up to 125% (Supplementary Figure 4). On the other hand, when gKv7/MTerminal was decreased, even a slight increase in g_Na(v)1.9 resulted in spontaneous firing, such that when gKv7/MTerminal was reduced by half, about 10% increase in g_Na(v)1.9 produced activation of modeled nociceptive neuron (Figure 8). As stated before, in these conditions, stimulus-evoked hyper-responsiveness was present even when g_Na(v)1.9 was intact (see Figure 6A, Supplementary Figure 4).

Altogether these data suggest that when terminal I_M is intact no spontaneous activity will occur even during substantial perturbation in depolarizing conductances in subthreshold range. It also implies that in pathological conditions spontaneous activation of nociceptive neurons could be more easily achieved by the cooperative effect of decrease in g_Kv7 and increase in g_Na(v)1.9.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors and states that the process nevertheless met the standards of a fair and objective review.