Prolonged action potential firing rapidly reduces membrane excitability in an activity-dependent manner
Excitability is a neuronal feature that reflects the contribution of several membrane properties, and was used as the initial measure for an effect of action potential firing on neuronal properties in vivo. Action potential firing caused by neuronal depolarization (10–20 Hz firing, 60s depolarization by direct current, to Vm −50 to −45 mV) caused a robust decrease of excitability (n=11/12 neurons, approximately 39.4 ± 8.1 % of baseline excitability after 1 min; ; p<0.001, one-way repeated-measures ANOVA, F(11,32)=21.27). This decrease of neuronal excitability lasted between 5–10 min, before returning to baseline levels (; baseline significantly different than post-firing values at 0 to 8.5 min after firing, post-hoc two-way t-test comparisons with Bonferroni corrections).
Neuronal activity causes a decrease of membrane excitability
This effect of neuronal firing depended upon the number of action potentials evoked during the period of prolonged action potential firing, as well as the duration of depolarization. A depolarization was less effective if it resulted in fewer action potentials (; 1–10 Hz; excitability reduced to 87.8 ± 3.6% of baseline 1 min after the end of activity, n=10; p=0.556, one-way repeated-measures ANOVA, F(9,32)=0.95). There was a significant correlation between the number of action potentials evoked during the 60 s depolarization and the decrease in measured excitability (; r2=0.675, p<0.01, n=23; slope=−0.10 ± 0.015; each additional AP leads to approximately −0.1% change of excitability). This effect of action potential firing relies upon concurrent action potential firing and depolarization, and is not caused solely by the amplitude of depolarization, as a large depolarization that resulted in initial robust firing (20–40 Hz during beginning) followed by strong firing rate accommodation (and therefore fewer action potentials, reduced to 0–10 Hz by the end) had a weaker effect on excitability (84.7 ± 10.2% of baseline excitability after 1 min, n=6; two-way repeated measures ANOVA comparison between 10–20 Hz and 20–40 Hz, p=0.004, main effect of firing rate F(1,34)=11.0) than prolonged firing in the presence of a smaller depolarization (as above, 39.4 ± 8.1% after 1 min).
Depolarization produced only transient changes if it was of shorter duration. Thus, 15 s depolarization resulted in decreased excitability only when measured immediately after depolarization (; 10–20 Hz firing; n=6; p<0.01, one-way repeated-measures ANOVA, F(5,32)=3.09; baseline significantly different than post-firing values at 0 to 30 s after firing, post-hoc two-tailed t-test comparisons with Bonferroni corrections), but this quickly returned to baseline, and no significant changes were observed when measured at greater than 1 min after depolarization (93.1 ± 7.4% of baseline excitability after 1 min). An intermediate effect was observed after 30 s depolarization (; 10–20 Hz firing; 67.9 ± 5.4% of baseline excitability after 1 min, n=5; p<0.001, one-way repeated-measures ANOVA, F(4,32)=6.89; baseline significantly different than post-firing values at 0 to 2 min after firing, post-hoc two-tailed t-test comparisons with Bonferroni corrections). This is consistent with an activity-dependent effect on neuronal membrane excitability.
Theta burst firing decreases membrane excitability in vivo
Activity-dependent effects of neuronal firing on excitability can be more precisely measured if the action potentials are evoked in a more predictable, more controllable manner. In addition, continuous firing for 60 s might not be a normal firing pattern in vivo
. Therefore, short depolarizing pulses were used to control the timing and frequency of action potentials. It has been shown that the BLA displays rhythmicity near theta frequencies (Pare and Collins, 2000
; Pape et al., 2005
), and that burst firing near theta frequencies can induce robust plasticity in other brain regions in vitro
(Narayanan and Johnston, 2007
). Therefore, short depolarizing pulses were used to induce burst firing of LAT neurons near theta rhythmicity, a procedure termed theta burst firing (TBF, 60 ms pulses repeated at 5 Hz, ). The amplitude of each pulse was set to evoke an average of 1 – 4 APs/burst. When evoked from resting membrane potentials, 60 s of TBF activity produced only a transient decrease of excitability (n=10, p<0.01, F(9,32)=6.68; baseline significantly different than post-firing values at 0 to 60 s after firing, post-hoc two-tailed t-test comparisons with Bonferroni corrections) that returned to baseline after 1 min (; 87.7 ± 8.7% of baseline excitability). However, when the membrane was depolarized to more closely mimic the membrane potential in awake animals ((Steriade et al., 2001
); here approximately −60 mV, subthreshold to significant spontaneous action potential firing), and theta burst firing was applied (5 Hz, 60 s), a robust decrease of excitability was observed (; 30.7 ± 2.6% of baseline excitability after 1 min, n=11/12 neurons, p<0.001, F(11,32)=35.99; baseline significantly different than post-firing values at 0 to 7 min after firing, post-hoc two-tailed t-test comparisons with Bonferroni corrections). There was a correlation between the average number of action potentials/burst and the observed decrease of excitability measured after 1 min (; r2
=0.66, p<0.01, n=16; slope=−0.189 ± 0.036). However, depolarization to −55 mV alone was insufficient to induce a long-term reduction of membrane excitability (; 103.2 ± 4.3% of baseline excitability after 1 min, n=7 neurons, p=0.007, F(6,32)=1.85; post-firing significantly greater
than baseline values at 0 to 30 s after depolarization, post-hoc two-tailed t-test comparisons with Bonferroni corrections). The effects of TBF on excitability are consistent with a rapid in vivo
reduction of membrane excitability following relatively short periods of activity, and this change is dependent upon the depolarization state of the neuron.
Theta burst firing activity causes a decrease of membrane excitability
In a subset of neurons, a more comprehensive measure of membrane excitability was obtained before and after TBF using a range of current steps (0 to 1000 pA, Methods) to formulate a complete input-output curve of excitability. A reduction of excitability after TBF activity was observed (quantified as the mean number of action potentials/current step, n=6, p=0.0003, F(1,56)=29.13, two-way repeated measures ANOVA). This reduction was seen across the entire range of measure (, left; reflected as a reduction of the slope of the input-output relationship, baseline 1.47 ± 0.096 AP for each 100 pA increase of current step input, post-TBF activity 1.06 ± 0.085 AP for each 100 pA increase of current step input, p=0.0028, F(1,56)=9.78, two-way repeated measure ANOVA).
The reduction of membrane excitability caused by TBF can be due to several membrane properties that influence neuronal excitability, including a shift in action potential threshold and input resistance. However, there was no significant change in action potential threshold (baseline −50.6 ± 1.9 mV, post-TBF −50.1 ± 1.7 mV, p=0.07, t=1.98, df=15, two-tailed paired t-test). There was a significant reduction of input resistance after TBF (; baseline 33.4 ± 1.6 MΩ, post-TBF activity 30.1 ± 1.0 MΩ, p=0.014, t=3.46, df=7, two-tailed paired t-test). This was accompanied by a significant shortening of the membrane time constant (; baseline 17.9 ± 1.7 ms, post-TBF activity 15.7 ± 1.6 ms, p=0.015, t=3.35, df=7, two-tailed paired t-test). A change of the resting membrane potential (Vm) is unlikely to explain the changes induced by TBF because these changes were compensated with offsetting DC prior to measurement of excitability (following TBF there was typically a small hyperpolarization of the Vm that returned to baseline in 1–2 s). This change of membrane properties has similar requirements to the reduction of membrane excitability induced by TBF. Thus, if the TBF was applied without depolarization, a condition that does not lead to a decrease of membrane excitability (above), there was no significant change of input resistance (; baseline 35.4 ± 2.24 MΩ, post-TBF 33.9 ± 2.52 MΩ, n=10, p=0.139, t=1.63, paired t-test) or membrane time constant (; baseline 16.9 ± 0.96 ms, post-TBF 16.7 ± 1.22 ms, n=8, p=0.913, t=0.114, paired t-test).
Theta burst firing modifies membrane properties of BLA neurons
Theta burst firing decreases synaptic integration in vivo
Modification of membrane properties by TBF, such as input resistance and membrane time constant, should lead to a change of the integrative properties of these neurons. To test this, parameters of spontaneous synaptic events that are sensitive to changes of membrane properties were measured. TBF caused a reduction in the amplitude of spontaneous synaptic events (; baseline 3.81 ± 0.20 mV, post-TBF activity 3.32 ± 0.18 mV, p=0.0029, t=3.80, df=11, two-tailed paired t-test) and a decrease in the half-width of events (; baseline 11.98 ± 1.12 ms, post-TBF activity 10.64 ± 0.77 ms, p=0.015, t=2.87, df=11, two-tailed paired t-test), without a change in the frequency of events (; inter-event interval; baseline 0.59 ± 0.03 s, post-TBF activity 0.58 ± 0.04 s, p=0.329, t=1.022, df=11, two-tailed paired t-test). These spontaneous synaptic events often occur in groups, leading to summation (). TBF caused a reduction of the peak amplitude of these summated events (; baseline 7.54 ± 0.40 mV, post-TBF activity 6.19 ± 0.33 mV, p=0.002, t=4.02, df=11, two-tailed paired t-test). As a measure of post-synaptic activity that is independent of event detection thresholds, the standard deviation (SD) of the mean membrane potential was also measured, and was significantly decreased after TBF activity (baseline 2.71 ± 0.24 mV, post-TBF activity 2.42 ± 0.29 mV, p=0.020, t=2.71, df=11, two-tailed paired t-test), observable as a narrowing of the distribution of the membrane potential (). This is consistent with a change in postsynaptic properties that influences integration of synaptic activity. However, these measured changes may be due to a retrograde modulation of presynaptic function. Therefore, the voltage response to EPSC-shaped current injection was also measured, a parameter strongly dependent upon postsynaptic membrane properties (αPSPs, Methods). TBF caused a reduction in the summation of αPSPs (; baseline 0.77 ± 0.10, post-TBF activity 0.67 ± 0.12, p=0.041, t=2.60, df=6, two-tailed paired t-test; there was no significant difference in the amplitude of the first PSP despite the previously-noted reduction of input resistance, probably because the time course of the PSC is rapid, and the peak of the PSP occurs more rapidly than the membrane time constant in response to a square step: baseline 5.90 ± 0.59 mV, post-TBF activity 5.78 ± 0.57 mV, p=0.767, t=0.311, df=6, two-tailed paired t-test). These data point to a post-synaptic change of synaptic integration caused by TBF.
Theta burst firing decreases synaptic integration
Plasticity of membrane excitability does not require NMDA receptors in vivo
Previous studies in vitro
have demonstrated that many forms of plasticity rely upon activation of postsynaptic NMDA receptors and Ca2+
influx through these channels or other sources. To determine what factors contribute to change of excitability caused by TBF, chemical blockers were included in the intracellular pipette to target the recorded neuron. In these experiments, a minimum of 25 min was utilized to allow time for the chemical blocker to diffuse into the recorded neuron, before neurons were exposed to a strong 5 Hz bursting protocol (2 – 4 APs/burst) paired with depolarization. Intracellular blockade of NMDA channels with administration of high concentrations of Mg2+
(20 mM; Mayer et al., 1984
; Nowak et al., 1984
; Johnson and Ascher, 1990
) or MK801 (2 mM; Berretta and Jones, 1996
; Humeau et al., 2003
) did not block TBF-dependent reduction of membrane excitability, though the effectiveness of TBF activity was reduced (; intracellular MK801 56.1 ± 5.3% of baseline excitability 1 min after TBF activity, n=6, intracellular Mg2+
48.3 ± 5.8%, n=6, control 30.7 ± 2.6%; significant effect of NMDA blockade compared to control, p=0.0068, F(2,21)=6.39, two-way repeated measures ANOVA). It is possible that MK801 failed to block the effects of TBF due to lack of sufficient intracellular diffusion of this drug, resulting in suboptimal blockade of NMDA channels. Therefore, experiments were replicated with systemic administration of a fairly high dose of MK-801 (0.2 mg/kg i.p.; dose higher than effective doses for blockade of amygdala-dependent behaviors; Venable and Kelly, 1990
; Baker and Azorlosa, 1996
; Langton et al., 2007
), and similar results were observed (systemic MK801 62.2 ± 7.4% of baseline excitability 1 min after TBF activity, n=6, significant effect of NMDA blockade compared to control, p=0.022, F(1,16)=5.12, two-way repeated measures ANOVA). Thus, when NMDA channels are blocked, TBF still induces a change of membrane excitability. Though it should be noted that, while TBF still induced a reduction of excitability in the presence of MK801, the effectiveness of TBF was reduced.
Plasticity of membrane excitability is not dependent upon NMDA receptors
In addition to the a reduction of membrane excitability after TBF in the presence of NMDA blockers, TBF still induced a change of membrane properties when NMDA channelss were blocked () including a reduction of Rn (baseline Rn 30.2 ± 2.7 MΩ, post-TBF 26.7 ± 2.6 MΩ, p<0.001, t=9.56, df=5, two-tailed paired t-test) and a faster membrane Tau (baseline 19.7 ± 1.4 ms, post-TBF 17.3 ± 1.3 ms, p=0.009, t=4.14, df=5, two-tailed paired t-test).
Plasticity of membrane excitability requires Ca2+ channel activity in vivo
The decrease of excitability observed after TBF was prevented by blockade of Ca2+ channels with Ni2+ (0.5 mM) or Cd2+ (0.5 mM) in the intracellular pipette (; intracellular Cd2+ 86.6 ± 2.0% of baseline excitability 1 min after TBF activity, n=7, intracellular Ni2+ 97.7 ± 2.4%, n=6; significant effect of Ca2+ channel blockade compared to control, p<0.001, F(2,20)=10.22, two-way repeated measures ANOVA). When examined with a more comprehensive measure of membrane excitability using a full input-output measurement (0 to 1000 pA) to verify the effectiveness of Ca2+ channel blockade, it was found that blockade of Ca2+ channels with Cd2+ prevented the impact of TBF on membrane excitability over the entire input-output curve (, left; change in the slope of the input-output relationship, baseline 1.31 ± 0.14 AP for each 100 pA increase of current step input, post-TBF activity 1.47 ± 0.15 AP for each 100 pA increase of current step input, p=0.71, t=0.38, two-tailed unpaired t-test).
Plasticity of membrane excitability is dependent upon calcium channels
In addition to disruption of the effects of TBF on excitability, intracellular Ni2+ or Cd2+ blocked the effects of TBF activity on input resistance (; Cd2+, n=7, baseline 35.9 ± 1.6 MΩ, post-TBF 36.0 ± 1.9 MΩ; Ni2+, n=6, baseline 38.22 ± 1.61 MΩ, post-TBF 39.49 ± 1.65 MΩ) and membrane time constant (; Cd2+, n=7, Tau baseline 16.07 ± 1.63 ms, post-TBF 15.50 ± 1.62 ms; Ni2+, n=6, baseline Tau 17.7 ± 1.45 ms, post-TBF 17.5 ± 1.61 ms).
In the presence of intracellular Ni2+ or Cd2+, there was also no observed effect of TBF on spontaneous EPSPs summation (; Cd2+, n=7, barrage peak amplitude, 7.38 ± 0.69 mV, post-TBF 7.49 ± 0.54 mV; Ni2+, n=6, baseline peak amplitude 7.11 ± 0.81 mV, post-TBF 7.35 ± 0.82 mV) or αPSP summation (; Cd2+, n=7, baseline 0.85 ± 0.14, post-TBF activity 0.81 ± 0.15; Ni2+, n=6, baseline 0.83 ± 0.17, post-TBF 0.86 ± 0.17). In addition, with intracellular Cd2 or Ni2 there was no significant effect of TBF on parameters of single EPSPs that are sensitive to a change of membrane properties () such as amplitude (Cd2+, n=7, baseline 3.94 ± 0.26 mV, post-TBF 4.15 ± 0.24 mV, p>0.05, paired t-test; Ni2+, n=6, baseline 4.08 ± 0.29 mV, post-TBF 4.18 ± 0.29 mV, p>0.05, paired t-test) and half-width (Cd2+, n=7, baseline 12.58 ± 1.37 ms, post-TBF 11.82 ± 1.21 ms, p>0.05, paired t-test; Ni2+, n=6, baseline 13.73 ± 1.22 ms, post-TBF 12.58 ± 1.21 ms, p>0.05, paired t-test). The blockade of the effects of TBF on membrane properties, membrane excitability and synaptic integration by blockers of Ca2+ channels indicates a common associated trigger for these effects of TBF activity on neuronal function in vivo. These data indicate that periods of robust in vivo firing lead to a change of membrane properties that is associated with decreased excitability and decreased synaptic integration, and that these changes are induced by Ca2+ influx through voltage-gated Ca2+ channels.
Plasticity of synaptic integration is dependent upon calcium channels
One potential concern is that intracellular blockade of Ca2+ channels can exert actions that indirectly influence the induction of TBF plasticity, such as increased basal firing rate leading to a ceiling effect for TBF induction, or decreased number of action potential firing during the TBF activity leading to reduced TBF induction. The basal spontaneous firing rate was very low under our recording conditions. Only 1/13 neurons with intracellular Ni2+/Cd2+ displayed spontaneous firing (0.067 Hz). The firing during the TBF activity was not disrupted (total number of action potentials during TBF: Cd2+ 956.2 ± 45.4, n=7, Ni2+ 863.3 ± 37.4, n=6, equivalent control 841.8 ± 56.0, n=12, p=0.41, F(2,20)=0.94, one-way ANOVA).
Hyperpolarization-activated ion channels contribute to expression
The observed reduction of excitability, input resistance and integration of synaptic inputs following TBF-activity is consistent with an increase of hyperpolarization-activated ion channel (h channel) function. To test whether h channels are involved in the expression of TBF-induced plasticity, h channels were pharmacologically blocked. To do this, microinfusions of ZD7288 (1mg/mL) into the LAT were performed. All recording sites included for analysis were within 1.5 mm of the infusion site. To confirm the effectiveness of ZD7288 and the time course of its actions, ZD7288 infusions were performed following a control period. The control period consisted of the usual repeated testing of membrane excitability (). ZD7288, but not aCSF, caused a rapid increase of excitability that did not reverse over the time course tested (, right; two-way repeated-measures ANOVA, significant effect of time F(1,32)=4.42, p<0.001; significant effect of infusion, F(1,32)=21.34, p<0.01, ZD7288 n=6, aCSF n=4).
Blockade of h channels disrupts the expression of plasticity of membrane properties
To test whether blockade of h channels disrupts plasticity induced by TBF, ZD7288 was infused immediately following a strong 5 Hz TBF protocol (3 – 4 APs/burst; ). Infusion of ZD7288 following TBF blocked TBF-induced changes of membrane excitability. Thus, infusion of ZD7288 rapidly induced an increase of excitability, above the diminished post-TBF levels, and above baseline levels (, middle), quantified as the increased excitability in response to a range of current steps (, mddle, 0–1000 pA; quantified as the mean number of action potentials/current step, n=5, p<0.01, F(1,24)=13.6, two-way repeated measures ANOVA). Infusions of aCSF did not block TBF-induced plasticity (n=5, p<0.01, F(1,24)=15.8, two-way repeated measures ANOVA), and TBF resulted in a decrease of membrane excitability when aCSF was infused (, left). Furthermore, ZD7288 infusion resulted in a similar final membrane excitability in control and TBF conditions, measured as the slope of the input-output relationship (; ZD7288 + TBF 1.69 ± 0.078 AP for each 100 pA increase of current step input, n=5, ZD7288 + control activity 1.73 ± 0.093 AP for each 100 pA increase of current step input, n=6, p=0.53, F(1,62)=0.39, two-way repeated measure ANOVA). Because TBF decreases membrane excitability, this is consistent with a greater degree of h channel activity following TBF activity. It further demonstrates that when the influence of h channels is removed, control and TBF groups are equal. In addition, after ZD7288 was infused, input resistance and integration of αPSPs was similar between control and TBF groups (), demonstrating that h channel activity underlies the expression of TBF-induced reduction of excitability, input resistance and PSP integration.
Activation of h channels leads to a relatively slow voltage sag during a hyperpolarizing pulse. To further verify a role for h channels in the expression of TBF-induced plasticity, the amplitude of the voltage sag was measured before and after a strong 5 Hz TBF protocol (3 – 4 APs/burst), following aCSF infusion. TBF caused an increase in the amplitude of the voltage sag (, calculated as [(peak amplitude-steady state)/peak]; baseline 0.094 ± 0.0075, post-TBF 0.11 ± 0.0050, p=0.0032, t=6.33, n=5, two-tailed paired t-test) after TBF-activity. The voltage sag was blocked by infusions of ZD7288, confirming the h channel origin of the voltage sag in the neurons in vivo ().
Theta burst firing leads to an increase of voltage sag, an indicator of h channel activity
Theta burst firing reduces synaptic plasticity in vivo
An important feature of neuronal function that contributes to learning is the capacity for activity-dependent changes in synaptic strength, exemplified as LTP of synaptic inputs. If TBF exerts a significant modulatory influence in vivo, it is expected to impact LTP. TBF was followed by an LTP protocol (Methods). In control experiments TBF was performed without concomitant depolarization from the resting membrane potential (which does not lead to a long-term change of membrane properties or excitability, above). Under these control conditions, LTP of synaptic inputs was readily observed (, top, B; 147.4 ± 5.4% of baseline after 5 min; n=7, p<0.001, F(2,108)=20.62, two-way repeated measures ANOVA). However, if parameters for effective TBF are used, with concomitant depolarization from the resting membrane potential (which leads to a change of membrane properties and reduction of excitability), subsequent LTP induction was much less effective (, bottom, B; 97.7 ± 2.3% of baseline after 5 min; n=7, p=0.51, F(2,108)=0.861, two-way repeated measures ANOVA). This result does not appear to depend upon EPSP size prior to LTP (mean EPSP amplitude control TBF 7.7 ± 0.9 mV, n=7, TBF with depolarization 7.1 ± 1.0 mV, n=7) and a similar number of action potentials were evoked during the TBF protocols (control TBF 2.8 ± 0.4 action potentials/burst, n=7, TBF with depolarization 2.6 ± 0.4 action potentials/burst, n=7). If TBF truly has a modulatory effect on LTP induction, it is expected that TBF procedures that lead to only moderate changes of excitability would have a smaller effect on LTP induction. A weaker 30 s TBF protocol (above) had a smaller impact on LTP induction (; 120.6 ± 5.7% of baseline after 5 min; n=5, p<0.001, F(2,72)=8.97, two-way repeated measures ANOVA). This association between the effectiveness of the TBF protocol on excitability and on subsequent LTP indicates that these changes may share a common mechanism. To further examine the link between the effects of TBF, associations between the various TBF-induced effects were examined within single neurons.
Theta burst firing decreases synaptic plasticity
Associated changes in excitability, synaptic integration and LTP
In a subset of experiments, the association between the effectiveness of TBF on excitability and synaptic integration, and excitability and LTP were examined. There was a strong correlation between the magnitude of the effects of TBF on excitability and on input resistance (; Rn measured after 3 and 5 min, excitability measured after 2.5 min, r2 = 0.91, significantly different than 0, F=49.0, p<0.001, n=5), and between the effects of TBF on excitability and synaptic integration, as measured by summation of αPSPs (; r2 = 0.73, significantly different than 0, F=13.2, p<0.05, n=7). Combined with the similar dependence on Ca2+ channel activity, this implies a strong relationship between these two outcomes of TBF. There was also a strong correlation between the magnitude of the effects of TBF on excitability and LTP (; LTP measured after 10 min, excitability measured immediately before LTP; r2 = 0.79, significantly different than 0, F=37.9, p<0.001, n=12). In particular, in those instances where TBF was only weakly effective, LTP was more robust.
Correlation between the change of excitability induced by theta burst firing and other measures