Mechanisms and consequences of action potential burst firing in rat neocortical pyramidal neurons

Abstract

1. Electrophysiological recordings and pharmacological manipulations were used to investigate the mechanisms underlying the generation of action potential burst firing and its postsynaptic consequences in visually identified rat layer 5 pyramidal neurons in vitro. 2. Based upon repetitive firing properties and subthreshold membrane characteristics, layer 5 pyramidal neurons were separated into three classes: regular firing and weak and strong intrinsically burst firing. 3. High frequency (330 +/- 10 Hz) action potential burst firing was abolished or greatly weakened by the removal of Ca2+ (n = 5) from, or by the addition of the Ca2+ channel antagonist Ni2+ (250-500 microm; n = 8) to, the perfusion medium. 4. The blockade of apical dendritic sodium channels by the local dendritic application of TTX (100 nM; n = 5) abolished or greatly weakened action potential burst firing, as did the local apical dendritic application of Ni2+ (1 mM; n = 5). 5. Apical dendritic depolarisation resulted in low frequency (157 +/- 26 Hz; n = 6) action potential burst firing in regular firing neurons, as classified by somatic current injection. The intensity of action potential burst discharges in intrinsically burst firing neurons was facilitated by dendritic depolarisation (n = 11). 6. Action potential amplitude decreased throughout a burst when recorded somatically, suggesting that later action potentials may fail to propagate axonally. Axonal recordings demonstrated that each action potential in a burst is axonally initiated and that no decrement in action potential amplitude is apparent in the axon > 30 microm from the soma. 7. Paired recordings (n = 16) from synaptically coupled neurons indicated that each action potential in a burst could cause transmitter release. EPSPs or EPSCs evoked by a presynaptic burst of action potentials showed use-dependent synaptic depression. 8. A postsynaptic, TTX-sensitive voltage-dependent amplification process ensured that later EPSPs in a burst were amplified when generated from membrane potentials positive to -60 mV, providing a postsynaptic mechanism that counteracts use-dependent depression at synapses between layer 5 pyramidal neurons.

Figure 1. Identification of burst firing neurons

Cell-attached somatic voltage-clamp recording (V_pipette = -65 mV) of neuronal firing in response to extracellular synaptic excitation (upper traces) demonstrates two groups of layer 5 pyramidal neurons: regular and burst firing. The lower traces demonstrate that the subsequent formation of whole-cell current-clamp recording did not modify action potential firing properties when evoked from the resting membrane potential (-64 and -63 mV, regular and burst firing neuron, respectively). The experimental arrangement is shown in the inset.

Figure 2. Action potential firing patterns of layer 5 pyramidal neurons in response to somatic current injection

Discharge patterns of three neurons in response to steps of positive current (lower superposed traces). The holding potential is shown below the lower voltage traces. Note that in the regular firing neuron a single action potential is evoked at threshold current intensity. In weak and strong burst firing neurons, however, a high frequency (> 250 Hz) burst of action potentials is the threshold response. Bursts of action potentials were repeated throughout the duration of current pulses in strong, but not weak, burst firing neurons. The number of action potentials composing a burst, however, decreased with time in strong burst firing neurons.

Figure 3. Voltage dependence of burst discharges in response to somatic current injection

A, incremental series of somatic current steps, delivered from the indicated values of holding potential, produce families of voltage responses. Note that the threshold discharge pattern is a stereotyped burst of action potentials irrespective of the prior holding potential. Also note the highly non-liner slope resistance of this neuron. B, repeated bursts of action potentials are evoked during sustained suprathreshold depolarisation produced by the tonic passage of current through the recording electrode (values beneath traces). Lower traces show segments of the data at a faster time base: note the periodic nature of burst discharges.

Figure 5. Ionic basis of burst firing

A, burst discharges evoked by a brief somatic current pulse (2 ms, 1.6 nA) recorded at the soma are significantly weakened by removal of calcium from the extracellular solution. Voltage traces obtained from control (2 mm calcium:1 mm magnesium), nominally zero calcium (3 mm magnesium) and following wash (holding potential -66 mV). B, burst discharges evoked by a long somatic current pulse (500 ms, 0.9 nA) recorded at the soma are blocked by the calcium-channel antagonist nickel (500 μm). Voltage traces obtained from control, nickel and wash conditions document the reversible blockade of burst firing (holding potential -65 mV). C, local apical dendritic application of tetrodotoxin blocks burst discharges. The experimental arrangement is shown in the inset. Traces recorded at the soma from control, following the transient local application of TTX (100 nm in pipette) to the apical dendrite 230 μm from the soma and following recovery. Note that TTX did not change the amplitude, threshold or waveform of the first somatic action potential. D, local apical dendritic application of nickel reduces the intensity of burst discharges. Traces from control, following the transient local application of nickel (1 mm in pipette) to the apical dendrite 260 μm from the soma and following recovery. Note the reduction of burst firing is not accompanied by a change in the amplitude, waveform or threshold of first action potential. Holding potential in C and D was -65 mV.

Figure 8. Action potentials are not decremental in the distal axon during a burst discharge

A, pairs of simultaneous recorded somatic whole-cell (top traces) and axonal cell-attached (V_pipette = -65 mV) (bottom traces) recordings at different distances from the soma during burst firing. During an action potential burst the amplitude of somatically recorded action potentials decreases, in some trials later somatically recorded action potentials show inflections in their rising phase (**) and can apparently fail (*). Simultaneous axonal recordings show that when action potential invasion of the soma almost fails (**) two axonal events are recorded, and that when somatic failure occurs the axonal action potential is largely unchanged (axonal recording 25 μm from the base of the soma, two consecutive records). The decrement in axonal action potentials during a burst is dependent upon the distance of the axonal recording from the soma. Note that each action potential of a burst is of a similar amplitude at the most distal axonal recording site (40 μm from the base of the soma). B. the relative amplitude of the second, third, fourth and fifth action potentials in a burst during cell-attached axonal recordings at different distances from the soma. Action potential amplitude was normalised to that of the first action potential in the burst. Total of 14 cells represented. C, simultaneous somatic and axonal whole-cell recording during burst firing. Note all action potentials in the burst are generated first in the axon.

Figure 4. Characteristics of the voltage-current relationship in neurons exhibiting different firing behaviour

Families of voltage (upper, superposed traces) and current (lower, superposed traces) records demonstrate the typical rectification properties of regular, weak burst and strong burst firing neurons. Recordings were made from similar holding potentials (-65 mV). Note, in the strong burst firing neuron a clear transient depolarising potential was generated at the onset of positive voltage responses. The lower graphs show voltage-current relationship when measured at the peak (○) and steady-state, 20 ms prior to the offset of current command steps (•). The subtraction of these measurements yielded the curves shown by ▵

Figure 6. Burst firing is promoted in regular firing neurons by dendritic depolarisation

A, all traces during simultaneous dendritic and somatic recording, as symbolised in the inset. The threshold discharge response to a somatic current pulse (I_inj) is a single action potential (left), while a burst of action potentials is evoked by threshold dendritic current injection 250 μm from the soma (right). B, somatic current injection results in a train of action potentials typical of a regular firing neuron (left), whereas dendritic current injection 200 μm from the soma evokes a single action potential at threshold; more intense dendritic depolarisation, however, leads to the generation of bursts of two action potentials later in the train (right). Note the progressive increase in the duration of dendritic action potentials during the dendritic current injection (right) is absent during somatic current injection (left). The values of injected current are shown at the left of voltage records.

Figure 7. Burst firing is strengthened by dendritic depolarisation

A, all traces during simultaneous dendritic and somatic recording, as represented in the inset. In a strong burst firing neuron, threshold somatic (left) or dendritic (right) current injection leads to a burst discharge. A larger somatic current step evoked a series of burst discharges that become weaker later in the pulse (left), whereas dendritic current injection 300 μm from the soma generates strong burst discharges throughout the duration of the current injection (right). B, in a weak bursting neuron, the transformation of the firing pattern from burst to single action potentials in response to somatic current injection (left) is replaced by the appearance of repeated burst discharges in response to dendritic current injection made 230 μm from the soma (right). C, a burst during dendritic current injection made 480 μm from the soma is shown on an expanded time base, note the presence of a large depolarising envelope in the dendritic recording and that each action potential in the burst is observed to occur first at the somatic recording site.

Figure 9. Each action potential in a burst can lead to transmitter release

Examples of single sweeps of unitary EPSPs recorded from synaptically coupled layer 5 pyramidal neurons during burst firing. Presynaptic action potentials are shown at the top (11 superposed trials), followed by the evoked postsynaptic potentials. The mean of 100 trails is shown at the bottom together with the mean synaptic current (150 trials). Note that both the mean EPSPs and EPSCs show step-wise use-dependent synaptic depression.

Figure 10. The postsynaptic consequence of varying the membrane potential and the number of action potentials in a burst

Overlayed mean (n = 50 trials) traces of EPSPs recorded at the indicated holding potentials, in response to one, two and three action potentials. Arrows indicate the peak of the first and second EPSP at the most depolarised holding potential. The number of action potentials evoked in a burst was controlled by applying positive- followed by negative-current sequences to the presynaptic neuron.

Figure 11. Postsynaptic amplification of burst discharges

A, superposed consecutive traces (n = 10) demonstrating the amplification of the second EPSP in a burst when evoked at near threshold membrane potentials (-55 mV), and its depression at more negative holding potentials (-75 mV). The lower action potential waveform is the mean of 20 trials. B, scatter-plot demonstrating the voltage dependence of the amplification of the second EPSP in a burst. Each point represents mean (n = 30-50) EPSP amplitude at a single holding potential, where EPSP₁ is the amplitude of the first EPSP in the burst and EPSP₂ is the peak amplitude of the response to a burst of two action potentials. Data pooled from 12 paired recordings. C, amplification of later EPSPs in a burst is tetrodotoxin sensitive. Simulated EPSPs produced by somatic current injection of the mean synaptic current generated during a burst (derived from the cell illustrated in A). The voltage dependence of simulated EPSP amplification is shown in the upper superposed traces at the indicated holding potentials. The addition of TTX (1 μm) abolished amplification (lower superposed traces). D, scatter plot demonstrating the pooled voltage dependence of amplification of the second simulated EPSP in a burst in control (n = 7) and in TTX (n = 5).