Intraspinally mediated state-dependent enhancement of motoneurone excitability during fictive scratch in the adult decerebrate cat

Abstract

This is the first study to report on the increase in motoneurone excitability during fictive scratch in adult decerebrate cats. Intracellular recordings from antidromically identified motoneurones revealed a decrease in the voltage threshold for spike initiation (V(th)), a suppression of motoneurone afterhyperpolarization and activation of voltage-dependent excitation at the onset of scratch. These state-dependent changes recovered within 10-20 s after scratch and could be evoked after acute transection of the spinal cord at C1. Thus, there is a powerful intraspinal system that can quickly and reversibly re-configure neuronal excitability during spinal network activation. Fictive scratch was evoked in spinal intact and transected decerebrate preparations by stroking the pinnae following topical curare application to the dorsal cervical spinal cord and neuromuscular block. Hyperpolarization of V(th) occurred (mean 5.8 mV) in about 80% of ipsilateral flexor, extensor or bifunctional motoneurones during fictive scratch. The decrease in V(th) began before any scratch-evoked motoneurone activity as well as during the initial phase in which extensors are tonically hyperpolarized. The V(th) of contralateral extensors depolarized by a mean of +3.7 mV during the tonic contralateral extensor activity accompanying ipsilateral scratch. There was a consistent and substantial reduction of afterhyperpolarization amplitude without large increases in motoneurone conductance in both spinal intact and transected preparations. Depolarizing current injection increased, and hyperpolarization decreased the amplitude of rhythmic scratch drive potentials in acute spinal preparations indicating that the spinal scratch-generating network can activate voltage-dependent conductances in motoneurones. The enhanced excitability in spinal preparations associated with fictive scratch indicates the existence of previously unrecognized, intraspinal mechanisms increasing motoneurone excitability.

Figure 1. Method of V_th determination

Upper trace: intracellular recording from an extensor motoneurone in the SmAB motor pool in discontinuous current clamp mode (10 kHz switching) and digitized at 10 kHz. The voltage threshold of the action potential (V_th) evoked by a rectangular current pulse (lower trace) was defined as the data point at which the change in membrane potential of subsequent points exceeds 1 mV (indicated by the arrow). Middle trace: the derivative of the intracellular trace shows that the V_th data point clearly corresponds to the fast rising portion of the spike where the voltage trajectory surpasses 10 V s⁻¹. The oscillating noise on the traces is the result of switching artifacts from the discontinuous current clamp.

Figure 7. Afterhyperpolarization amplitude is reduced during fictive scratch

A illustrates extensor (PBSt) and flexor (TA) ENG recordings, the intracellular recording from a GS motoneurone and the current monitor trace. Records in A labelled B, D and * show on an expanded timescale, action potentials evoked by ramp-shaped current injection prior to (B) and following (D and *) ipsilateral fictive scratch. The AHP amplitude remained decreased immediately following fictive scratch (D) and was fully recovered within 10 s of scratch cessation (denoted by *), when compared to control (B). Full recovery of AHP amplitude is clearly illustrated by the overlay of the action potentials in the inset. C and E show an expanded timescale of action potentials evoked by the scratch CPG. AHP amplitude was partially reduced prior to (C) scratch and was almost completely absent during scratch (E), when compared to control (B).

Figure 2. V_th is hyperpolarized during fictive scratch

A and B show extensor (GS) and flexor (TA) ENG recordings, the intracellular recording from a GS motoneurone and the current monitor trace in a spinal cord intact preparation. Records in A taken about 7 s before those in B show action potentials evoked under control conditions by a ramp-shaped current injection. The ENG traces in B show the approach and rhythmic phases of fictive scratch evoked by rubbing the ipsilateral ear. A portion of the intracellular trace and TA ENG trace (indicated by the shaded boxes) are shown expanded in C. Spikes evoked by current injection just prior to the onset of tonic TA ENG activity are shown in D. The spikes occurring spontaneously on the first supra-threshold depolarizing SDP during scratch are shown in E at a more expanded time base than those in C and D. The V_th of this motoneurone hyperpolarized by −7.4 mV prior to the onset of ENG activity during scratch and remained hyperpolarized during the rhythmic activity associated with the bout of scratch. The duration of the first inter-spike intervals are shown below the firing in panels C, D and E.

Figure 3. V_th is hyperpolarized during the active and inactive portions of the fictive scratch cycle

A shows extensor (GS) ENG activity during ipsilateral fictive scratch (top trace) and the intracellular membrane potential recording and current injection (middle traces) for a GS motoneurone in a preparation with an intact spinal cord. The shaded area denotes the portion illustrated in C on an expanded timescale. The V_th for action potentials before fictive scratch was −39.4 mV and was elicited by a 19 nA rectangular current pulse. The current was increased to 28 nA to ensure spiking during the hyperpolarized phase of the SDP. C shows the thresholds of the current-evoked action potential and the action potential evoked by the SDP were similar and hyperpolarized by −2.1 mV compared to control (B). Note the spikes are truncated in panels B and C.

Figure 4. Similar patterns of fictive scratch activity before and after spinal transection

The 2 upper traces in A and B show episodes of fictive scratch in the same preparation before (A) and after (B) an acute complete transection of the spinal cord at the C1 level. ENG recordings from other nerves are shown at a faster time base below. The similarity of the pattern of ENG activities, duration of motoneurone pool discharges and cycle periods before and after spinalization are evident in the records in C.

Figure 5. V_th is hyperpolarized during fictive scratch after spinal transection

A shows the SmAB ENG and an intracellular recording from a MG motoneurone during the rhythmic portion of an episode of fictive scratch in an acutely spinalized cat. Intracellular and current monitor traces are shown in B and C on an expanded time base. The V_th of spikes occurring on the SDP was hyperpolarized by −4.7 mV during scratch (C) compared to those evoked by current injection during control conditions (B). The current monitor trace in B shows the small hyperpolarizing current pulse used to assess membrane conductance.

Figure 6. V_th is depolarized during fictive weight support and hyperpolarized during ipsilateral scratching

A shows continuous ENG records, first during ipsilateral rhythmic scratching evoked by rubbing the ipsilateral ear (8–15 s in the run), and then during contralateral scratch evoked by rubbing the other ear (48–58 s) in a decerebrate cat without spinal transection. During the approach phase of contralateral scratch (i.e. during tonic activity in the contralateral flexor, coTA) the ipsilateral extensor ipSmAB displays tonic ‘fictive weight support’ activity (48–52 s). The intracellular record from a SmAB motoneurone is shown below the ENGs, and representative values of the V_th measured throughout the run are shown below. Expanded records of the regions indicated by the vertical shaded areas are shown in B and C. Depolarizing current pulses were delivered throughout the run to evoke firing in addition to the spontaneous, scratch-induced firing. The control V_th of −45.6 mV (left-most value) is hyperpolarized by −1.8 mV (to −47.4 mV) during the rhythmic phase of ipsilateral scratch (see B) and recovers a few seconds after the end of ipsilateral scratch activity. During the fictive weight support that begins at about 48 s (see C), V_th depolarized by +6.4 mV from the control value immediately before contralateral scratch began (i.e. from −43.3 to −36.9 mV; see C for values from individual spikes).

Figure 8. Conductance changes during fictive scratch

Bars represent the average conductance determined for the depolarized (filled bars) and hyperpolarized (open bars) phases of the fictive scratch cycle in a sample of 10 motoneurones. Values are normalized to control (100% as indicated by the horizontal, dashed black line). The narrow grey bar separates the motoneurones in which conductance assessment was done before (n= 3) or after spinal transection (n= 7). All data are from one cat.

Figure 9. Voltage-dependent amplification of scratch drive potentials in an acutely spinalized preparation

A illustrates the membrane potential recording of a Tib motoneurone (upper trace) in which action potentials were blocked by inclusion of QX314 in the microelectrode. Prior to the onset of fictive scratch, two 10 nA ramps of depolarizing current (middle trace) were delivered which caused ∼15 mV depolarizations of the motoneurone membrane potential. After the initiation of fictive scratch indicated by the alternating activity in the MG and EDL ENGs (lower traces) as well as SDPs in the motoneuron, the delivery of the current ramps was repeated. The dashed lines on the first current injection delivered during fictive scratch approximate the peak-to-peak amplitude of the SDPs on the ascending portion of the current ramp. Note that divergence of the slopes of the dashed lines indicates that SDPs occurring at more depolarized membrane potentials were larger than those occuring at more hyperpolarized membrane potentials. On the second current ramp during fictive scratch, the 2 SDPs occurring during no current injection (indicated with the grey box and ‘B’) are compared to the 2 SDPs occurring during the maximum depolarization induced by the current injection (also indicated with a grey box and ‘B’). These SDPs are shown superimposed on an expanded time scale in B, aligned at the most hyperpolarized portion of the SDP. The largest SDP (denoted by *) occurred at the most depolarized membrane potential.

Figure 10. Voltage-dependent amplification of scratch drive potentials can occur spontaneously during fictive scratch in an acutely spinalized preparation

The upper traces show the MG and TA ENGs prior to, and during a bout of ipsilateral scratch in an acutely spinalized preparation. The middle trace shows the membrane potential recording from a Tib motoneurone (same cell as in Fig. 9) in which the action potentials were blocked by including QX314 in the microelectrode. Note the hyperpolarization of the motoneurone occurring during the tonic activity in the TA ENG and which is typical for extensor motoneurones during the approach phase. With the onset of rhythmic scratch and alternating ENG activity, prominent SDPs are evident in the motoneuron. After approximately 5 rhythmic scratch cycles, −8 nA of hyperpolarizing current was delivered to the motoneurone (see current trace at bottom), which was terminated at approximately 4.2 s. At approximately 4.9 s, +8 nA of depolarizing current was delivered. The shaded bars on the membrane potential traces during the −8 nA, 0 nA and +8 nA current periods indicate the peak-to-peak amplitude of the SDP. The inset shows those amplitudes side by side on the same scale and indicate that depolarization of the motoneurone caused the largest SDPs, but also that hyperpolarization of the motoneurone could reduce the amplitude of the SDP compared to those occurring in the absence of current injection. This indicates that the scratch network was able to induce a degree of voltage-dependent amplification of the SDP in the absence of current injection.