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Pinch-current injection defines two discharge profiles in mouse superficial dorsal horn neurones, in vitro - PubMed

  • ️Mon Jan 01 2007

Pinch-current injection defines two discharge profiles in mouse superficial dorsal horn neurones, in vitro

B A Graham et al. J Physiol. 2007.

Abstract

Neurones in the superficial dorsal horn (SDH) are a major target for nociceptive afferents and play an important role in pain processing. One approach to understanding the role of SDH neurones has been to study their action potential (AP) discharge in spinal cord slices during injection of depolarizing step-currents. Four or five neurone subpopulations are typically identified based on AP discharge, with various roles proposed for each in pain processing. During noxious peripheral stimulation in vivo, however, SDH neurones are activated via synaptic inputs. This produces a conductance change with different somato-dendritic distributions and temporal characteristics to that provided by a somatic step-current injection. Here we introduce an alternative approach to studying SDH neurone discharge under in vitro conditions. We recorded voltage-clamp responses in SDH neurones, in vivo, during noxious mechanical stimulation of the hindpaw (1 s pinch, approximately 100 g mm(-2)). From these recordings a representative 'pinch-current' was selected and subsequently injected into SDH neurones in spinal cord slices (recording temperature 32 degrees C). Pinch-current-evoked discharge was compared to that evoked by rectangular step-current injections. Pinch- and step-current-evoked AP discharge frequency was highly correlated (r2 = 0.61). This was also true for rheobase current comparisons (r2 = 0.61). Conversely, latency to discharge and discharge duration were not correlated when step- and pinch-current responses were compared. When neurones were grouped according to step-current-evoked discharge, five distinct patterns were apparent (tonic firing, initial bursting, delayed firing, single spiking, and reluctant firing). In contrast, pinch-current responses separated into two clear patterns of activity (robust and resistant firing). During pinch-current injection, tonic-firing and initial-bursting neurones exhibited robust AP discharge with similar characteristics. In contrast, single-spiking and reluctant-firing neurones were resistant to AP discharge. Delayed-firing neurones exhibited pinch-current responses that were transitional between those of tonic-firing/initial-bursting and single-spiking/reluctant-firing neurones. Injection of digitally filtered pinch-currents indicated that transient current fluctuations are necessary for robust repetitive discharge in initial-bursting neurones. These data suggest the functional significance of the diverse step-current-evoked firing patterns, previously reported in SDH neurones remains to be fully understood. When a 'facsimile' current profile or pinch-current is used in place of step-currents, AP discharge diversity is much reduced.

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Figures

Figure 1
Figure 1

In vivo current- and voltage-clamp recordings from SDH neurones during noxious mechanical stimulation of the hindpaw A, recordings from three neurones during the application of a 1 s pinch to the hindpaw. Current-clamp recordings (upper black traces) show that responses range from robust AP discharge throughout the pinch stimulus (left), to a single AP at pinch onset (right). Arrowheads denote −60 mV. Voltage-clamp recordings (lower grey traces) show that peak amplitude, area under the curve (total charge), and decay of the inward current evoked by pinch is qualitatively and quantitatively similar in all neurones (also see Table 1). B, Expanded view of selected pinch-current (asterisk in A), after conversion to a depolarizing current-stimulus for injection into SDH neurones under in vitro conditions. This current was scaled to provide a series of five pinch-currents (20%, 40%, 60%, 80% and 100% of the original amplitude). C, the mean current delivered during each pinch-current appears in the left bar plot. Dashed lines extending from the depolarizing step-currents indicate the mean current delivered by each step. The five pinch-currents are best matched by the first five step-currents (P1, 20 pA; P2, 40 pA; P3, 60 pA; P4, 80 pA; and P5, 100 pA). D, the total charge delivered during each pinch-current appears in the left bar plot. Dashed lines extending from the depolarizing step-currents indicate the total charge delivered by each step. The five pinch-currents are best matched by the first three, fifth, and sixth step-currents (P1, 20 pA; P2, 40 pA; P3, 60 pA; P4, 100 pA; and P5, 120 pA).

Figure 2
Figure 2

SDH neurones show variable responses to pinch- and step-currents in vitro A and B, recordings from two SDH neurones showing responses during pinch- (upper traces) and step-currents (lower traces) of increasing magnitude (arrowheads denote −60 mV). The neurone in A responded during pinch- and step-currents with robust AP discharge that increased in frequency and duration as pinch- and step-current magnitude increased (lower traces). In contrast, the neurone in B did not discharge APs during pinch-currents, and only discharged a single AP at the highest step-current intensity. C, plots comparing AP discharge characteristics elicited by step- versus pinch-currents. The first plot compares the minimum step- and pinch-currents required to elicit AP discharge (rheobase-steps versus rheobase-pinches). This relationship was highly correlated. The remaining plots compare data from pinch 5 (P5) with the 120 pA step-current. Mean AP discharge frequency, like rheobase, was significantly correlated. The latency between stimulus onset and AP discharge and the discharge duration were not significantly correlated. Note there is extensive overlap of data points in the plot of AP latency.

Figure 3
Figure 3

Pinch-current-evoked discharge in SDH neurones expressing different step-current-evoked discharge patterns A–E, pinch-current data for recorded SDH neurones were grouped according to the discharge pattern exhibited during step-current injections (left, overlayed voltage traces, arrowheads denote −60 mV). Averaged peristimulus histograms (100 ms bins) of AP discharge were constructed for each pinch-current (pinch 1 to pinch 5, left to right). A, tonic-firing neurones typically exhibited responses to most pinch-currents with increasing discharge frequency and duration as pinch-current intensity increased. B, initial-bursting neurones also exhibited responses to most pinch-currents with increasing discharge frequency and duration as pinch-current intensity increased. Moreover, these responses had similar characteristics to those of tonic-firing neurones. C, delayed-firing neurones did not respond to low-intensity pinch-currents, but exhibited responses to high-intensity pinch-currents with discharge characteristics similar to tonic-firing and initial-bursting responses during low-intensity pinch-currents. D, single-spiking neurones only responded to the highest-intensity pinch-currents, and these responses were restricted to a brief discharge of one or two APs. E, reluctant-firing neurone responses during pinch-currents were similar to those of single-spiking neurones, showing a brief discharge of one or two APs during the high-intensity pinch-currents.

Figure 4
Figure 4

Reducing pinch-current complexity Three alternative pinch-current stimuli were generated to determine how the overall level of depolarization versus the transient fluctuating nature of the pinch-current baseline influenced AP discharge. The original pinch-current (top left) is essentially a triangular depolarization with a fluctuating baseline (inset shows segment of each pinch-current on an expanded time scale; indicated by black bar). The original pinch-current was filtered at two levels to smooth its baseline, producing two pinch-currents with progressively reduced baseline fluctuations (smoothed-pinch 1, bottom left; and smoothed-pinch 2, top right). A ramp-pinch current was also generated that followed the underlying ‘triangular’ depolarization of the original pinch-current (bottom right).

Figure 5
Figure 5

Initial-bursting, but not tonic-firing, neurones require pinch-current complexity (transient fluctuations) for sustained AP discharge A, representative voltage recordings from tonic-firing (upper traces) and initial-bursting (middle traces) neurones during injection of pinch-currents of decreasing complexity (lower traces, left to right). Arrowheads denote −60 mV. Tonic-firing neurone responses are unchanged as transient fluctuations are progressively removed from pinch-currents. In contrast, initial-bursting neurone responses are decreased as transient fluctuations are removed. B, group data comparing the number of APs (left) and the discharge duration (right) in tonic-firing and initial-bursting neurones during pinch-current injections of decreasing complexity. For tonic-firing neurones, AP number and discharge duration were similar for all pinch-currents. Alternatively, AP number and discharge duration were significantly reduced during pinch-current injections of decreasing variance (SP2 and RP) in initial-bursting neurones. *Significant difference from original (OP) pinch-current response.

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