Action potentials recorded with patch-clamp amplifiers: are they genuine?

Magistretri, Jacopo; Mantegazza, Massimo; Guatteo, Ezia; Wanke, Enzo

doi:10.1016/s0166-2236(96)40004-2

Cited by 121 publications

(99 citation statements)

References 29 publications

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“…Standard patch amplifiers are limited by voltage distortion in current-clamp mode, and may not obtain accurate recordings of excitability parameters [59]. However, using intracellular microelectrode recordings from intact, excised DRGs, we have previously noted that axotomy substantially increases AP duration and decreases the amplitude, duration and area of AHP in Aδ neurons, compared with other groups [80].…”

Section: Discussionmentioning

confidence: 99%

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Sarantopoulos¹,

McCallum²,

Rigaud³

et al. 2007

Brain Research

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Calcium-activated potassium channels regulate AHP and excitability in neurons. Since we have previously shown that axotomy decreases I Ca in DRG neurons, we investigated the association between I Ca and K (Ca) currents in control medium-sized (30-39 μM) neurons, as well as axotomized L5 or adjacent L4 DRG neurons from hyperalgesic rats following L5 SNL. Currents in response to AP waveform voltage commands were recorded first in Tyrode's solution and sequentially after: 1) blocking Na + current with NMDG and TTX; 2) addition of K (Ca) blockers with a combination of apamin 1μM, iberiotoxin 200 nM, and clotrimazole 500 nM; 3) blocking remaining K + current with the addition of 4-AP, TEA-Cl, and glibenclamide; and 4) blocking I Ca with cadmium. In separate experiments, currents were evoked (HP −60 mV, 200 ms square command pulses from −100 to +50 mV) while ensuring high levels of activation of I K(Ca) by clamping cytosolic Ca 2+ concentration with pipette solution in which Ca 2+ was buffered to1μM. This revealed I K(Ca) with components sensitive to apamin, clotrimazole and iberiotoxin. SNL decreases total I K(Ca) in axotomized (L5) neurons, but increases total I K(Ca) in adjacent (L4) DRG neurons. All I K(Ca) subtypes are decreased by axotomy, but iberiotoxin-sensitive and clotrimazole-sensitive current densities are increased in adjacent L4 neurons after SNL. In an additional set of experiments we found that small sized control DRG neurons also expressed iberiotoxin-sensitive currents, which are reduced in both axotomized (L5) and adjacent (L4) neurons.Conclusions: Axotomy decreases I K(Ca) due to a direct effect on K (Ca) channels. Axotomy-induced loss of I Ca may further potentiate current reduction. This reduction in I K(Ca) may contribute to elevated excitability after axotomy. Adjacent neurons (L4 after SNL) exhibit increased I K(Ca) current.

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Section: Discussionmentioning

confidence: 99%

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Sarantopoulos¹,

McCallum²,

Rigaud³

et al. 2007

Brain Research

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“…Action potentials recorded with patch-clamp amplifiers can be distorted significantly, because the current-clamp mode of such amplifiers requires a feedback circuit for which the bandwidth is limited (Magistretti et al, 1996(Magistretti et al, , 1998. The extent of the distortion can be estimated from the deviation from zero in the actual current flowing in the headstage, which can be large (several nanoamps) when feedback bandwidth is limited.…”

Section: Action Potential Clamp In Small Drg Neuronsmentioning

confidence: 99%

“…Because of the imperfection in current clamp, the upstroke of the action potential is somewhat less steep than it would be if measured perfectly (Magistretti et al, 1996), thus reducing the peak ionic current calculated from ϪC m dV/dt. In addition, when this action potential is used as the command in voltage clamp, the slower rate of rise would allow more complete activation of the sodium current.…”

Section: Action Potential Clamp In Small Drg Neuronsmentioning

confidence: 99%

Roles of Tetrodotoxin (TTX)-Sensitive Na⁺Current, TTX-Resistant Na⁺Current, and Ca²⁺Current in the Action Potentials of Nociceptive Sensory Neurons

2002

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Nociceptive sensory neurons are unusual in expressing voltage-gated inward currents carried by sodium channels resistant to block by tetrodotoxin (TTX) as well as currents carried by conventional TTX-sensitive sodium channels and voltagedependent calcium channels. To examine how currents carried by each of these helps to shape the action potential in smalldiameter dorsal root ganglion cell bodies, we voltage clamped cells by using the action potential recorded from each cell as the command voltage. Using intracellular solutions of physiological ionic composition, we isolated individual components of current flowing during the action potential with the use of channel blockers (TTX for TTX-sensitive sodium currents and a mixture of calcium channel blockers for calcium currents) and ionic substitution (TTX-resistant current measured by the replacement of extracellular sodium by N-methyl-D-glucamine in the presence of TTX, with correction for altered driving force). TTX-resistant sodium channels activated quickly enough to carry the largest inward charge during the upstroke of the nociceptor action potential (ϳ58%), with TTX-sensitive sodium channels also contributing significantly (ϳ40%), especially near threshold, and high voltage-activated calcium currents much less (ϳ2%). Action potentials had a prominent shoulder during the falling phase, characteristic of nociceptive neurons. TTXresistant sodium channels did not inactivate completely during the action potential and carried the majority (58%) of inward current flowing during the shoulder, with high voltage-activated calcium current also contributing significantly (39%). Unlike calcium current, TTX-resistant sodium current is not accompanied by opposing calcium-activated potassium current and may provide an effective mechanism by which the duration of action potentials (and consequently calcium entry) can be regulated.

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“…All membrane potential values were corrected for a 10-mV calculated liquid junction potential (Barry and Lynch 1991). We note that the different amplifiers used for our in vitro and in vivo experiments have been reported to filter and distort voltage signals differently (Magistretti et al 1996). The extent of this distortion was quantified using the same model cell (500 M⍀, 16.5-ms time constant, 10-M⍀ series resistance) on the Axopatch 200B and Axoclamp 2B amplifiers.…”

Section: In Vivo Electrophysiologymentioning

confidence: 99%

Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro

Graham

Brichta²,

Callister³

2008

Journal of Neurophysiology

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Graham BA, Brichta AM, Callister RJ. Recording temperature affects the excitability of mouse superficial dorsal horn neurons, in vitro. J Neurophysiol 99: 2048 -2059, 2008. First published February 20, 2008 doi:10.1152/jn.01176.2007. Superficial dorsal horn (SDH) neurons in laminae I-II of the spinal cord play an important role in processing noxious stimuli. These neurons represent a heterogeneous population and are divided into various categories according to their action potential (AP) discharge during depolarizing current injection. We recently developed an in vivo mouse preparation to examine functional aspects of nociceptive processing and AP discharge in SDH neurons and to extend investigation of pain mechanisms to the genetic level of analysis. Not surprisingly, some in vivo data obtained at body temperature (37°C) differed from those generated at room temperature (22°C) in spinal cord slices. In the current study we examine how temperature influences SDH neuron properties by making recordings at 22 and 32°C in transverse spinal cord slices prepared from L3-L5 segments of adult mice (C57Bl/6). Patch-clamp recordings (KCH 3 SO 4 internal) were made from visualized SDH neurons. At elevated temperature all SDH neurons had reduced input resistance and smaller, briefer APs. Resting membrane potential and AP afterhyperpolarization amplitude were temperature sensitive only in subsets of the SDH population. Notably, elevated temperature increased the prevalence of neurons that did not discharge APs during current injection. These reluctant firing neurons expressed a rapid A-type potassium current, which is enhanced at higher temperatures and thus restrains AP discharge. When compared with previously published whole cell recordings obtained in vivo (37°C) our results suggest that, on balance, in vitro data collected at elevated temperature more closely resemble data collected under in vivo conditions.

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Action potentials recorded with patch-clamp amplifiers: are they genuine?

Cited by 121 publications

References 29 publications

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Roles of Tetrodotoxin (TTX)-Sensitive Na⁺Current, TTX-Resistant Na⁺Current, and Ca²⁺Current in the Action Potentials of Nociceptive Sensory Neurons

Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro

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Action potentials recorded with patch-clamp amplifiers: are they genuine?

Cited by 121 publications

References 29 publications

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Roles of Tetrodotoxin (TTX)-Sensitive Na+Current, TTX-Resistant Na+Current, and Ca2+Current in the Action Potentials of Nociceptive Sensory Neurons

Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro

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Roles of Tetrodotoxin (TTX)-Sensitive Na⁺Current, TTX-Resistant Na⁺Current, and Ca²⁺Current in the Action Potentials of Nociceptive Sensory Neurons