Methodology for quantifying excitability of identified projection neurons in the dorsal horn of the spinal cord, specifically to study spinal cord stimulation paradigms

https://doi.org/10.1016/j.jneumeth.2019.108479Get rights and content

Highlights

  • In vivo electrophysiological recordings struggle with stringent identification of neuronal subtypes.

  • Recording from the same neuron over time is challenging due to the dense neuroanatomy of the spinal cord.

  • We present refinements of established techniques to identify dorsal horn projection neurons to record for over 3 h.

  • A Matlab algorithm generates a template from precise stimulation to allow accurate and fast quantification of excitability.

  • These refinements can be used to explore the mechanism of action of neuromodulation, such as spinal cord stimulation.

Abstract

Background

Using in and ex vivo preparations, electrophysiological methods help understand the excitability of biological tissue, particularly neurons, by providing microsecond temporal resolution. However, for in vivo recordings, in the context of extracellular recordings, it is often unclear precisely which type of neuron the tip of the electrode is recording from. This is particularly true in the densely-populated central nervous system, such as the spinal cord dorsal horn at both superficial and deep levels.

New Method

Here, we present a detailed protocol for the identification of superficial dorsal horn spinal cord neurons that receive peripheral input and project to the brain, using multiple surgical laminectomies and the careful placement of electrodes. Once a superficial projection unit was found, quantification to electrical peripheral stimulation was performed using a Matlab algorithm to form a template of projection neuron response to controlled C2 stimulation and accurately match this to the responses from peripheral stimulation.

Results

These superficial spinal projection neurons are normally activated by noxious peripheral stimuli, so we adopted a well-characterised wind-up protocol to obtain a neuronal excitability profile. Once achieved, this protocol allows for testing specific interventions, either pharmacological or neuromodulatory (e.g., spinal cord stimulation) to see how these affect the neuron’s excitability. This preparation is robust and allows the accurate tracking of a projection neuron for over 3-h.

Comparison with existing method(s)

Currently, most existing methods record from dorsal horn neurons that are often profiled based on their excitability to different peripherally-applied sensory modalities. While this is well-established, it fails to discriminate between interneurons and projection neurons, which is important as these two populations signal via distinctly different neuronal networks. Using the approach detailed here will result in studies with improved mechanistic understanding of the signal integration and processing that occurs in the superficial dorsal horn.

Conclusions

The refinements detailed in this protocol allow for more comprehensive studies to be carried out that will help understand spinal plasticity, in addition to many considerations for isolating the relevant neuronal population when performing in vivo electrophysiology.

Introduction

The human somatosensory nervous system is responsible for minimising tissue damage to external stimuli by conveying the sensation of pain from the periphery to highly complex neuronal networks in the central nervous system (CNS) (Basbaum et al., 2009; Peirs et al., 2015). However, a myriad of factors such as injury, genetic abnormalities, or neuronal plasticity can result in this warning system maladapting. This regularly results in chronic pain, which is common across society and cost an estimated $635 million in 2010 (Gaskin and Richardy, 2012), but remains poorly treated due to incomplete efficacy and dose-limiting effects of pharmacological compounds (Gilron et al., 2013). Understanding the nervous system’s activation, integration and response to peripheral stimuli will facilitate the development of analgesics to provide specific and robust therapeutic options. While substantial progress has been made, the mechanistic role of specific subtypes of neurons has not been thoroughly characterized, particularly using in vivo experiments. The mammalian nervous system shows remarkable heterogeneity amongst neurons, which include interneurons (INs) and projection neurons (PNs), and understanding the firing properties of these different types will improve our understanding and hopefully assist the development of better treatment therapies.

Within the spinal cord is the superficial dorsal horn, which consists of neuroanatomically distinct lamina in the cat (Rexed, 1952), the rat (Molander and Grant, 1986) and humans (Schoenen, 1982). The dorsal horn is neuronally dense with an incredible heterogeneity of neurons, resulting in 15 different inhibitory INs and 15 different excitatory INs, in addition to PNs (Häring et al., 2018). In particular, Lamina I, the most superficial, is comprised of 95% INs and 5% PNs (Spike et al., 2003), which results in ∼400 PNs at the L4 segment (Polgár et al., 2010). Amongst PN types, the neurokin-1 receptor positive PNs are critical in pain processing, as knock-out of neurons expressing this receptor using a Saporin toxin conjugate prevents the development of chronic inflammatory pain (Mantyh et al., 1997; Nichols et al., 1999). However, developing a pharmacological treatment to specifically target these neurons has been a significant challenge and has not yet been accomplished (Hill, 2000). However, whether chronic pain treatments, either pharmacological or neuromodulatory, are acting on these superficial dorsal horn PNs has not been robustly characterized and requires the ability to accurately record from these PNs.

Over past decades, to examine neuronal excitability, electrophysiology has been commonly used. There are many different variations, but extracellular recording protocols were first used to record from single neurons (Hubel, 1957) and this was followed by single-unit recordings in the superficial dorsal horn (Wall, 1965). More recently, there have been dramatic improvements in the temporal and spatial resolution of extracellular electrophysiology (Buzsáki, 2004; Harris et al., 2016).

While there are excellent protocols for performing in vivo electrophysiology of dorsal horn neurons, these do not differentiate between either PNs or the many different types of INs in both the rat (Svendsen et al., 1999; Urch and Dickenson, 2003) and mouse (Cuellar et al., 2004). There have been many informative in vivo electrophysiological studies to examine the effect of therapies such as neuromodulation on the excitability of dorsal horn neurons (Yakhnitsa et al., 1999; Shechter et al., 2013), but, again, these have not differentiated between INs and PNs. There are published methodologies for a rat preparation to identify superficial PNs using antidromic stimulation (McMahon and Wall, 1983), though they haven’t been widely adopted. This technique is reliable for PNs as INs are unable to follow 100 Hz reliably (Lipski, 1981; McMahon and Wall, 1983). However, by combining the nuances of these techniques, experiments can be far more informative and neurons can be identified by their functional neuroanatomy.

In addition, technological improvements offer incredible opportunity for more robust methodologies. For extracellular electrophysiology, spike sorting (Medrano et al., 2016) has improved significantly using software algorithms (Barnett et al., 2016) and this helps to track multiple units from a single recording position over time, especially in response to various stimuli. Therefore, this study introduces Matlab code to robustly quantify neuronal activity from neurons that receive input from the periphery and project from the spinal cord to the brain. Additionally, this preparation allows for the examination of different types of therapeutic intervention; in this case, a specific type of neuromodulation for pain: spinal cord stimulation (SCS) (Verrills et al., 2016). However, the principles of this extracellular recording and identification technique can be used both with different equipment and for neurons in other neuroanatomical structures.

Neuromodulation offers many advantages over traditional pharmacological interventions, since neuromodulatory therapy can be far more specific through careful placement of the electrodes and a vast range of stimulation protocols to differentially affect neuronal activity (Verrills et al., 2016; Shamji et al., 2017). In this methodology, SCS was focussed on, as this is a widely-used therapy to treat pain, with two different protocols depending on the frequency: low-frequency and high-frequency (Chakravarthy et al., 2017). Currently, it is not clear whether these treatments act to affect the excitability of superficial dorsal horn neurons. Improvements in the understanding of this should help to offer dramatically improved therapies for individuals that suffer from pain.

Section snippets

Materials and methods

The methodology presented here offers refinements to well-established techniques to help improve the quality of the information obtained from in vivo extracellular dorsal horn electrophysiology. The workflow is summarised below (Fig. 1A) and each step has a subsection offering details. Additionally, while this methodology uses specific equipment, most of it is interchangeable for similarly functioning hardware.

Preparation

The methodology presented here aims to improve current in vivo electrophysiological extracellular recordings from single neurons in the dorsal horn of the spinal cord. An advantage of this preparation is the stability, with a PN being tracked for 3-h and continuing to wind-up over 16 peripheral stimuli (Fig. 4). Most importantly, the C2 antidromic stimulation allows for verification of the same unit being recorded from throughout the entire preparation: 0-min (Fig. 4A), 45-min (Fig. 4E), 90-min

Discussion

In order to offer better treatment for chronic diseases, especially illnesses involving maladaptive plasticity of the nervous system, such as pain, there needs to be better understanding of the precise mechanisms involved in the development of these illnesses. Therefore, techniques are required to have with greater resolution with accurate tracking of the neuronal signals of interest, so that more power is provided from the results of each study. This methodology offers the following

Conclusion

Using repetitive antidromic stimulation at the C2 spinal vertebrae level of the spinal cord, superficial dorsal horn PNs were activated and stably recorded for up to 3-h, which allowed the formation of templates of the unit’s response. With these templates, accurate quantification of peripherally-evoked activity from hind limb nerve stimulation was easily and robustly performed. We found our template-matching technique was a rapid, reliable, semi-automated technique for consistently identifying

Acknowledgements

This work was funded by a contract between King’s College London and Nevro. The authors are grateful to the staff in the Biomedical Services Unit. In addition, the expertise, both administrative and technical, of John Grist, Caroline Abel, Vivien Cheah, Gary Fulcher and Claire Pearce has been hugely appreciated throughout this project. For creating figures, the help and advice of Dr. Christopher Chapman has been instrumental. Finally, we’d like to thank Simon Townsend and Duncan Farquharson

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