Material below is adapted from the SfN Short Course Local Circuit Analysis With Integrated Large-Scale Recording of Neuronal Activity and Optogenics, by György Buzsáki, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

Understanding all the parts of a neuronal circuit will ultimately help explain how the circuit — and the brain — gives rise to behavior. To investigate how a circuit functions, different types of neurons spread across the brain need to be monitored simultaneously, and the contribution of each neuron determined. This requires hardware capable of recording many individual neurons, methods to separate spikes, and a way to identify different neuronal types.

When placed close enough to an individual neuron, electrodes can specifically record from that particular neuron. However, placing sharp electrodes so close to the cell’s body presents problems: the presence of the electrode can disrupt the cell’s firing, long-term recording is not practical as slight movements can disturb the electrode, and recording from multiple neurons (each with its own electrode) creates too much tissue damage.

On the other hand, monitoring neurons from farther away has problems as well. With only one electrode, different neurons at the same distance to the electrode may give identical signals, making it difficult to tell them apart. Using multiple recording sites (often four wires bunched together into a “tetrode”) can help separate neurons by triangulating the distances. But again, multiple electrodes create more damage.

An alternative to sharp electrodes is a micro-electro-mechanical system with silicon probes. Silicon probes are smaller than tetrodes, so they create less damage. Each probe has multiple recording sites in a known arrangement, allowing for the determination of various neurons’ locations and for simultaneous recording across different cortical layers. Silicon probes can also measure extracellular current, which (along with action potentials) can help calculate both inputs and outputs of a group of neurons.

Spikes can generally be sorted in two ways: either by their amplitude and waveform or by their location. But neither method is ideal. When based on amplitude and waveforms, one strongly activated neuron may appear to be multiple neurons. Alternatively, using triangulation to separate signals based on location assumes all signals from a neuron come from a single point, but signals may come from many places along the membrane. Again, because of this, a single neuron may appear as multiple neurons in different locations. Recording from multiple sites might help reduce these problems, but better, automatic methods and algorithms are needed to reduce human error.

Finally, coupling silicon probe recordings with optogenetics allows a way to classify the neurons. When analyzing a circuit, it’s important to know what types of neurons contribute and how. By integrating recording with optogenetics, neurons can be identified genetically, characterized physiologically, and tested for their roles within the circuit. Hybrid devices with both silicon probes and optical fibers reduce the light power required, minimizing secondary effects of stimulation and shrinking the size of the equipment needed, allowing for more freedom of movement in experimental animals.

Currently, optogenetics can only identify one or two neuron types in a single experiment. In the future, better understanding of the physiology of different cell types may lead to better identification of neurons without the need for optogenetics.  In the meantime, the combination of extracellular recording and optogenetics enables better understanding of the roles of individual neurons within local circuits, and ultimately how perception, memory, and behavior are created.

Material below summarizes the article Optogentetic Activation of Non-nociceptive Aβ Fibers Induces Neuropathic Pain-Like Sensory and Emotional Behaviors After Nerve Injury in Rats, published on February 5, 2018, in eNeuro and authored by Ryoichi Tashima, Keisuke Koga, Misuzu Sekine, Kensho Kanehisa, Yuta Kohro, Keiko Tominaga, Katsuyuki Matshushita, Hidetoshi Tozaki-Saitoh, Yugo Fukazawa, Kazuhid Inoue, Hiromu Yawo, Hidemasa Furue, and Makoto Tsuda.

Somatosensory information from the periphery is conveyed to the spinal dorsal horn (SDH) via primary afferent sensory neurons. The incoming sensory information is processed by complex circuits in the SDH, integrated to projection neurons relaying to several regions of the brain.

Primary afferents are broadly divided into two classes: nociceptive (mainly unmyelinated C, and thinly myelinated alpha delta (Aδ) fibers), and non-nociceptive (myelinated alpha beta (Aβ) fibers), which respond to noxious and innocuous stimuli, respectively.

Excitatory signals via nociceptive, which produces pain, and non-nociceptive fibers, which under normal healthy conditions do not cause pain, activate distinct SDH neuronal circuits. However, after injury to the nervous system from cancer, diabetes, chemotherapy, or trauma, even innocuous mechanical stimuli, such as the light touch of clothing, may cause pain.

This abnormal pain is known as mechanical allodynia and is a serious symptom of neuropathic pain, which is a debilitating chronic pain condition following nerve damage.

Neuropathic allodynia is refractory to the currently available treatments, including opioids. Thus, the clarification of mechanisms for neuropathic allodynia and the development of new therapeutic drugs are major challenges.

The mechanisms underlying mechanical allodynia have been investigated in rodent models of neuropathic pain. Previous studies have proposed the role of non-nociceptive Aβ fibers. However, it remains obscure.

Furthermore, it is unknown whether stimulation of non-nociceptive Aβ fibers produces allodynia-like behaviors in models of neuropathic pain. This might be due to the lack of research tools able to selectively manipulate Aβ fibers.

For elucidating the mechanistic basis of neuropathic mechanical allodynia, this study established a new approach that enables selective stimulation of non-nociceptive Aβ fibers in awake and freely moving animals using optogenetics.

We used a transgenic rat line, W-TChR2V4, within which the dorsal root ganglia (DRG)( the blue light-sensitive cation channels channelrhodopsin-2 (ChR2)) are selectively expressed in innocuous mechanoreceptive A fibers, including Aβ fibers.

We applied blue laser light illumination to the plantar skin of the hind paw of W-TChR2V4 rats after peripheral nerve injury (PNI) and found that the light illumination to the PNI side of the hind paw produced pain-like behaviors of lifting and flinching.

In contrast, the illumination to the non-PNI side produced no reaction or only mild movement without any lifting or flinching. Furthermore, these light-induced neuropathic pain behaviors were resistant to morphine.

These results indicate the light illumination to the plantar surface of the hind paw of rats with PNI elicited morphine-resistant pain-like behaviors.

We further investigated input of optogenetically evoked signals to the SDH. In immunohistochemistry, using neuronal activity markers (c-Fos and phosphorylated ERK), we observed the number of superficial SDH neurons positive to these markers increased following light illumination to the hind paw of W-TChR2V4 rats after PNI.

Furthermore, we performed whole-cell patch-clamp recordings using spinal cord slices of W-TChR2V4 rats and investigated synaptic activity of lamina I SDH neurons (presumably nociceptive).

The optogenetic stimulation of Aβ fibers resulted in excitation of lamina I SDH neurons of PNI rats, but not naive rats. These results suggest that optically stimulated Aβ fiber signals after PNI could be conveyed to the SDH and excite lamina I SDH neurons that normally respond to noxious stimuli.

Pain has sensory and emotional aspects. Using a place-aversion apparatus, this study also demonstrates light-induced Aβ fiber stimulation after PNI induces aversive behavioral symptoms.

Furthermore, Aβ fiber stimulation activated central amygdaloid neurons, considered the core brain region that processes aversive information of the pain experience.

This study established a novel approach to investigate neuronal circuits and behaviors responsible for Aβ fiber-mediated neuropathic allodynia. An advantage of this method is the assessment of Aβ fiber-derived neuropathic pain behaviors in awake, freely moving animals without direct contact of the light probe to the skin.

This is in stark contrast to tests using von Frey filaments, brushes, and electrical stimuli. An additional advantage over previous methods is this method can be performed without extensive training. This approach is easier to perform, and we expect it will improve accuracy, and reproducibility between investigators.

Light illumination could also enable stimulation of the ChR2⁺ Aβ fibers in in vitro and in vivo experimental conditions. A technical limitation of previous methods using filaments and brushes is the inability of mechanical stimuli to activate afferent subpopulations in in vitro studies, such as electrophysiological experiments using spinal slices with dorsal roots.

This model may enable future in vivo and in vitro studies to elucidate the mechanistic underpinnings for Aβ fiber-evoked neuropathic pain.

This study demonstrates for the first time that optogenetic stimulation of non-nociceptive Aβ fibers after PNI produces neuropathic pain-like behaviors with sensory and aversive emotional aspects.

Morphine resistance may correlate with the clinical situation in which neuropathic pain patients are often refractory to treatment. This method could provide a new approach for investigating the mechanisms underlying neuropathic mechanical allodynia with sensory and emotional features, and for developing new drugs to treat neuropathic pain.

Visit eNeuro to read the original article and explore other content. Read other summaries of JNeurosci and eNeuro papers in the Neuronline collection SfN Journals: Research Article Summaries.

Optogenetic Activation of Non-Nociceptive Aβ Fibers Induces Neuropathic Pain-Like Sensory and Emotional Behaviors after Nerve Injury in Rats. Ryoichi Tashima, Keisuke Koga, Misuzu Sekine, Kensho Kanehisa, Yuta Kohro, Keiko Tominaga, Katsuyuki Matsushita, Hidetoshi Tozaki-Saitoh, Yugo Fukazawa, Kazuhide Inoue, Hiromu Yawo, Hidemasa Furue and Makoto Tsuda. eNeuro Feb 2018 5 (1). DOI: 10.1523/ENEURO.0450-17.2018

About the Contributor

Ryoichi Tashima

Ryoichi Tashima is a PhD candidate in the Graduate School of Pharmaceutical Sciences at Kyushu University and studies molecular and cellular mechanisms in the peripheral and central nervous system underlying chronic pain.

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