A new minimally-invasive method for microinjection into the mouse spinal dorsal horn

Kohro, Yuta; Sakaguchi, Emi; Tashima, Ryoichi; Tozaki‐Saitoh, Hidetoshi; Okano, Hideyuki; Inoue, Kazuhide; Tsuda, Makoto

doi:10.1038/srep14306

Cited by 71 publications

(55 citation statements)

References 53 publications

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“…The viral vector microinjection was performed as in previous studies (Inquimbert et al 2013, Kohro et al 2015). Briefly, the mice were anesthetized with 2% isoflurane inhalation, and the lumbar spinal cord was exposed via hemilaminectomy.…”

Section: Methodsmentioning

confidence: 99%

Regulating nociceptive transmission by VGluT2‐expressing spinal dorsal horn neurons

Wang

Chen

et al. 2018

Journal of Neurochemistry

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Vesicular glutamate transporter-2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Adeno-associated viral vectors expressing double-floxed Cre-dependent Gαq-coupled human M3 muscarinic receptor DREADD (hM3D)-mCherry or Gαi-coupled κ-opioid receptor DREADD (KORD)-IRES-mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2-Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2-expressing neurons with clozapine N-oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2-expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2-expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2-expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2-expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory pain and neuropathic pain.

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Section: Methodsmentioning

confidence: 99%

Regulating nociceptive transmission by VGluT2‐expressing spinal dorsal horn neurons

Wang

Chen

et al. 2018

Journal of Neurochemistry

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“…We injected RV‐GFP into the spinal cord without laminectomy as described previously (Kohro et al, ). Mice were anesthetized with subcutaneous injection of ketamine/xylazine solution (125 mg/kg and 10 mg/kg, respectively) and the back skin was incised at Th12‐L3.…”

Section: Methodsmentioning

confidence: 99%

Rabies virus‐mediated oligodendrocyte labeling reveals a single oligodendrocyte myelinates axons from distinct brain regions

Osanai

Shimizu

Mori

et al. 2016

Glia

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Oligodendrocytes myelinate neuronal axons during development and increase conduction velocity of neuronal impulses in the central nervous system. Neuronal axons extend from multiple brain regions and pass through the white matter; however, whether oligodendrocytes ensheath a particular set of axons or do so randomly within the mammalian brain remains unclear. We developed a novel method to visualize individual oligodendrocytes and axon derived from a particular brain region in mouse white matter using a combinational injection of attenuated rabies virus and adeno-associated virus. Using this method, we found that some populations of oligodendrocytes in the corpus callosum predominantly ensheathed axons derived from motor cortex or sensory cortex, while others ensheathed axons from both brain regions, suggesting heterogeneity in preference of myelination toward a particular subtype of neurons. Moreover, our newly established method is a versatile tool for analyzing precise morphology of each oligodendrocyte in animal models for demyelinating disorders and addressing the role of oligodendrocyte in higher brain functions. GLIA 2016. GLIA 2017;65:93-105.

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“…Male ddY mice weighing 20–25 g and C57BL/6 J mice weighing 20 g (virus study) were used. The viral injections of the C57BL/6 J mice were conducted according to a previous protocol (Kohro et al, ). Five animals were housed in each cage in a room maintained at 23 ± 2 °C with an alternating 12‐h light–dark cycle.…”

Section: Methodsmentioning

confidence: 99%

“…Male ddY mice weighing 20-25 g and C57BL/6 J mice weighing 20 g (virus study) were used. The viral injections of the C57BL/6 J mice were conducted according to a previous protocol (Kohro et al, 2015).…”

Section: Animalsmentioning

confidence: 99%

“…Repeated pharmacological inhibition of astrocytes reportedly alleviates mechanical hyperalgesia in neuropathic pain (Raghavendra, Tanga, Rutkowski, & DeLeo, ). In a study of the specific role of spinal dorsal horn (SDH) astrocytes, Kohro et al () have shown that the long‐term specific activation of L4 SDH astrocytes is not only necessary for maintaining tactile allodynia but also sufficient for the induction of allodynia. Despite these findings, the role of astrocytes in the processing of spinal nociceptive transmission is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

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Astrocyte‐neuron lactate shuttle sensitizes nociceptive transmission in the spinal cord

Miyamoto

Ishikura

Kume

et al. 2018

Glia

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Astrocytes play a key role in the maintenance of synaptic transmission by producing L‐lactate via the astrocyte‐neuron lactate shuttle (ANLS). Astrocyte activation in the spinal cord is involved in the expression of neuropathic pain. We investigated the role of the ANLS in the spinal cord on hyperalgesia in neuropathic pain in mice. Specific activation of dorsal horn astrocytes induced mechanical hyperalgesia, which was attenuated by α‐cyano‐4‐hydroxycinnamate (4‐CIN), an inhibitor of monocarboxylate transporters that deliver L‐lactate from astrocytes to neurons. Intrathecal L‐lactate administration lowered the mechanical nociceptive threshold, which was attenuated by pretreatment with 4‐CIN and isosafrole (a lactate dehydrogenase inhibitor), but not gliotoxin. Intrathecal L‐lactate administration significantly upregulated c‐Fos and cofilin phosphorylation, which was reversed by 4‐CIN. The lowered mechanical nociceptive threshold was significantly attenuated by intrathecal fluorocitrate (an astrocyte‐specific Krebs cycle inhibitor), 4‐CIN, and isosafrole treatment. Thus, these results suggested that, in neuropathic pain, mechanical hyperalgesia was maintained by excessive L‐lactate supplied by activated astrocytes via an aberrant ANLS.

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A new minimally-invasive method for microinjection into the mouse spinal dorsal horn

Cited by 71 publications

References 53 publications

Regulating nociceptive transmission by VGluT2‐expressing spinal dorsal horn neurons

Regulating nociceptive transmission by VGluT2‐expressing spinal dorsal horn neurons

Rabies virus‐mediated oligodendrocyte labeling reveals a single oligodendrocyte myelinates axons from distinct brain regions

Astrocyte‐neuron lactate shuttle sensitizes nociceptive transmission in the spinal cord

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