Name: In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons
Uploaded: 2024-02-09T13:35:00
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in vivo imaging of gcamp6s to study rat trigeminal ganglion neurons

Visualização in vivo do gânglio trigêmeo de ratos com indicadores de cálcio

Somatosensation, the neural encoding of mechanical, thermal, and chemical stimuli impinging on the skin or other bodily structures, including muscles, bone, and viscera, starts with activity in primary afferent neurons that innervate these structures1. Single unit based electrophysiological approaches have provided a wealth of information about the afferent subtypes involved in this process as well as how their stimulus-responses properties may change over time1,2,3. However, while there remains strong evidence in support of the labeled line theory, which suggests specific sensory modalities are conveyed by specific subpopulation(s) of neurons, the ability of many subpopulations of neurons to respond to the same types of mechanical, thermal, and chemical stimuli suggests the majority of somatosensory stimuli are encoded by multiple subpopulations of neurons4,5. Thus, a better understanding of somatosensation will only come with the ability to study the activity of 10&#39;s, if not hundreds, of neurons simultaneously.
Advances in optical approaches with the relatively recent advent of confocal and, subsequently, multiphoton and digital imaging techniques have facilitated the ability to perform relatively non-invasive population-level analyses of neuronal activity6,7. One of the last hurdles in the application of this technology has been the development of tools to enable the optical assessment of neural activity. Given the speed of an action potential that can start and end in less than a millisecond, a voltage-sensitive dye with the capacity to follow changes in membrane potential at the speed of an action potential would be the ideal tool for this purpose. But while there has been tremendous progress in this area7,8,9,10, the signal-to-noise ratio for many of these dyes is still not quite high enough to enable a population analysis of hundreds of neurons at the single cell level. As an alternative approach, investigators have turned to monitoring changes in intracellular Ca2+ concentration ([Ca2+]i). The limitations with this strategy have been clear from the start and include the fact that an increase in [Ca2+]i is an indirect measure of neural activity11; that an increase in [Ca2+]i may occur independently of Ca2+ influx associated with the activation of voltage-gated Ca2+ channels (VGCCs)12,13; that the magnitude and duration of a Ca2+ transient may be controlled by processes independent of VGCC activity11,12,14; and that the time-course of Ca2+ transients far exceeds that of an action potential15. Nevertheless, there are a number of significant advantages associated with the use of Ca2+ as an indirect measure of neural activity. Not the least of these is the signal-to-noise ratio associated with most Ca2+ indicators, reflecting both the magnitude of the change in intracellular Ca2+ and the fact that the signal is arising from the three-dimensional space of the cytosol rather than the two-dimensional space of the cell membrane. Furthermore, with the development of genetically encoded Ca2+ indicators (GECI&#39;s), it is possible to take advantage of genetic strategies to drive the expression of the Ca2+ indicators in specific subpopulations of cells, facilitating population-level analyses in intact preparations (e.g., see16).
Given the number of genetic tools now available in mice, it should be no surprise that GECI&#39;s have been used most extensively in this species. Mouse lines with constitutive GECI expression in subpopulations of sensory neurons have been developed7,16,17. With the development of mouse lines expressing recombinases in specific cell types, it is possible to use even more sophisticated strategies to control GECI expression15. However, while these tools are ever more powerful, there are a number of reasons why other species, such as rats, might be more appropriate for some experimental questions. These include the larger size, facilitating a number of experimental manipulations that are difficult, if not impossible, in the smaller mouse; the ease of training rats in relatively complex behavioral tasks; and at least some evidence that biophysical properties and expression patterns of several ion channels in rat sensory neurons may be more similar to that observed in human sensory neurons than are&#160;the same channels in mouse relative to the human18.
While the transduction of somatosensory stimuli generally occurs in the peripheral terminals of primary afferents, the action potential initiated in the periphery must pass through the structure that houses primary afferent somata, referred to as dorsal root (DRG) or trigeminal (TG) ganglia before reaching the central nervous system19. While there is evidence that not every action potential propagating along a primary afferent axon will invade the cell body20, a consequence of the fact the primary afferent somata are connected to the main afferent axon via a T-junction19, the majority of action potentials initiated in the periphery appear to invade the soma21. This confers three experimental advantages when using GECIs to assess population coding in primary afferents: the large size of the cell body relative to the axons further increases the signal to noise when using [Ca2+]i as an indirect measure of afferent activity; the DRG are generally easy to access; and assessing activity at a site that is spatially remote from the afferent terminals minimizes the potential impact of the surgery needed to expose the ganglia on the stimulus-response properties of the afferent terminals. However, because TG are located beneath the brain (or above the palette), they are far more difficult to access than DRG. Furthermore, while there are many similarities between DRG and TG neurons, there is a growing list of differences as well. This includes the roughly somatotopic organization of neurons in the TG22, unique structures innervated, different central terminal termination patterns23,24,25,26, and now a growing list of differences in both gene expression27,28 and functional receptor expression29. In addition, because we are interested in the identification of peripheral mechanisms of pain, the relatively large number of pain syndromes that appear to be unique to the trigeminal system (e.g., migraine, trigeminal neuralgia, burning mouth syndrome) that appear to involve aberrant activity in primary afferents30,31,32, suggests that the TG needs to be studied directly.
Thus, while stimulus-response properties of TG neurons have been studied with GECIs in the mouse16, because the reasons listed above suggest that the rat may be a more appropriate species to address a variety of experimental questions, the purpose of the present study was to develop an approach to use GECIs to study TG neurons in the rat. To achieve this, we utilized a viral approach to drive the expression of the GECI GCaMP6s in the peripheral nervous system. We then removed the forebrain to allow access to the TG. Finally, mechanical and thermal stimuli were applied to the face while neuronal responses were assessed under fluorescent microscopy. Together, these data support a role for utilizing the rat to investigate changes in the TG under many states, expanding the toolkit for investigators interested in sensory coding in the trigeminal system.

Autor em destaque: Explorando os mecanismos periféricos da dor neuropática na lesão do nervo trigêmeo 

In-Vivo Imagem de cálcio de neurônios sensoriais no gânglio trigeminal de ratos

The general goal of our lab is to understand the peripheral mechanisms of pain, and my project specifically investigates pain associated with trigeminal nerve injury. The preparation that I'm showing today allows us to characterize changes in sensory coating and identify afferent subpopulations contributing to neuropathic pain. So we have shown that there are differences in between the trigeminal and somatic afferents in response to nerve injury.NaV1.1 specifically is preferentially upregulated in the trigeminal nerve, suggesting that selective blockers for NaV1.1 can be used to identify different afferent subpopulations that contribute to neuropathic pain. So transgenic mice are used most extensively at the moment in combination with a variety of imaging and omic based strategies. Translating data generated in mice to other species, if not humans.Mice are really small, they're difficult to train, and there's a growing list of differences between mice, rats, and humans. So using rats may help address several of these challenges. Pain mechanisms and potential therapeutic targets differ in craniofacial structures, as in those above the neck, and other somatic and visceral structures, i.e.those below the neck. Results with this preparation suggest that there's a component of neuropathic pain, which is previously thought to reflect changes in the central nervous system, but may actually also reflect changes in the peripheral nervous system.

Watch this Scientific Journal Video about In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons at JoVE.com

In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons  

Imagem in vivo de GCaMP6s para estudar neurônios do gânglio trigeminal de rato

Para começar, use uma seringa Hamilton de 25 microlitros para injetar filhotes de ratos anestesiados por intraperitoneal com 15 microlitros de Addgene. Em seguida, raspe o cabelo e os bigodes de filhotes anestesiados de seis a oito semanas de idade. Monte o rato em uma armação estereotáxica com barras auriculares.E coloque uma almofada de aquecimento por baixo para manter a temperatura corporal do animal. Próximo local de gaze mergulhada em soro fisiológico gelado na cabeça do rato para minimizar o sangramento. Com bisturi tamanho 15, faça uma incisão na linha média da pele e músculo sobre o crânio.Puxe a pele e o músculo para expor o crânio. Com uma broca redonda de um por quatro, perfure cuidadosamente a calota craniana para expor o prosencéfalo. Em seguida, use rongeurs para cortar o crânio.Com bisturi tamanho 15, faça uma incisão no cérebro e bulbo olfativo. Use uma espátula para desconectar cuidadosamente a dura-máter do crânio. Em seguida, levante suavemente o cérebro cortado para exibir o gânglio trigeminal e a base do crânio.Cauterize qualquer sangramento com uma caneta cauterizada. Para obter imagens do GCaMP6-X, coloque a cavidade craniana abaixo da objetiva do microscópio. Em seguida, coloque o gânglio terminal em foco.Em seguida, aplique estímulos mecânicos em um centímetro quadrado da face. Use uma objetiva de 20X para visualizar os neurônios do gânglio trigeminal que respondem ao estímulo. Para coletar o stato fluorescente ao longo do tempo com o software de aquisição, pressione o botão de aquisição de fluxo no menu de aquisição.Em seguida, defina o número de quadros para 300 e o tempo de aquisição para 90 segundos. Defina os parâmetros da câmera para adquirir imagens na taxa de quadros. Obtenha gravações de linha de base estáveis ao longo de três minutos.Um maior volume de título viral resultou em aumento da infecção neuronal dos filhotes de ratos. A estimulação mecânica luminosa ativou apenas a região V2 do gânglio trigeminal. A fluorescência de repouso foi relativamente baixa na maioria dos neurônios, com poucos aumentos espontâneos.Os estímulos naturais, especificamente a estimulação pontual, provocaram as respostas neuronais mais robustas. Os estímulos com escova em modelos de lesão por constrição crônica provocaram um aumento de duas vezes na magnitude do pico de resposta do lesado em relação ao lado contralateral. Quando dois estímulos foram aplicados em série, houve amplificação significativa em resposta ao segundo estímulo.

Research

JoVE Journal

Neuroscience

In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...