Atualização em Optogenética

O Grupo de Estudo do IEA-RP, “Reflexões em Neurociência Contemporânea”, realizará no dia 13 de julho, às 14h a mesa redonda “Atualização em optogenética”. O evento ocorrerá no Salão de Eventos do CeTI-RP/USP e as palestras serão em inglês.

O evento reunirá pesquisadores nacionais e internacionais para discutir como grupos específicos de células associadas ou não a patologias do sistema nervoso, podem ser manipuladas, ativadas ou bloqueadas.

O termo Optogenética refere-se a técnicas que combinam luz, genética e bioengenharia e permitem o estudo de circuitos neuronais e comportamentos atuando em células específicas, comenta o Professor Norberto Garcia Cairasco (FMRP-USP), Coordenador do Grupo 30de Estudo e Moderador da Mesa Redonda.

Inicialmente o Professor Merab Kokaia (Universidade de Lund – Suecia), durante sua palestra “Inhibitory Interneurons Studied by Optogenetics” explorará maneiras de ativar ou inativar interneurônios inibitórios, usualmente GABAérgicos, e determinará seu impacto em processos de aumento anómalo de excitabilidade cerebral.

Na sequência, o Professor Patrick Forcelli (Georgetown University – Estados Unidos da América), fará a palestra “Optogenetic Interrogation of Nigrotectal Circuitry for Broad Spectrum Seizure Control” demonstrando como a ativação por optogenética de uma via específica no tronco cerebral de ratos pode se comportar como anticonvulsivante em quatro modelos experimentais de epilepsia.

Encerrando as apresentações, o Pós-Doutorando Cleiton Aguiar (FMRP-USP), discorrerá, na sua palestra “Optogenetic Investigation of Gamma Oscillations in the Medial Prefrontal Cortex of Urethane-Anesthetized Mice”, sobre as possibilidades da optogenética para caracterizar a participação de interneurônios positivos para parvalbumina na expressão de deficits cognitivos e nas mudanças nas atividades de redes com relevância para a sintomatologia da esquizofrenia.

Resumo das palestras:

“Inhibitory Interneurons Studied by Optogenetics”
Merab Kokaia – Lund University Hospital

Optogenetic techniques provide powerful tools for bidirectional control of neuronal activity and investigating alterations occurring in excitability disorders, such as epilepsy. In particular, the possibility to specifically activate by light determined interneuron populations expressing channelrhodopsin-2 provides an unprecedented opportunity of exploring their contribution to physiological and pathological network activity. There are several subclasses of interneurons in cortical areas with different functional connectivity to the principal neurons (e.g., targeting their perisomatic or dendritic compartments). Therefore, one could optogenetically activate specific or a mixed population of interneurons and dissect their selective or concerted inhibitory action on principal cells. We chose to explore a conceptually novel strategy involving simultaneous activation of mixed populations of interneurons by optogenetics and study their impact on ongoing epileptiform activity in mouse acute hippocampal slices. Here we demonstrate that such approach results in a brief initial action potential discharge in CA3 pyramidal neurons, followed by prolonged suppression of ongoing epileptiform activity during light exposure. Such sequence of events was caused by massive light-induced release of GABA from ChR2-expressing interneurons. The inhibition of epileptiform activity was less pronounced if only parvalbumin- or somatostatin-expressing interneurons were activated by light. Our data suggest that global optogenetic activation of mixed interneuron populations is a more effective approach for development of novel therapeutic strategies for epilepsy, but the initial action potential generation in principal neurons needs to be taken in consideration.

“Optogenetic Interrogation of Nigrotectal Circuitry for Broad Spectrum Seizure Control”
Patrick A Forcelli  – Georgetown University Department of Pharmacology & Physiology

A long-standing role for the substantia nigra pars reticulata (SNpr) and its principal projection target, the superior colliculus (SC) has been described in the control of experimental seizures. These prior studies have relied on microinjection, lesion, and electrical stimulation approaches. While informative, these approaches suffer from several confounds and limitations including drug spread, poor temporal control, activation of fibers of passage, and permanent damage. The optogenetic approach, by contrast, allows for selective activation or silencing of these structures in real-time. Here, we report our recent work examining optogenetic activation of SC, optogenetic inhibition of SNpr, as well as optogenetic silencing of nigrotectal projections. We examined the effect of these manipulations on experimental seizures evoked by pentylenetetrazole, gamma butyrolactone, focally from Area Tempestas, or in audiogenic-seizure susceptible rats (GEPRs). These models trigger secondarily generalized, absence, complex partial, and primary brainstem seizures, respectively. We found that silencing SNpr or nigrotectal terminals, or activation of SC exerted broad spectrum seizure suppressive effects in these models. Effects were detected with frequencies of stimulation as low as 5Hz. These results provide definitive evidence that the nigrotectal pathway is sufficient to account for seizure suppression evoked from SNpr. Moreover, these data indicate that low frequency stimulation within this pathway may be sufficient for seizure control.

“Optogenetic Investigation of Gamma Oscillations in the Medial Prefrontal Cortex of Urethane-Anesthetized Mice” 
Cleiton Lopes-Aguiar1,2, Nicolas Guyon1, Calvin Young1, Hoseok Kim1, Marie Carlén1
1 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
2 Department of Neuroscience, Ribeirão Preto School of Medicine, University of São Paulo, Brazil.

Perturbed excitatory-inhibitory balance occurs in multiple psychiatric and neurological conditions and might be a critical link between schizophrenia positive and negative symptoms, aberrant neural oscillatory activity, cognitive deficits and psychiatric comorbidities in epilepsy. Recently, Carlén’s group showed that loss of NMDAR-mediated drive of parvalbumin positive interneurons (PV+) results in cognitive deficits and changes in network activities highly relevant to schizophrenia symptomatology. Here, we aimed to investigate the role of NMDAR specifically in PV+ during basal and optogenetically-evoked gamma oscillations (40 – 100 Hz) in the medial prefrontal cortex (mPFC) of urethane-anesthetized mice. First, adult PV-Cre and NR1-PV-Cre mice (knockout for the NR1 subunit specifically in PV+ cells) were injected with AAV2-DIO-ChR2-mCherry or AAV5-DIO-EYFP (control-virus). Four to twelve weeks later, the animals were anesthetized with urethane and submitted to electrophysiological recordings with a 32 channels silicon probe (A4x2-tet-5mm-150-200-312-A32, Neuronexus) and to optogenetic stimulation of the prefrontal PV+ cells. Our preliminary findings suggest that photostimulation for activation of ChR2-expressing PV+ neurons imposes transitory decrease in the firing rate of nearby neurons and specific enhancement of gamma band power in the mPFC. PV-Cre and NR1-PV-Cre groups will be compared regarding oscillatory patterns and firing rate in the mPFC. These data analyses are ongoing.