A Genetic Method for Selective and Quickly Reversible Silencing of Mammalian Neurons

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Bötzinger Complex (preBötC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBötC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 ± 45 neurons stopped spontaneous respiratory rhythm, and our data suggest ≈82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBötC can sustain insults that destroy no more than ≈18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.central pattern generator | calcium imaging | brainstem C entral pattern generator (CPG) networks produce coordinated motor patterns that underlie behaviors such as breathing, locomotion, and chewing (1, 2). A key issue is not just which cell types generate these patterns of activity, but how many? This question pertains to whether and how cumulative cell loss will impair and, possibly preclude, network function.Breathing consists of inspiratory and expiratory phases. The preBötzinger Complex (preBötC) of the medulla putatively contains the inspiratory CPG (3, 4). Over several days in vivo, saporinmediated destruction of preBötC neurons that express neurokinin-1 receptors (NK1Rs) causes sleep-disordered breathing and fatal respiratory pathology (5, 6). Similarly, acute cell-silencing of a subset of the same NK1R-expressing population of preBötC neurons stops spontaneous breathing in awake adult rats (7). These studies helped to determine the cellular composition of the preBötC and confirmed that it was essential for breathing in an otherwise intact animal, but could not measure the resiliency of the preBötC when faced with silencing or deleting its core piecewise.We examined this issue by using slice preparations that capture the preBötC and spontaneously generate fictive breathing-related activity in vitro. We developed a computer-automated system that detects rhythmically active interneurons via two-photon calcium imaging, stores their physical locations in memory, and then laser ablates the targets sequentially, while electrophysiologically monitoring the motor output of the circuit. We applied this system to the preBötC to test the hypothesis that piecewise destruction of the CPG core would cause graded loss of motor activity up to rhythm cessation. We surmised that this effect ...

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“…Viral transfection can be used to silence neuron populations (21). Knockout mice also remove cell classes wholesale rather than piecewise.…”

Section: Discussionmentioning

confidence: 99%

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

Hayes

Wang²,

Negro³

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Last, our ablation technique could be targeted in a spatially selective manner to a subset of a neuronal population (e.g., ablation of mossy cells in the temporal end of the slice). This spatial selectivity was crucial in determining the effect of mossy cells on extra-lamellar granule cell populations, and it could not have been achieved with other cell-specific ablation techniques (Lechner et al, 2002).…”

Section: Rapid and Spatially Selective Nature Of The Ablation Techniquementioning

confidence: 99%

Rapid Deletion of Mossy Cells Does Not Result in a Hyperexcitable Dentate Gyrus: Implications for Epileptogenesis

Ratzliff¹,

Howard²,

Santhakumar³

et al. 2004

J. Neurosci.

109

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Loss of cells from the hilus of the dentate gyrus is a major histological hallmark of human temporal lobe epilepsy. Hilar mossy cells, in particular, are thought to show dramatic numerical reductions in pathological conditions, and one prominent theory of epileptogenesis is based on the assumption that mossy cell loss directly results in granule cell hyperexcitability. However, whether it is the disappearance of hilar mossy cells from the dentate gyrus circuitry after various insults or the subsequent synaptic-cellular alterations (e.g., reactive axonal sprouting) that lead to dentate hyperexcitability has not been rigorously tested, because of the lack of available techniques to rapidly remove specific classes of nonprincipal cells from neuronal networks.We developed a fast, cell-specific ablation technique that allowed the targeted lesioning of either mossy cells or GABAergic interneurons in horizontal as well as axial (longitudinal) slices of the hippocampus. The results demonstrate that mossy cell deletion consistently decreased the excitability of granule cells to perforant path stimulation both within and outside of the lamella where the mossy cell ablation took place. In contrast, ablation of interneurons caused the expected increase in excitability, and control aspirations of the hilar neuropil or of interneurons in the presence of GABA receptor blockers caused no alteration in granule cell excitability.These data do not support the hypothesis that loss of mossy cells from the dentate hilus after seizures or traumatic brain injury directly results in hyperexcitability.

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“…For example, the regional expression of a genetically modified K ϩ channel in Drosophila was able to reduce the excitability of targeted cells (i.e., muscle, neurons, photoreceptors) (1). Silencing of cortical neurons was achieved by binding of the peptide allostatin to its exogenously expressed receptor (2). Recently, Zemelman et al (3) elegantly demonstrated that light activation of the protein complex, encoded by the Drosophila photoreceptor genes (i.e., arrestin-2, rhodopsin, and G protein ␣ subunit), could induce action potential firing of hippocampal neurons.…”

mentioning

confidence: 99%

Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin

Gutierrez

Hanson

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

501

420

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Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi͞o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltagegated Ca 2؉ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.A major challenge in understanding the relationship between neural activity and development and between neuronal circuit activity and specific behaviors is to be able to control the activity of large populations of neurons or regions of individual nerve cells simultaneously. Recently, it was demonstrated that neuronal circuits can be manipulated by expressing mutated ion channels or G protein-coupled receptors (GPCRs). For example, the regional expression of a genetically modified K ϩ channel in Drosophila was able to reduce the excitability of targeted cells (i.e., muscle, neurons, photoreceptors) (1). Silencing of cortical neurons was achieved by binding of the peptide allostatin to its exogenously expressed receptor (2). Recently, Zemelman et al. (3) elegantly demonstrated that light activation of the protein complex, encoded by the Drosophila photoreceptor genes (i.e., arrestin-2, rhodopsin, and G protein ␣ subunit), could induce action potential firing of hippocampal neurons. Activation and deactivation of neuronal firing could also be achieved when ligand-gated ion channels, such as the capsaicin receptor, menthol receptor, purinergic receptors, or lightcontrollable K ϩ channel blockers, were used to control firing in hippocampal neurons (4, 5). However, the application of these techniques to control neuronal function especially in neural circuits and living animals is limited by their relatively slow time course, the complex...

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A Genetic Method for Selective and Quickly Reversible Silencing of Mammalian Neurons

Cited by 137 publications

References 21 publications

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

Rapid Deletion of Mossy Cells Does Not Result in a Hyperexcitable Dentate Gyrus: Implications for Epileptogenesis

Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin

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