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1.
J Vis Exp ; (203)2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38314798

RESUMEN

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.


Asunto(s)
Células Amacrinas , Retina , Ratones , Animales , Colodión , Retina/fisiología , Células Fotorreceptoras , Vertebrados
2.
Nat Commun ; 14(1): 1492, 2023 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-36932080

RESUMEN

Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, social behaviors, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2Cre mouse line, we show a subtype of ipRGCs restricted to the dorsal retina that selectively projects to the SON. These ipRGCs tile a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate in multiple regions, including the suprachiasmatic nucleus (SCN) and SON. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field to influence diverse behaviors.


Asunto(s)
Retina , Núcleo Supraóptico , Femenino , Ratones , Animales , Núcleo Supraóptico/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/fisiología , Opsinas de Bastones/genética
3.
Sci Rep ; 11(1): 2117, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33483520

RESUMEN

Parvalbumin-containing projection neurons of the medial-septum-diagonal band of Broca ([Formula: see text]) are essential for hippocampal rhythms and learning operations yet are poorly understood at cellular and synaptic levels. We combined electrophysiological, optogenetic, and modeling approaches to investigate [Formula: see text] neuronal properties. [Formula: see text] neurons had intrinsic membrane properties distinct from acetylcholine- and somatostatin-containing MS-DBB subtypes. Viral expression of the fast-kinetic channelrhodopsin ChETA-YFP elicited action potentials to brief (1-2 ms) 470 nm light pulses. To investigate [Formula: see text] transmission, light pulses at 5-50 Hz frequencies generated trains of inhibitory postsynaptic currents (IPSCs) in CA1 stratum oriens interneurons. Using a similar approach, optogenetic activation of local hippocampal PV ([Formula: see text]) neurons generated trains of [Formula: see text]-mediated IPSCs in CA1 pyramidal neurons. Both synapse types exhibited short-term depression (STD) of IPSCs. However, relative to [Formula: see text] synapses, [Formula: see text] synapses possessed lower initial release probability, transiently resisted STD at gamma (20-50 Hz) frequencies, and recovered more rapidly from synaptic depression. Experimentally-constrained mathematical synapse models explored mechanistic differences. Relative to the [Formula: see text] model, the [Formula: see text] model exhibited higher sensitivity to calcium accumulation, permitting a faster rate of calcium-dependent recovery from STD. In conclusion, resistance of [Formula: see text] synapses to STD during short gamma bursts enables robust long-range GABAergic transmission from MS-DBB to hippocampus.


Asunto(s)
Hipocampo/fisiología , Neuronas/fisiología , Parvalbúminas/metabolismo , Tabique del Cerebro/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Algoritmos , Animales , Calcio/metabolismo , Hipocampo/citología , Potenciales de la Membrana/fisiología , Ratones Transgénicos , Modelos Neurológicos , Neuronas/citología , Neuronas/metabolismo , Optogenética/métodos , Técnicas de Placa-Clamp , Tabique del Cerebro/citología
4.
Front Cell Neurosci ; 14: 277, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33093823

RESUMEN

Determining biophysical details of spatially extended neurons is a challenge that needs to be overcome if we are to understand the dynamics of brain function from cellular perspectives. Moreover, we now know that we should not average across recordings from many cells of a given cell type to obtain quantitative measures such as conductance since measures can vary multiple-fold for a given cell type. In this work we examine whether a tight combination of experimental and computational work can address this challenge. The oriens-lacunosum/moleculare (OLM) interneuron operates as a "gate" that controls incoming sensory and ongoing contextual information in the CA1 of the hippocampus, making it essential to understand how its biophysical properties contribute to memory function. OLM cells fire phase-locked to the prominent hippocampal theta rhythms, and we previously used computational models to show that OLM cells exhibit high or low theta spiking resonance frequencies that depend respectively on whether their dendrites have hyperpolarization-activated cation channels (h-channels) or not. However, whether OLM cells actually possess dendritic h-channels is unknown at present. We performed a set of whole-cell recordings of OLM cells from mouse hippocampus and constructed three multi-compartment models using morphological and electrophysiological parameters extracted from the same OLM cell, including per-cell pharmacologically isolated h-channel currents. We found that the models best matched experiments when h-channels were present in the dendrites of each of the three model cells created. This strongly suggests that h-channels must be present in OLM cell dendrites and are not localized to their somata. Importantly, this work shows that a tight integration of model and experiment can help tackle the challenge of characterizing biophysical details and distributions in spatially extended neurons. Full spiking models were built for two of the OLM cells, matching their current clamp cell-specific electrophysiological recordings. Overall, our work presents a technical advancement in modeling OLM cells. Our models are available to the community to use to gain insight into cellular dynamics underlying hippocampal function.

5.
PLoS One ; 13(7): e0199589, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29975722

RESUMEN

Synaptic plasticity, the cellular basis for learning and memory, is mediated by a complex biochemical network of signaling proteins. These proteins are compartmentalized in dendritic spines, the tiny, bulbous, post-synaptic structures found on neuronal dendrites. The ability to screen a high number of molecular targets for their effect on dendritic spine structural plasticity will require a high-throughput imaging system capable of stimulating and monitoring hundreds of dendritic spines in various conditions. For this purpose, we present a program capable of automatically identifying dendritic spines in live, fluorescent tissue. Our software relies on a machine learning approach to minimize any need for parameter tuning from the user. Custom thresholding and binarization functions serve to "clean" fluorescent images, and a neural network is trained using features based on the relative shape of the spine perimeter and its corresponding dendritic backbone. Our algorithm is rapid, flexible, has over 90% accuracy in spine detection, and bundled with our user-friendly, open-source, MATLAB-based software package for spine analysis.


Asunto(s)
Espinas Dendríticas , Aprendizaje Automático , Neuronas/citología , Programas Informáticos , Algoritmos , Animales , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía , Plasticidad Neuronal
6.
PLoS One ; 12(1): e0170586, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28114380

RESUMEN

Long-term structural plasticity of dendritic spines plays a key role in synaptic plasticity, the cellular basis for learning and memory. The biochemical step is mediated by a complex network of signaling proteins in spines. Two-photon imaging techniques combined with two-photon glutamate uncaging allows researchers to induce and quantify structural plasticity in single dendritic spines. However, this method is laborious and slow, making it unsuitable for high throughput screening of factors necessary for structural plasticity. Here we introduce a MATLAB-based module built for Scanimage to automatically track, image, and stimulate multiple dendritic spines. We implemented an electrically tunable lens in combination with a drift correction algorithm to rapidly and continuously track targeted spines and correct sample movements. With a straightforward user interface to design custom multi-position experiments, we were able to adequately image and produce targeted plasticity in multiple dendritic spines using glutamate uncaging. Our methods are inexpensive, open source, and provides up to a five-fold increase in throughput for quantifying structural plasticity of dendritic spines.


Asunto(s)
Automatización , Espinas Dendríticas/efectos de la radiación , Luz , Plasticidad Neuronal/fisiología , Animales , Espinas Dendríticas/fisiología , Ratones , Ratones Endogámicos C57BL
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