Strategies for Identification of Medial Olivocochlear Neurons for Patch-Clamp Studies of Synaptic Function Using Electrical Stimulation and Optogenetics

2022 ◽  
pp. 339-356
Author(s):  
Kirupa Suthakar ◽  
Lester Torres Cadenas ◽  
Catherine Weisz
Keyword(s):  
Electrical Stimulation ◽  
Patch Clamp ◽  
Synaptic Function ◽  
Medial Olivocochlear

scholarly journals Long range channelrhodopsin-assisted circuit mapping of inferior colliculus neurons with blue and red-shifted channelrhodopsins

2019 ◽  
Author(s):  
David Goyer ◽  
Michael T. Roberts
Keyword(s):  
Electrical Stimulation ◽  
Patch Clamp ◽  
Inferior Colliculus ◽  
Long Range ◽  
Cochlear Nucleus ◽  
Auditory Brainstem ◽  
Dorsal Cochlear Nucleus ◽  
Multiple Sources ◽  
Patch Clamp Recording

ABSTRACTWhen investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM) this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.SUMMARYChannelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

Effects of preventing reinnervation on axotomized spinal motoneurons in the cat. II. Changes in group Ia synaptic function

1989 ◽  
Vol 62 (2) ◽  
pp. 325-333 ◽  
Author(s):  
S. Vanden Noven ◽  
M. J. Pinter
Keyword(s):  
Electrical Stimulation ◽  
Rise Time ◽  
Mean Amplitude ◽  
Synaptic Function ◽  
Medial Gastrocnemius ◽  
Lateral Gastrocnemius ◽  
Epsp Amplitude ◽  
Time To Peak ◽  
Postoperative Time ◽  
Stimulation Of

1. Composite excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of heteronymous group Ia afferents have been studied at various postoperative times in axotomized motoneurons that were denied the opportunity to reinnervate muscle. 2. The medial gastrocnemius (MG) nerve was transected and sutured onto the surface of the normally innervated lateral gastrocnemius (LG) muscle. The denervated MG muscle was excised thereby eliminating access of regenerating MG motor axons to vacant end-plates. 3. The mean amplitude of monosynaptic Ia EPSPs evoked by electrical stimulation of the LG-soleus (LGS) nerve and recorded in axotomized MG motoneurons showed an initial decline at 20 days postoperative (DPO) that was not significant. At 44 DPO, mean amplitude had declined significantly to 43% of the control mean amplitude. At 90 DPO, mean EPSP amplitude was not significantly different from control. At the latest postoperative time (150-180 DPO), mean amplitude was significantly less than the control amplitude. 4. Mean EPSP rise time (time-to-peak) was significantly increased (27%) at the earliest postoperative times (20-44 DPO). At later postoperative times (90-180), mean EPSP rise time was not significantly different from mean control rise time. 5. "Partial responses" superimposed on EPSPs were not observed at any postoperative time. 6. Mean posttetanic potentiation (PTP) of the LGS EPSP was significantly depressed at 20 DPO. At later postoperative times, PTP did not differ significantly from mean control PTP. 7. The possibility is considered that postaxotomy alterations in the electrical properties of motoneurons may explain these complex variations of mean EPSP amplitude and rise time.

Postsynaptic neuronal geometry and synaptic effectiveness

1987 ◽  
Vol 45 ◽  
pp. 690-697
Author(s):  
C.J. Wilson
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Synaptic Function ◽  
Postsynaptic Neuron ◽  
Synaptic Inputs ◽  
Partial Analysis ◽  
Postsynaptic Cell ◽  
Neuronal Geometry ◽  
The Relationship ◽  
Complex Scale

Most central nervous system neurons receive synaptic input from hundreds or thousands of other neurons, and the computational function of such neurons results from the interactions of inputs on a large and complex scale. In most situations that have yielded to a partial analysis, the synaptic inputs to a neuron are not alike in function, but rather belong to distinct categories that differ qualitatively in the nature of their effect on the postsynaptic cell, and quantitatively in the strength of their influence. Many factors have been demonstrated to contribute to synaptic function, but one of the simplest and best known of these is the geometry of the postsynaptic neuron. The fundamental nature of the relationship between neuronal shape and synaptic effectiveness was established on theoretical grounds prior to its experimental verification.

High-voltage electron microscopy of patch-clamped membranes

1987 ◽  
Vol 45 ◽  
pp. 582-583
Author(s):  
F. Sachs ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Patch Clamp ◽  
High Voltage Electron Microscopy ◽  
Small Patch ◽  
Cellular Electrophysiology ◽  
Voltage Electron ◽  
Electron Microscopes ◽  
Patch Technique ◽  
Membrane Patch

Cellular electrophysiology has been revolutionized by the introduction of patch clamp techniques. The patch clamp records current from a small patch of the cell membrane which has been sucked into a glass pipette. The membrane patch, a few micons in diameter, is attached to the glass by a seal which is electrically, diffusionally and mechanically tight. Because of the tight electrical seal, the noise level is low enough to record the activity of single ion channels over a time scale extending from 10μs to days. However, although the patch technique is over ten years old, the patch structure is unknown. The patch is inside a glass pipette where it has been impossible to see with standard electron microscopes. We show here that at 1 Mev the glass pipette is transparent and the membrane within can be seen with a resolution of about 30 A.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

Three-dimensional structure of dendritic spines, neuronal specializations that impart both stability and flexibility to synaptic function

1993 ◽  
Vol 51 ◽  
pp. 92-93
Author(s):  
Kristen M. Harris
Keyword(s):  
Dendritic Spines ◽  
Three Dimensional ◽  
Synaptic Function ◽  
Dimensional Structure ◽  
Excitatory Synapses ◽  
Concept Of Function ◽  
Section Electron ◽  
High Quality Information ◽  
The Central Nervous System ◽  
Mathematical Analyses

Dendritic spines are the tiny protrusions that stud the surface of many neurons and they are the location of over 90% of all excitatory synapses that occur in the central nervous system. Their small size and variable shapes has in large part made detailed study of their structure refractory to conventional light microscopy and single section electron microscopy (EM). Yet their widespread occurrence and likely involvement in learning and memory has motivated extensive efforts to obtain quantitative descriptions of spines in both steady state and dynamic conditions. Since the seminal mathematical analyses of D’Arcy Thompson, the power of establishing quantitatively key parameters of structure has become recognized as a foundation of successful biological inquiry. For dendritic spines highly precise determinations of structure and its variation are proving themselves as the kingpin for establishing a valid concept of function. The recent conjunction of high quality information about the structure, function, and theoretical implications of dendritic spines has produced a flurry of new considerations of their role in synaptic transmission.

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