Primary Neurons Culturing Expertise

hippocampal_neurons_1_weekI recently featured Dr. Evanna Gleason.  As part of this, we highlighted her lab’s epertise in culturing our E18 Primary Rat Hippocampal Neurons

I recently received impressive data and protocols from Emily Mcmains, a lab member.hippocampal_neurons_4-days

Please note the excellent image of the cells 1 week after culturing and images taken after 4 days.

Courtesy of Emily McMains (Gleason Lab), LSU.

Featuring Dr. Evanna Gleason

 Spotlighting How Retinal Neurons Communicate

About Dr. Evanna Gleason

Dr. Evanna Gleason

Dr. Evanna Gleason

Background:

1996-Current-Associate Professor, LSU

1993-1996-Post Doc, UC San Diego

1991-1992-Post Doc, UC Davis-Wilson Lab

1990 Ph. D- UC San Diego

1984 Undergrad-ASU

How do neurons communicate across synapses? Finding answers to this is of central interest to many of our customers and colleagues. After all, it is the transmission of signals across synapses that collectively orchestrate our perceptions.

Abnormal transmission is at the root of many neuro-disorders that plague society. Research in the cell and molecular biology of synapse transmission is a piece of the puzzle in discovering cures.

This leads to why I am honored to feature Dr. Evanna Gleason and her work on how Retinal Neurons Communicate. She and her team focus on how retinal synapses are specialized to transmit visual information. Her work adds to the body of understanding of the processes that enable us to see.

Beginnings

Assembling the pieces of Evanna’s research begin with her graduate work in Dr. Martin Wilson’s lab at UC Davis. Here she developed the culturing techniques required to study transmission between isolated pairs of amacrine cells. These techniques enabled the lab to study the firing of individual neurons and created the platform for her current research Here are related publications:

E Gleason, S Borges and M Wilson. Synaptic transmission between pairs of retinal amacrine cells in culture. Journal of Neuroscience, Vol 13, 2359-2370, Copyright © 1993 by Society for Neuroscience.

Gleason E., Borges S., Wilson M. Control of transmitter release from retinal amacrine cells by Ca2+ influx and efflux. Neuron 1994.  Nov;13(5):1109-17.

More on Amacrine Cells-Amacrine cells operate at the inner plexiform layer (IPL), the second synaptic retinal layer where bipolar cells and retinal ganglion cells synapse. There are about 40 different types of amacrine cells, most lacking axons. Like horizontal cells, amacrine cells work laterally affecting the output from bipolar cells, however, their tasks are often more specialized. Each type of amacrine cell connects with a particular type of bipolar cell, and generally has a particular type of neurotransmitter. One such population, AII, ‘piggybacks’ rod bipolar cells onto the cone bipolar circuitry. It connects rod bipolar cell output with cone bipolar cell input, and from there the signal can travel to the respective ganglion cells.They are classified by the width of their field of connection, which layer(s) of the stratum in the IPL they are in, and by neurotransmitter type. Most are inhibitory using either GABA or glycine as neurotransmitters.

 Developmental Neurobiology 

Evanna did her post doc in Dr. Nick Spitzer’s lab at UC-San Diego. She studied the development of voltage-dependent ion channels and neurotransmitter receptors in the embryo. The focus in the lab was more on systems assembly and differentiation vs the study of synaptic transmission between individual neurons.

Although an interesting sidetrack, Evanna shared with me that her passion is the study of synaptic transmission in retinal neurons. This bring us to her current work.

From San Diego to Baton Rouge

I became acquainted with Evanna in a phone follow up concerning use of our E18 Primary Rat Hippocampal Neurons. This conversation proved enlightening as she provided specific insight on what she did with the cultures. Growing healthy an robust cultures was the easy part.

I learned that she and her team at LSU have the experience and expertise required to indentify and isolate amacrine cells. She shared how the cells were then used for studying the role of Nitric Oxide (NO) and Chloride (Cl-) in synaptic modulation and provided me with related data. This data proved to be of interest to a cross section of customers and colleagues studying synaptic transmission. Here’s the resulting publication and sample data:  

gaba_currentsFigures: Higher concentrations of NO promote a positive shift in EGABA. A and B, top traces: raw data from ruptured-patch voltage-clamp recordings of GABA-gated currents from a representative cell before and after NO application. GABA pulses (20 µM) were 300 ms in duration and are indicated by horizontal bars. A: whole cell, voltage-clamp recordings (Cs+-A internal and TEA-A external) of GABA-gated currents reveal that higher concentrations of NO induce a transient, several-fold enhancement of GABA-gated currents. *, NO-dependent current observed prior to the 2nd GABA application. B: same experiment as in A, using air-exposed NO solution. Raw data in A and B are from same cell. Scale bars are 150 pA, 1 s. C: amacrine cell is held at the predicted EGABA. GABA is applied for 300 ms during each trace. No GABA-gated currents are observed until application of NO. *, NO-dependent current. Scale bars are 25 pA, 5 s. D: voltage ramps in GABA were delivered before and after addition of NO. Leak-subtracted currents reveal a shift in EGABA after NO application (gray trace). Inset: subtraction of the NO-induced shift in reversal potential reveals an increase in the slope of the GABA-gated current-voltage relationship after NO injection (gray trace). Scale bars are 100 pA, 20 mV. E: mean EGABA values are plotted over time. F: representative GABA-gated currents from voltage ramps delivered after a 11-min treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 2 µM). Black trace, before NO injection; Gray trace, after NO injection. G: ODQ did not block the NO-induced shift in EGABA (P = 0.83, n = 5).

As I learned from from this and my interview with Evanna: she and her team are assembling a clearer picture of the relationship between NO, Cl-  and what is happing at  GABAergic synapses.

I plan to keep my eyes on how the puzzle grows and communicate the discoveries that bring the picture into clearer focus.  We will specifically be focused on the impact of Evanna’s research contributions to the overall understanding. of  how messages are communicated in the CNS and PNS.

What’s Next

Evanna indicated to me the potential of her using siRNA to do gene expression analysis.  As outlined in previous News Behind the Neuroscience News postings, this is near and dear to me. The ability to switch on and off genes involved in transmission will undoubtedly enhance the platform and drive new discoveries. 

March Profile-Dr. Evanna Gleason

Coming soon…evanna_gleasonNow for something completely different…Synaptic Transmission Research.

I am pleased to be featuring Dr. Evanna Gleason.  I selected her because She and her team’s research is a basic component of most areas  Neuroscience Research including pain, neurodegeneration, vision, TBI, SCI, drug addiction, neuro-disorders and more…This base component is how neurons communicate with other cells at synapses. 

She focuses on synaptic transmission in the vertebrate retina. Retinal neurons have distinctive anatomical and physiological properties that suggest they employ unique synaptic mechanisms. The long term objective her research is to understand how retinal synapses are specialized to transmit visual information.

More to come in March!