# What do healthy spinal neurons look like in slices (electrophysiology)? **URL:** \ **Category:** Uncategorized\ **Created:** [September 1, 2017, 8:29pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263 "2017-09-01T20:29:28Z")\ **Posts on this page:** 8\ **Page:** 1
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 1, 2017, 8:29pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/1 "2017-09-01T20:29:29Z")
Does anyone have exemplary images of what good, healthy spinal neurons looks like that would be suitable for electrophysiological recording? As a newbie to spinal e-phys, this has proved to be one of the hardest parts, that is, identifying which cells are good to patch. I know which ones I _shouldn’t_ patch: ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/66d57a6105613b8e043fb6d9d987952b997c4c1f.jpg) These are big and swollen and you can see the nuclei. So avoid these. But finding the good ones is a bit more challenging. The features I’ve been advised to look for are cells with smooth surfaces, no internal darkening. Can anyone post some exemplary images of what good dorsal horn neurons look like and also maybe comment on the type of optics you use? @cedric.peirs @lfqueme @MGradwell ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 4, 2017, 12:30pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/2 "2017-09-04T12:30:51Z")
I also posed this question at ResearchGate and there are some good answers there: [https://www.researchgate.net/post/How\_do\_you\_identify\_healthy\_neurons\_in\_slices\_brain\_spinal\_cord\_for\_Patch-Clamp\_recording?view=59ad378dcbd5c2a9e86d4f15#59ad47365b495237f778e56a](https://www.researchgate.net/post/How_do_you_identify_healthy_neurons_in_slices_brain_spinal_cord_for_Patch-Clamp_recording?view=59ad378dcbd5c2a9e86d4f15#59ad47365b495237f778e56a) ---
**Author:** ![MGradwell](https://avatars.discourse-cdn.com/v4/letter/m/a183cd/32.png) [@MGradwell](https://forum.painresearcher.net/u/MGradwell)\ **Post date:** [September 9, 2017, 1:50am UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/3 "2017-09-09T01:50:27Z")
Hi, I’ve attached an image of what some normal spinal tissue looks like. You are right, you really should be avoiding any cells that look big and round, often with a visible nucleus - the classic fried egg. The ones to look for, you can see a few in my image should have nice edges and hold their shape - when I’m teaching ppl to patch I usually tell the ‘jelly beans’ are good ones to start with. Having a pipette with positive pressure in the slice will also really help you identify the healthy cells with crisp edges. I’ve sealed onto a cell in the image here, there’s another good one just to the left up, left and below, a bit further right next to a nasty looking round one, and on the right below the myelin running horizontal. I use standard DIC imaging. ![160503_Thy1_070705_002](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/4fe18fc68dd90313146f4db1695ea939e5737747.JPG) Hopefully this is of some help! Cheers, Mark ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 10, 2017, 1:37pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/4 "2017-09-10T13:37:39Z")
Thanks @MGradwell. That’s extremely helpful. Would you be willing to share your slicing protocol too? ---
**Author:** ![MGradwell](https://avatars.discourse-cdn.com/v4/letter/m/a183cd/32.png) [@MGradwell](https://forum.painresearcher.net/u/MGradwell)\ **Post date:** [September 13, 2017, 4:39am UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/5 "2017-09-13T04:39:53Z")
I would point you towards a previous publication from our group: > **[Morphological, neurochemical and electrophysiological features of...](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476641/)** > > Axo-axonic synapses on the central terminals of primary afferent fibres modulate sensory input and are the anatomical correlate of presynaptic inhibition. Although several classes of primary afferents are under such inhibitory control, the origin of... Any specific questions let me know. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 13, 2017, 2:14pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/6 "2017-09-13T14:14:56Z")
Hi @MGradwell. Thanks. Actually, I used another paper from your lab as the basis for a lot of my buffers this summer: > > > The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs. Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin-expressing neurons. Most calretinin cells (85%) exhibited large A-type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization-activated cation currents and low threshold T-type calcium currents, and tonic- or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord. Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin-expressing neurons suggest that they play distinct roles in spinal sensory processing circuits.Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation. Overall it was pretty good and standard (sucrose cutting, and then regular aCSF). Some specific points though about the slicing and lead-up. - Do you do a full agarose embedding with melted agarose? Or do you make a cut-out from a solid block of agarose to hold the cord in place while slicing? And do you make any modifications to keep the cord from sliding out of the cut-out? - Do you ever do transcardial perfusion with ice-cold aCSF or do you go right into the cord? I was using transcardial perfusion so that I could make the cord cold quickly, and that afforded me time to dissect the cord in the animal while still in the body. I know some people just cut out the whole vertebral column and put it in ice/slurry sucrose solution and then dissect in that, but I find it easier to take the vertebrae off when the cord is still in the intact animal. - Up to what age animal do you use the sucrose cutting method? For animals older than 5 weeks, I know some have tried the NMDG recovery method ([https://www.brainslicemethods.com/](https://www.brainslicemethods.com/)). Have you tried this or do you always use sucrose? Thanks for your help. ---
**Author:** ![MGradwell](https://avatars.discourse-cdn.com/v4/letter/m/a183cd/32.png) [@MGradwell](https://forum.painresearcher.net/u/MGradwell)\ **Post date:** [September 14, 2017, 5:42am UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/7 "2017-09-14T05:42:25Z")
The buffers will be the same throughout all the group’s papers. We don’t use the agarose technique. I’ll point you toward another of my supervisor’s papers - [http://jn.physiology.org/content/99/5/2048.long](http://jn.physiology.org/content/99/5/2048.long). You will see in that paper that for transverse slices we use a styrofoam block cut at about the length of the cord, sitting up. We find the cord ‘sticks’ to this pretty well and allows for slicing. For this method it is important to put the thicker end of the cord down, or you have a teetering tower situation. We don’t use transcardial perfusion. We dissect out the column as you mention and remove the cord in a sACSF slurry. I did a 2.5 yr old mouse the other day - though this is at the extreme end we typically don’t use particularly young mice, ranging anywhere from 1 month - 2 yrs on avg. I have not tried the NMDG method, always use the same cutting solutions. Cheers, Mark ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 14, 2017, 1:01pm UTC](https://forum.painresearcher.net/t/what-do-healthy-spinal-neurons-look-like-in-slices-electrophysiology/263/8 "2017-09-14T13:01:23Z")
Thanks again @MGradwell for the information. The styrofoam sounds interesting. I’ve never heard of that. I’ll give it a try. So just a block of styrofoam behind the cord to give support? No cut out? That’d be an easy solution, easier than the agarose block. Also good to know that you can use older animals with sucrose and still get good results.