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Minocycline treatment rescues IAsynapse structure but not function following peripheral nerve injury

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Dr. Travis Rotterman details his research on Minocycline its effects following nerve injury.

Well good afternoon everyone my name is travis roderman i’m a postdoc in tim cope’s lab at georgia tech in atlanta georgia uh so today i’m going to tell you a story about how preserving synaptic structure connectivity doesn’t always lead to a recovery of function after nerve injury and first i just want to thank all the individuals who contributed to this work

Including the funding sources all right so every single spinal motor neuron receives somewhere on the order of 30 to 50 000 synapses and a proportion of those express the vesicular glutamate transporter isoform 1 or v glu 1 as indicated there by the green boutons and these synapses are uh predominantly if not exclusively coming from the proprioceptor 1a afferents

That monosynaptically project to the motor neurons and um so i reconstructed these motor neurons and i found that most of these v-glu ones reside in the most proximal dendritic arbor of the cells and interestingly after nerve cut where both the sensory and motor axons are severed majority of these synapses are lost and they never come back even well after peripheral

Regeneration is complete so now surprisingly this leads to some deficits in sensory motor encoding so one strategy is to to attempt to preserve these synapses and hope that that would result in some behavioral improvements but first we have to really uh understand uh how these synapses are are lost so in the spinal cord you have these microglial cells uh that

Are the native macrophages of the cns they have these small cell bodies and these dynamic processes that extend and retract as i’ll show you with this two photon live imaging experiment in the adult uh spinal cord so you can see what the microglia cells look like in real time so as you sit there and listen to me talk in your brain and in your spinal cord these

Cells are constantly moving and in fact uh every synapse in the cns is contacted multiple times throughout the day by these microglial cells so interestingly after nerve cut these microglia cells respond both in the dorsal horn and in the ventral horn and they take on this pro-inflammatory-like phenotype where their cell body increases in size their processes

Retract and they undergo proliferation within the first few days two weeks after injury and if you look at one of these activated microglial cells you really see a unique phenotype here compared to the the surveying state and so what we know is that in neurodevelopment during synaptic pruning and in other neurodegenerative conditions these microglia cells either

Directly or indirectly are involved in synaptic removal so we really wanted to know if this microglia reaction corresponded to the loss of the v glu1 synapses so what i found was that that microglia reaction uh is induced by the release of a signaling molecule called colony stimulating factor one or csf1 that’s released specifically from exotomized motor neurons

And in the cns csf1 is specific for microglial cells so when we eliminated that csf1 molecule using a kreloc system from motor neurons we see that we prevented the microglia reaction uh from occurring there in the ventral horn but we really wanted to know on the motor neurons were we able to preserve those synapses so we quantified uh the synaptic density uh

V-glut1 synapses uh and compared that to controls so the wild-type animals which are the white bars you see there’s uh synaptic loss both on the cell body and the dendrites not surprisingly but in the mutant animals where we eliminated csf1 we were able to preserve those synapses both on the soma and dendrites so structurally we can preserve them but it doesn’t

Really tell us anything about function so in order to address that i transitioned to the cope lab where they study motor circuits in the rat spinal cord now unfortunately i didn’t have the transgenics available but i took a pharmacological approach using monocycline to suppress inflammation with the hope that we could preserve synaptic structure and ultimately

Synaptic function now minocycline is not specific for microglial cells it impacts multiple uh components of the inflammatory cascade but it basically it kind of tunes down inflammation so the hope was that that would result in the restoration of the synapses so i did this in adult rats both male and female and we completely transected the mg nerve repaired it

And then we treated the rats with minocycline for 14 days after injury and we first looked structurally to see if we could preserve those synapses at three key time points so at 14 days you have this dynamic phase of inflammation you have synaptic loss occurring uh three months the synaptic losses plateaued but the motor axons have regenerated but the sensory

Axons at least for the 1a’s have not and at six months both sensory and motor axons have regenerated uh at least to the uh the best they’re gonna get so then we quantified the dendritic of the v glut1 dendritic linear density on these motor neurons and compared that to controls so every single dot you see up there is an individual motor neuron and they’re color

Coded based on the animal that they came from so in the 14 days we see that there’s uh synaptic loss not surprisingly by three months that’s significantly down from control and that persists even at six months so those synapses don’t come back that’s not surprising we’ve shown that previously but with the minor cycling treatment at 14 days let’s see if this

Works uh we were able to preserve those synapses and now again the treatment stops at 14 days but at uh three months uh it’s down from control but it’s not significant and at six months uh it’s also you know slightly down from control but it didn’t reach a level of significance but we do see a lot of variability uh in those motor neurons being that some preserve

The synapses quite well while others lose a fair amount so we wanted to know really is there a difference in synaptic density at this time point um because this is when we can really investigate uh function so what we did we generated a generative statistical model that is conditioned on the observed data so basically what that allows us to do is we can run 10 000

Simulations and which is included in this uh distribution so if you look at the control which is the green that fits really well with the observed data and then in the regenerated animals that are treated with vehicle there’s this far shift to the left not surprisingly but with the minus cycling which is the purple we were able to show that we were in fact better

Able to preserve those synapses compared to vehicle treated alone so what about function so in order to test that we recorded synaptic potentials in vivo intra intracellularly in response to muscle stretch so you get this response to that type of stretch and in controls 100 of motor neurons produce a stretch evoke synaptic potential in response to to the ramp

With the cut vehicle that connectivity drops to around sixty percent where sixty percent of motor neurons respond where the remaining forty percent have no stretchy vote uh potential and with minus cycling we see that that uh connectivity we’ve only lost about 10 percent of cells uh not responding to to the ramp and when we simulated that data it matches quite well

With what you see in the the dendritic linear density and that minus cycling has improved the uh the connect functional connectivity now we went on to look at the amplitudes of those responses and they’re kind of three main components that we look at the initial burst that the that occurs at the onset of the ram the dynamic phase while it’s being stretched and

Then this uh static component uh and when we looked in the cut vehicle and the cut minus cycling animals we see that the amplitudes have significantly declined in all three components suggesting that the synaptic efficacy has not recovered with minor cycling treatment and then finally uh we went on to record the stretch reflex where we can actually look at the

Muscle force being produced with a corresponding emg and if i just kind of blow oh then nope okay well all right blow it up a little bit uh you’ll see uh the passive component there in red and then the the black trace is showing the the active force and then in blue i’ve just kind of highlighted the the reflex uh but with cut vehicle uh no no reflex and with

Cut monocycling we weren’t able to elicit a reflex either in those animals um so just to uh uh highlight the contributions here is the minor cycling has improved connectivity uh both structurally and functionally but we were not able to preserve synaptic efficacy or restore the stretch reflex so just to cut in on a positive note you know the next step is going

To be looking at using some sort of movement therapy or neurotrophic support that both of which has been shown to improve the stretch reflex so with that uh and and just want to say thank you to the organizers especially monica it’s really been fantastic so thank you great talk travis are there any questions

Transcribed from video
Minocycline treatment rescues IAsynapse structure but not function following peripheral nerve injury By Cope Lab