The Neurosurgery — Neuroanatomy Research Axis: Future Perspectives to Study Synaptic Transmission Free

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- Colleagues and friends, thank you for joining us for another session of the virtual operating room. My name is Aaron Cohen. We have a special guest today, Dr. Maximilian Lenz. He's a professor and chair of Neuroanatomy at Hanover Medical School. Tremendous knowledge of what makes our job lobby easier as neurosurgeons to have great knowledge of what we're working with in the operating room regarding the anatomy every day. So Max, I wanna really thank you for being with us, and very much looking forward to learning from you. So please go ahead.

- Okay, so thank you very much for the introduction. It's a great pleasure for me to be here and give this lecture today, and thereby contribute to Neurosurgical Atlas. So it's a great opportunity, so thank you very much again for the invitation. So dear colleagues, what I would like to share with you in this lecture is a perspective surgery neuroanatomy research access that allows us to study the synaptic transmission in the human brain, and therefore gain a better understanding on how the human brain actually works. So this year is the organ that we are basically interested in, the human brain, and one of its most amazing feature is the ability for structural functional and molecular adaptations upon specific stimuli at individual contact sites of neurons. And this is summarized by the term of synaptic plasticity. So synaptic plasticity is relevant for very basic daily functions such as temporal and spatial orientation, learning, and then also the implementation of complex behavior. And therefore synaptic plasticity has been under intense investigation over the last decades in various animal and cell culture models. But nevertheless, it remains unclear how actually synaptic plasticity works in the human brain, and therefore this aspect where the investigation if we want to understand how the human brain actually works. And from this point on the major research question that we have arises, and this is the question that is guiding me today in this talk. How can we investigate synaptic transmission and plasticity in the adult human neocortex? And this led me to the agenda of today which I would like to share with you. So in the first part I would like to talk about synaptic transmission and plasticity in general to give you a quick overview on what we are actually talking about, what different forms of plasticity we know in the human brain, and how this helps us to balance our sense to stay in a physiological range. Then we come to the neurosurgery and neuroanatomy research axis, and I would like to introduce you to a strategy how we can study synaptic transmission in the human neocortex at the single cell level. And this just works with the synergies that we can create operative efforts. And from this on I would like to share with you some recent data and future perspectives on what we can do with these strategies, and there I show you some data on the effects of anti-epileptic drugs, and also retinoids in the human brain. Now when we talk about synapsis here and this is a synapse type that I'm basically talking about today, this is the electrochemical synapse. So a contact sides between two neurons in the central nervous system, while we have actually irrespective of it is an inhibitory on excitatory synapse where we have a common architecture with different components. We have a presynapse, we have a postsynapse and this is divided by a synaptic left. When an action potential is coming in it releases neurotransmitters from the presynaptic side and the neurotransmitters here come to the synaptic cleft, diffuses to the other postsynaptic side, and then binds to neurotransmitter receptors, and thereby elicits post-synaptic D or hyperpolarization. The synaptic cleft here is not just an empty space, so here we can have cellular interactions that are shaping further neurotransmission. For example, the astrocytic N feed that can take up neurotransmitters here, they regulate the diffusion of the neurotransmitter to the postsynaptic side. And what is not quite important is that this synapse, this connection side between individual neurons is not a static thing. It's highly flexible and can adapt to the environment. And this term has been called synaptic plasticity. And what we understand by this is a stimulus driven and long-lasting structural, functional, and molecular adaptation of these contact sites. So neuroplasticity has been actually defined in its word by two people, and Jerzy Konorski has said that, "Neuroplasticity is actually a permanent transformation arising in particular systems of neurons as a result of appropriate stimuli, and this shall then be called plasticity." Later on rules have been identified that can lead to plastic changes, and one of the most famous ones is Hebbs theory that, "Neurons that fire together should also wire together," which is a simplified version of a longer mechanistic description. And this theory has then been actually proven by similar work by Bliss and Lomo in 1973, where from the so-called long-term potentiation of the perforant path. So when you think about this kind of Hebbien type of synaptic plasticity, we here have a classic positive feedback loop in a biological system, where stronger connections between nerve cells are getting stronger and weakens are getting weaker. And the result of this will be that synaptic plasticity can also change neural network activity that might also exceed physiological levels. And therefore we need another mechanism that is keeping neural network activity within a dynamic, and this is a physiological range. And one of the concepts that have been established in the last years is the concept of homeostatic synaptic plasticity. And this concept of homeostatic synaptic plasticity aims at keeping the activity of individual neurons within a dynamic range upon perturbations of network activity, and therefore uses now a negative feedback mechanism. So what does that mean, when for example neural network activity drops down as a consequence of a pharmacological inhibition of network activity by a pharmacological intervention with anti-epileptic drugs for example, that is frequently seen in your surgical patients, synaptic strength is changed in a compensatory way that is now rebalancing the system, and to wall and reestablishing a homeostatic state. So how can we now study the concept of homeostatic synaptic plasticity? And here I now do a quick introduction into classic concepts on how we can study this. So this here is actually work that is used to study homeostatic synaptic plasticity. Here for example, tissue cultures can be used in the modulation of neural activity. And this neural activity then is, for example, changed by the application of pharmacological inhibitors of voltage-gated sodium channels such as tetrodatoxin. And this tetrodatoxin is then reducing network activity in its forcing a system to compensate for this change in network activity. And we can now for example study this by whole cell patch clamp recordings in the electrophysiology that allows us to study synaptic transmission on excitatory and inhibitory synapse. And in classical experiments we can also assess this in different cell types, for example, dentate gyrus cells, and CA1 pyramidal cells of organotypic tissue cultures of the enteral hippocampal complex. And if we challenge the system by a TTX treatment we see actually a compensatory excitatory synaptic strengthening. Furthermore, this excitatory synaptic strengthening in both cell types, so dentate gyrus cells, and CA1 pyramidal neurons is then a component but changes in the gene expression which can be analyzed by a transcriptome analysis. In this transcriptome analysis we actually find that especially those genes are changed in their expression, which can be dedicated to distinct synaptic compartments, such as presynapse synaptic vesicles, and also to the postsynapse compartment. So if you can conclude now this, you can say actually that activity deprivation causes homeostatics structural, functional and molecular adaptations of synapsis in animal research models. But the question is still open that I posed in the beginning and this is, is this relevant for the human brain, and how can we investigate this? And to investigate this, we teamed up with the neurosurgeons and established a workflow that allows us to study synaptic transmission in the adult human neocortex. For this we are using surgical access material from tumor and epilepsy patients after informed consent, where a little, in those patients, a little piece of the cortex has to be removed from procedural or medical indications, for example, as in this patient here, to ensure a proper tumor resection. These little pieces of cortex, and you can see here a dissection of one of these little pieces, these are then further processed and we prepare acute slice preparations along the pia white matter axis which allows us to maintain the cortical lamination but also preserve the cellular architecture of highly complex cells such as neurons. And now with the basic research experiments that I just have shown you a few slides ago, we can now match this with the medical record of our patients. So how are we preparing these slices? We here have usually tissue blocks from the cortical resections that are five to seven millimeters big. And then we prepare these slice preparations along the pia white matter axis, and the slice thickness is usually 300 to 400 micrometers. These sections then have here neurons in it that are still living and their architecture is preserved. And now we can study different neuron types in the various layers of the human layer cortex. We have now optimized the the surgical procedures as well that allows us to take optimal material to also do or to answer our research questions. And this has all be summarized in a technical report that we have just published a few months ago. We can not just use very small tissue samples but also bigger tissue samples, for example, from patients that require a bigger resection. And here we can do ex situ tissue resections that we then are further processing. And here you see a typical size of the tissues that are then sliced to obtain good quality material for our basic research experiments. And here you see these little tissue samples where we do our sections from this side, and now we are visualizing, for example, the cortical lamination, or the cellular architecture. And here you see how this looks like in our validation experiment when we have assessed if the cortical lamination is really preserved. For example, we can stain these tissue sections with an antibody against a neuronal marker. And this is NeuN in this case, and you can nicely see the cellular architecture when you stain four neurons in these sections. What you can also do is you can visualize individual neurons that you have just electrophysiological assessed and filled with a dye that can be later on visualized. And here you see that the cellular architecture of this superficial parameter neuron, for example is preserved, and allows us also to study the morphology of individual cells in these slice preparations. What can we then do as a summary? We can do electrophysiological recordings, we can do, for example, here intrinsic membrane properties, when we look at input resistances and action potential firing properties. We can now look at individual synaptic events that we have from these superficial neurons, where one of these individual events here reflects an action at an individual synapse. Furthermore, we can analyze the dendritic trees and even here the morphological correlates of synapses. As you can see here in this end right here you see these little protrusions, the dendritic spines where one of these spines reflects an excitatory synapse. So when you look at this picture what we can definitely do with that still living tissue is the electrophysiology and the post-hoc light microscopy from the very same neuron, and this allows us to correlate synaptic structure and function. Since the tissue is still preserved in its ultra structure, we can also do electro microscopy analysis. And here in this electro microscopy images you can now study the ultra structural composition of the synapsis. You can analyze the role of distinct organelles that you can visualize by electronic microscopy. For example here a stacked and a plasmic reticulum that is located near synaptic sites, and these here is the so-called spine apparatus organ. So in summary, we can actually use the neurosurgery neuroanatomy research axis with the collaborative effort to match all the medical records with the basic research findings, and identify now factors that might have an influence on excitatory neurotransmission and therefore induce synaptic plasticity. And one of the first questions that we have answered with this approach was the question, if maybe anti-epileptic medication is changing excitatory neurotransmission in the adult human neocortex. And thereby we are mimicking experiments that have been done already in cell cultures and also in vivo to induce homeostatic synaptic plasticity. And this would actually be the first time that homeostatic synaptic plasticity can be demonstrated in the adult human neocortex. So as already announced, the anti-epileptic drugs induce homeostatic synaptic plasticity in animal modes, when we have a very standardized and controlled environment, where we give anti-epileptic drugs to adult mice, we see that an administration of the anti-epileptic drug, lamotrigine, is inducing plasticity at excitatory synapsis as measured here in spontaneous excitatory post-synaptic currents on the level of the sEPSC frequency. And now we want to know whether also this is true in patients that receive an anti-epileptic medication. So what we did is we looked in the medical records of the samples that we had recorded, and now defined two subgroups where we had actually patients that received an anti-epileptic medication and those who have not received an antiepileptic medication, which was then defined as our control group. We now performed all cell patch clamp recordings in superficial parameter neurons of the adult human neocortex. And here we recorded excitatory postsynaptic currents, and one of these events that you can just see here is reflecting the action of an excitatory synapse. And indeed we found that associated with the anti-epileptic treatment there was a strengthening in excitatory neuro transmission, which can be seen as an increase in the sEPSC amplitude half worth, and also frequency in those samples from these patients. This is also actually the first experimental evidence that indeed there is a type of homeostatic synaptic plasticity happening in the adult human neocortex. Now we also looked at the structural level and analyzed the neurons that we have just electrophysiological assessed. And here you can see one of these post hoc visualized neurons. And here we looked at the morphological correlate of excitatory synapsis, the dendritic spines, and compared the two groups, the untreated samples, and the samples from patients that have received anti-epileptic drugs. And here again we see that the spine head size which is some kind of a correlate of the excitatory synaptic strength was increased in the samples from patients that have received the anti-epileptic medication. Furthermore, you see that this increase did not happen in all parts of the neurons, but just very localized in the epical dendritic segment but was absent in basal dendrites. So we can conclude from these findings that indeed anti-epileptic treatment is changing excitatory neurotransmission and recruits coordinated adaptations of synaptic structure and function. This actually also means that here we have maybe a biological explanation for some effects of anti-epileptic drugs, like neuropsychiatry effects, or that might give us an explanation why we see recurrent seizures after an anti-epileptic medication is discontinued. Furthermore, we also looked at the molecular biology level. And in the molecular biology, we again performed a transcriptome analysis of a cortico samples from patients that received the medication or have not the medication in their medical records. And again, we found a similar change as we have seen in our other research models from cell cultures and adult animals, where we see a change in gene expression, basically from those genes that can be dedicated to synaptic compartments as well. So taking together we can say that indeed the anti-epileptic medication induces also transcriptomic changes, and this comes along with these strengthening of excitatory neurotransmission in superficial parameter neurons of the adult human neocortex. So this is a good example of how we can actually match the medical records with our basic research findings. But it leaves one question open, and this is the question that was actually said as a prerequisite that we can study synaptic plasticity. And this is now the question, can we also test with these samples distinct stimulus driven changes in neurotransmission? So where we define an exact stimulus across patients that is very standardized, and that we can actually then study plasticity later on with. And here in this in this question we have used the strategy of an ex situ stimulus application with the slices, and a subsequent assessment of neuro transmission in our neocortical sections. So how do we do this? We perform the resection, we perform the slice preparations, and then ex situ we apply a distinct stimulus that is very well defined in controlled conditions, and this stimulus application can last up to 12 hours since this timeframe is suitable to maintain a good quality of the slices. And later on we do the experimental assessment which is in our case the electrophysiology, the microscopy, and also the molecular biology assessment that I've just introduced to you. And as a first stimulus that we apply here we have used the treatment with All-trans retinoic acid which has been well-defined as a stimulus for synaptic plasticity induction. So to give you a brief overview of retinoic acid and vitamin A metabolites, I can just say that vitamin A metabolites play an important role in mediating crucial body functions in various organisms, where it plays a role in development, growth, and also vision. And among these metabolites, all-trans retinoic acid can be also used in clinical practice for the treatment of severe acne and promyelocyte leukemia. All-trans retinoic acid has also been, as I just said, linked to synaptic plasticity and the physiological and pathological conditions, and therefore it was also discussed as a potential treatment option for neurological diseases that come along with changes in the ability of a neural network to express synaptic plasticity, such as Alzheimer's disease. And therefore we now study the effects of an all-trans retinoic acid treatment on our human neocortical sections ex situ. And what came out of this testing, first of all, we did a functional assessment of all-trans retinoic acid mediated plasticity ex situ. And here we found that after the all-trans retinoic acid exposure there were no change in basic membrane properties of these neurons. But what we found is that indeed there was a significant strengthening of excitatory neurotransmission on the level of the sEPSC amplitude in those slices that have been exposed to all-trans retinoic acid. One major advantage of these ex situ stimulus application is that we can also test now the very same stimulus across individuals. And here in this study we have included seven neurosurgical resections, and in all of them you can see that all-trans retinoic acid indeed is increasing excitatory postsynaptic strength. And this actually means that this is a phenomenon that is valid, or that is present across individuals, and therefore seems to be evolutionary conserved. Again, we also looked at the structural level and here we looked at, specifically again at the properties of post-synaptic morphological correlates of excitatory synapses, and these are again dendritic spine features. And indeed all-trans retinoic acid was able to increase the spine head size, which is a sign for a coordinated adaptation of structure and function at individual synapsis in the human brain. With these sections we can also go one level further to the subcellular level. When we look at now individual organelles, and specifically in this study that we have just published, we looked at synaptopodin and the spine apparatus organelle. So synaptopodin can be linked to synaptic plasticity, and to both types, Hebbian and homeostatic synaptic plasticity. And it sits when you do an immuno stand here at very strategic locations near synaptic sites in the spine heads and necks. When you now do an all-trans retinoic acid treatment, again, we see that there was a significant change in the cluster sizes of immunostain synaptopodin clusters, and also a change in the spine head sizes again but irrespective on the presence of synaptopodin. Since this spine apparatus organelle is something that you can visualize by electro microscopy, you can also see here how we can study morphological changes on the subcellular level by the in situ application. And what you can see after the all-trans retinoic acid treatment is that there was a significant increase in the spine apparatus size that we have just analyzed here, as also a correlate of the strengthening of excitatory synapsis. So if you summarize this, indeed all-trans retinoic acid treatment recruits coordinated structural and function adaptations which is also something that has been validated in animal and cell country models. So to summarize, this is what I have shown you in this talk is that we are indeed able to investigate the synaptic structure and function in translational research approaches at the axis of neurosurgery and neuroanatomy where we can create the synergies to find out how synaptic transmission in the human brain and synaptic plasticity is working. We start usually with tissue culture preparations where we identify mechanisms, we validate them in vivo, and then test how this is working in the human neocortex. And furthermore, these findings help us to develop research models that allow us to obtain findings with a certain relevance for the human neocortex, which helps to better understand what is happening in our brain, and how can we tackle crucial diseases that we would like to find a cure for. So with this, I'm at the end of my lecture and I would like to take the opportunity to thank all the people involved in this, especially in the labs where this procedure have been established in my time at the University of Freiburg, especially in the neuroanatomy headed by Andreas Vlachos, together with our great neurosurgeons at the University of Freiburg, Jurgeb Beck and Jakob Strahle. And now my team moved to Hanover Medical School which is a great environment for translational research and is providing an excellent scientific landscape, where we also have now teamed up with the neurosurgeons and also with the neuropathologists to also further develop this translational research approach. And with this, I would like to thank you all for your attention.

- Max, I wanna thank you for your great lecture talking about this synaptic transmission and the updates. You talked fairly about plasticity, which obviously as neurosurgeon so much we depend on to allow the patient to recover. There was something I wanted to ask you. You know we remove a glioma close to the mortal cortex or speech areas, and the patient has some deficits after surgery, they recover nicely, and then we come back for a recurrence a few years later, and we find out that the function has actually significantly moved from where we thought it used to be. How do you see your knowledge here relates to the finding we have in the operating room?

- Yeah, I think that's a very great question, and something that actually shows us how important it is to study synaptic plasticity because the plasticity of the network is what is enabling a reorganization of exactly, which is leading to the phenomenon that you have just mentioned. I think the cortical reorganization is something that is enabling our work in neuroanatomy but also neurosurgery. And it is very important since there are differences between the animal brain and between the human brain to understand what is going on there, and how we can maybe improve the recovery process, and also improve rehabilitation after, for example, neurosurgical resections of parts of the brain.

- That's very helpful. So with that, I want to thank you for your lecture, something that as neurosurgeons will now learn more about. And wish you all the best, and hope to have you with us. Again.

- Thank you very much, and thank you very much again for your invitation. It is a pleasure for me.

- You are welcome. Thank you. That's the official closure then. Max thank you so much for your time. Wish you great evening and we'll be in touch.

- Yeah, thank you very much, and wish you a great evening as well.

- Same here, take care.

- Take care, bye

- Bye.

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