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Characterization of Identified Dopaminergic Neurons in the Mouse Forebrain and Midbrain

Maggy Yu Hei Lau, Sana Gadiwalla , Susan Jones, Elisa Galliano

Posted on: 4 December 2023 , updated on: 8 December 2023

Preprint posted on 31 August 2023

Are all dopaminergic neurons the same? Lau and colleagues played a round of Spot the Difference with dopaminergic neurons in different brain regions and found two functionally defined groups.

Selected by Ana Dorrego-Rivas, Emily Winson-Bushby

Categories: neuroscience

preLight authors: Emily Winson-Bushby, Haoming You and Ana Dorrego-Rivas

 

Background

Historically, neurons have been classified into different cell types based on their gross features, including their morphology, location and biomarker expression. However, with the emergence and employment of modern technologies for study of the nervous system, there is accumulating evidence to suggest that what we usually think of as a “classical, single type” of neuron, is in fact a highly heterogeneous group of neurons, containing functionally different cells. An example of within-cell-type functional heterogeneity is seen in the population of dopaminergic (DA) neurons, which are found in midbrain structures such as the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA), as well as in forebrain structures, including in the glomerular layer of the olfactory bulb (OB).

DA neurons located in different parts of the brain are involved in diverse brain functions such as action selection, reward processing and olfactory perception. In addition, DA neurons in some of these regions have been implicated in various brain functions and pathologies, including the DA neurons of the SNC, which degenerate in Parkinson’s Disease. Although forebrain DA neurons have been presented as a potential candidate for cellular replacement in pathologies with loss of DA neurons in the midbrain (Cave et al., 2014), a functional comparison of the electrophysiological properties of forebrain and midbrain dopaminergic neurons has not yet been performed.

In this study, Lau et al. (2023) leverage the transgenic DAT-tdTomato (DAT-tdT) mouse, in which DA neurons are fluorescently labelled, and combine immunohistochemistry and whole-cell patch-clamp recordings to understand and compare the electrophysiological heterogeneity of DA neurons in the forebrain and midbrain.

 

Key findings of the study

Forebrain and midbrain dopaminergic neurons show molecular and anatomical differences.

In the first step of this study, the authors assessed the expression profile of DAT in the DAT-tdT mouse as compared with tyrosine hydroxylase (TH), a well-established marker for dopaminergic neurons. They found that (DAT-)tdT and TH staining varied between forebrain and midbrain, with the most colocalisation of tdT and TH expression found in the SNC (~99%), with less in the VTA (~85%) and the least in the OB (~72%). A comparison of DA neuron soma sizes showed that DA neurons in the SNC had the largest soma area, followed by VTA DA cells, with OB DA cells showing the smallest soma area.

 

Forebrain and midbrain neurons differ in specific electrophysiological parameters.

Next, Lau and colleagues assessed the passive electrical properties of DA neurons in the SNC, VTA and OB with whole-cell patch clamp recordings in labelled neurons in the DAT-tdT mouse. They found that the resting membrane potential of DA neurons was similar when comparing cells in the SNC and the VTA. However, consistent with previous findings, anaxonic DA neurons in the OB (OB DA neurons which lack an axon) were more hyperpolarized in resting state than the axon-bearing OB DA neurons. Moreover, because anaxonic neurons had a smaller soma than their axon-bearing counterparts, anaxonic neurons had the smallest capacitance amongst the forebrain and midbrain DA neurons. Lastly, the authors assessed DA neuron membrane resistance (Ri). Although there were no significant differences in Ri i) between VTA and SNC and ii) between midbrain and forebrain DA neurons, anaxonic neurons did have a larger Ri than axon-bearing neurons in the OB.

Having established standard passive membrane properties, the authors used recordings of sag potentials to assay the capability of the DA cells to generate hyperpolarisation-induced depolarisation. Midbrain DA neurons displayed a much stronger sag than forebrain DA neurons overall, and, within the midbrain, SNC neurons showed a stronger sag than VTA neurons. This indicates that midbrain neurons are able to more effectively return to resting membrane potential following hyperpolarisation than OB neurons, suggesting that they may be able to fire spikes at a higher frequency.

Next, the authors found action potential waveforms to be similar between forebrain and midbrain DA neurons, with some within-group differences found between regional subtypes. For example, axon-bearing OB DA cells showed a lower action potential threshold than anaxonic cells, whilst VTA DA cells displayed a greater action potential peak amplitude than SNC DA cells.

Despite these similarities in action potential waveform and properties between midbrain and forebrain DA neurons, repetitive action potential firing was quite different. Under current injections of increased intensity, DA neurons in the forebrain were able to fire more action potentials within the same injection window and displayed a higher firing frequency than midbrain DA neurons. This difference may relate to an increased need for firing limits in the midbrain, where dopamine is co-released with glutamate, meaning high-frequency firing may risk causing excitotoxic damage. In the forebrain, dopamine is co-released with GABA, and thus such limits are possibly not needed.

Forebrain and midbrain dopaminergic neurons represent two functionally defined groups.

Finally, Lau and colleagues performed principal component analysis to better understand the electrophysiological differences between forebrain and midbrain neurons and investigate whether they represent different groups of defined cells. This analysis revealed an evident clustering of the two neuron types, and the segregation found was mostly driven by differences in single and repetitive action potential firing parameters and sag voltage. So, while there are similar electrophysiological features between both populations, when all measurements were considered as a whole, the two neuron types give rise to two functionally distinct groups.

 

Why did we choose this preprint?

Dopaminergic neurons in the mammalian midbrain and forebrain alike have long been considered a homogeneous group of cells unified by their expression of common markers, like DAT and TH. Recent evidence suggests that these cells are rather heterogeneous, in terms of, for example, neurogenesis and morphology. Lau and colleagues show here the first attempt at defining the electrophysiological differences between midbrain and forebrain dopaminergic neurons using a genetic-labelling strategy. The results reveal clear functional differences between dopaminergic neurons in these two brain areas, adding a layer of complexity to our understanding of their overall heterogeneity.

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References

Cave, John, Meng Wang, and Harriet Baker. 2014. ‘Adult Subventricular Zone Neural Stem Cells as a Potential Source of Dopaminergic Replacement Neurons’. Frontiers in Neuroscience 8. https://doi.org/10.3389/fnins.2014.00016.

Galliano, Elisa, Eleonora Franzoni, Marine Breton, Annisa N. Chand, Darren J. Byrne, Venkatesh N. Murthy, and Matthew S. Grubb. 2018. ‘Embryonic and Postnatal Neurogenesis Produce Functionally Distinct Subclasses of Dopaminergic Neuron’. eLife, April. https://doi.org/10.7554/eLife.32373.

Lau, Maggy, Sana Gadiwalla, Susan Jones, and Elisa Galliano. 2023. ‘Characterization of Identified Dopaminergic Neurons in the Mouse Olfactory Bulb and Midbrain’. Preprint. bioRxiv. https://doi.org/10.1101/2023.08.29.554772.

 

 

doi: https://doi.org/10.1242/prelights.36089

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Author's response

The author team shared

Questions to the authors

Q1: From your electrophysiological findings, which classes of ion channels would you speculate might be consistent between midbrain and forebrain, and which would you speculate may be different? Is there any transcriptomic or immunohistochemical evidence of differences in the expression of any of these ion channels?

This is a fascinating question which would require another full study (or possibly more!). There is a substantial body of work done in the midbrain but relatively less in the OB, especially in terms of transcriptomics. We do not want to speculate too much, but if we had to start a new project on this, we would probably give a close look to the various potassium channels responsible for the afterhyperpolarization shape, especially SK.

Q2: Do you think that the fact that midbrain DA neurons co-release glutamate, whilst forebrain DA neurons co-release GABA, is a more functionally significant difference than the differences in repetitive firing properties, sag potentials, and passive properties?

We think that we have just started to scratch the surface of neurotransmitter co-release. We know that OB DA neurons also release GABA, but apparently so do some midbrain DA neurons. Interestingly, they do so by unconventional synthesis and/or by scavenging it from the extracellular space, accumulating it, and then releasing it to inhibit striatal neurons. How interesting – and so not text-book like – is that? We do not know how all this translates to function per se and in relation to intrinsic excitability, but surely we believe that it is a very important topic that needs to be further studied. For anyone interested, we recommend starting with the excellent reviews from the Westbrook and Sabatini’s labs (Vaaga et al 2014 PMID: 24816154; Wallace et al 2023 PMID: 37463580).

Q3: In your paper, Galliano et al. 2018, you showed that there are clear differences in the dendritic arborisations of axon-bearing and anaxonic OB DA cells. To your knowledge, does a comparison between the dendritic and axonal arborisations of forebrain and midbrain DA neurons exist, and why did you choose not to pursue this question in this study?

As always with the midbrain there is so much work out there! Some of our favourite comes from the labs of Paul Bolam, Pablo Henny and Jochen Roeper, and we would recommend reading Montero et al 2021 PMID: 3486721. It’s a big topic, but in summary yes, we would expect different dendritic arborizations between OB and midbrain, and quite a lot of variability within the midbrain. And of course, axonal arborization is widely different, especially between the SNC and anaxonic DA bulbar neurons!

Q4: In your study the resting membrane potential of the axon-bearing and anaxonic OB DA cells were very depolarised, but in your older study Galliano et al., 2018, you showed that resting membrane potential of anaxonic and axon-bearing cells were -78 mV and –74 mV, respectively. What do you think is the reason for such a large difference?

We were quite surprised by this, but of course changing the setup (including temperature controller!) could have a large impact on all these properties. Overall, we are confident of both sets of data because in either instance we were comparing parameters across different cells which were acquired using the exact same experimental conditions. What is the “real” resting membrane potential of a DA neuron? Probably only careful in vivo work in awake animals can answer this question by providing the real ground truth.

Q5: Your experiments assaying intrinsic properties, ie passive, single action potential and repetitive firing properties were done in the absence of synaptic blockers. Do you think it’s likely that spontaneous neurotransmitter release in your slices may have been influencing the responsivity of DA cells to electrical stimulation via the patch pipette? If yes, how should your results be interpreted?

Absolutely! In fact, we are planning a follow-up with a full pharmacological sequential blockade of excitatory and inhibitory synapses to see how the firing changes. Is any of you willing to come do the experiments?

Q6: Hypothetically speaking, and momentarily disregarding the actual results, what would have been the most interesting set of results to obtain from these experiments, for you? Were you surprised by any of the results of the experiments you performed?

We would have loved to find that anaxonic OB cells, the regenerating ones, fire in a similar manner to SNC DAs, because this could have opened up windows into potential transplants in the striatum of Parkinsonian animals. Unfortunately we found the opposite. The most surprising result was realising that axon-bearing bulbar DA cells are more similar to the anaxonic counterparts than to axon-bearing midbrain DA neurons. Totally unexpected, yet it clearly indicates how different the two groups are.

Q7: How do you hope the characterisation of cellular properties you’ve presented in this study will be useful to and built upon by other researchers in the field?

We hope that our work will contribute to the growing body of evidence showing that a “standard” dopaminergic neuron does not exist – instead there are many diverse flavours of dopaminergic cells. And of course this can be generalised to all and any cell types.

 

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