Characterization of Identified Dopaminergic Neurons in the Mouse Forebrain and Midbrain

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 (R_i). Although there were no significant differences in R_i i) between VTA and SNC and ii) between midbrain and forebrain DA neurons, anaxonic neurons did have a larger R_i 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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