Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes

Dense microcircuit reconstruction techniques have begun to provide ultrafine insight into the architecture of small-scale networks. However, identifying the totality of cells belonging to such neuronal modules, the “inputs” and “outputs,” remains a major challenge. Here, we present the development of nanoengineered electroporation microelectrodes (NEMs) for comprehensive manipulation of a substantial volume of neuronal tissue. Combining finite element modeling and focused ion beam milling, NEMs permit substantially higher stimulation intensities compared to conventional glass capillaries, allowing for larger volumes configurable to the geometry of the target circuit. We apply NEMs to achieve near-complete labeling of the neuronal network associated with a genetically identified olfactory glomerulus. This allows us to detect sparse higher-order features of the wiring architecture that are inaccessible to statistical labeling approaches. Thus, NEM labeling provides crucial complementary information to dense circuit reconstruction techniques. Relying solely on targeting an electrode to the region of interest and passive biophysical properties largely common across cell types, this can easily be employed anywhere in the CNS.

The released current is more evenly distributed between the holes at shallower angles. Accordingly, the current density coefficient of distant holes to the tip is much smaller for shallower angles.

Supplementary Figure 12: Two-photon visualization of electroporated neurons in vivo
Two-photon projection of the MOR174-9-GFP glomerulus labeled by local electroporation of Alexa594 hydrazide. Scale bar = 50 μm.

The Total Number of Neurons per Glomerulus:
While neuron number likely varies significantly among glomeruli, many studies have tried to provide quantitative morphometric descriptions of various elements in the olfactory bulb across species [1][2][3][4][5][6][7][8][9][10][11][12] . However, most of these studies used global quantification methods in which the total population of glomeruli and/or the overall number of certain cell types were estimated.
The composition of the glomerular domain was then calculated as an average ratio of cells per glomerulus. Moreover, cell type identification has only been based on bulbar layer identity of the cells and not on morphological parameters as in our study 7 . More specific quantitative approaches [12][13][14] have only become feasible in recent years with the arrival of targeted electroporation.
Since no systematic assessment of the quantitative extent of this technique has been undertaken to date, the exhaustiveness of the method is unclear but numbers between 7 and 16 MCL cells per glomerulus were reported in these studies. Other cell types have not yet been assessed quantitatively by this or a similarly direct technical approach. Importantly, the total numbers of cells as well as the numbers of MCL cells per glomerulus of the earlier, global estimates and the specific targeted approaches differ substantially, i.e. at least by a factor of two. This difference might be attributable to an incomplete delineation of the population of glomerulus-associated neurons by the targeted electroporation approach, but relevant inter-observer variability in the assessment of global estimates may also play a role.
However, an interesting recent study 6 provided convincing evidence that earlier global estimates in mice must be challenged due to an about twofold higher re-estimation of the total number of glomeruli per bulb compared to earlier studies 1, 2, 4 when using a new, potentially more rigorous approach based on immunohistochemistry that allows for reliable detection of small glomeruli. Such glomeruli had most likely been neglected in earlier studies resulting in a systematic underestimation of the number of glomeruli 1, 2, 4 . Thus, existing global estimates of the number of cells per glomerulus are likely to be inaccurate and a 'gold standard' to achieve a reliable quantitative description of the neuronal elements of the glomerular circuitry does not exist. Additionally, some cell types of the bulb such as short axon cells and other local interneurons do not extend any cellular process into the glomeruli but would likely be contained in average neuron counts.
Taking these limitations into account, we feel confident that the average cell number per MOR174-9 glomerulus of around 200 cells we find in our electroporation data (taking into account a 'missed' number of 20 %) might be a most direct estimate of cell number. This is lower than estimated previously 7 but their reported figure of 441 cells was based on an earlier total number of glomeruli 2 and when adjusted by the most recent count by Richard et al. 6 , their global estimation lies in a similar range (~ 220 cells).
The impact of animal age may also have to be taken into account here: PGs are continuously replaced by adult born neurons in the SVZ 15 . While earlier studies have claimed neuronal stability with a balanced turnover rate of neurons in the GL 16 , it was reported more recently that at least the subpopulation of dopaminergic PGs showed a relatively strong increase with age 17 .
The authors of that study did not explicitly address the question whether this finding was due to a dynamic remodelling of subpopulations within an overall stable total population, or whether this was hinting at an overall growing population. Also, environmental sensory enrichment did enhance proliferation of neuronal precursors in the SVZ already 18 as well as survival of newborn neurons 19 . Therefore, the number of around 200 glomerulus-associated cells in the relatively young animals used here (< 2 months) might increase further with age and olfactory experience.
Taken together, while we acknowledge that a 'ground truth' neuronal number per glomerulus does not exist to date and our independent retest-approach provides only a simplifying