The elaborate axonal and dendritic extensions from neuronal somas provide the anatomical substrate for neuronal connections and the formation of neural networks. The neuronal dendritic field is responsible for the integration of the vast majority of synaptic inputs received by the neuron, playing a large role in determining neuronal activity [1
]. Since the late 19th century, Golgi, Cajal and others have developed techniques to classify and quantitate neuronal structures en masse in the central nervous system [2
The widespread application of Golgi staining methods has provided insights into the morphological characteristics of several neuronal subpopulations. More recently, the development of analytical software to evaluate neuronal properties, combined with newer methodological approaches, including intracellular dye-filling methods, genetic labeling and confocal microscopy [6
] are now being implemented to quantitatively assess structural differences in neuronal subtypes, and to determine the genetic, environmental, behavioural and chemical effects on neuronal morphology.
The recent rise in quantitative analysis of neuronal dendritic length and complexity [9
] has resulted in considerable disparities within the literature. A particularly interesting example is the difference in quantitative measurements of principal neurons in the basolateral amygdala (BLA). Using a modified rapid Golgi–Cox staining method, total dendritic arbor lengths have ranged from 1822 µm for principal neurons in 8–10-week-old Wistar rats [10
], 1338 µm in 9–10-week-old Sprague-Dawley rats [11
] to 2900 µm in 5-week-old Long-Evans rats [12
]. We recently used neurobiotin™ (NB) filling and confocal microscopy to report the mean total dendritic arbor length as 2034 µm for NB-filled principal neurons within the BLA in 8-week-old Wistar rats [8
]. Past studies using similar dye-filling methods have provided estimates for total dendrite length of 6299 to 6722 μm for principal neurons in the lateral amygdala in 17–20-day old Wistar rats [13
], and 7908 μm for BLA principal cells from 8.5-week-old Sprague-Dawley rats [14
]. While this wide range of arbor lengths may be due to age- or strain-related differences, it may also be due to methodological variation.
In this study, we used Golgi–Cox staining and NB-filling to provide a direct quantitative comparison of BLA principal neuron morphology. We traced BLA principal cells of 8-week-old Wistar rats, obtained using both methods, and compared dendritic arbor general morphology, branch order and Sholl [15
] characteristics of BLA principal neurons. We show that the dendrites of BLA principal neurons filled with NB are significantly longer and display greater dendritic complexity, compared to Golgi–Cox-stained neurons. Additionally, we found that spine densities varied significantly in proximal and distal apical dendrites of NB-filled and Golgi–Cox-stained BLA principal neurons. Our data highlights quantitative differences in BLA principal neuron morphology, obtained with two different routinely used methods.
Analysis of neuronal dendritic structure can provide insight into neuronal inputs and function. Varieties of methods have been used to delineate dendritic structures, but it remains unclear how the methods used may contribute to variability in structural quantification. We have addressed this issue by quantifying the morphological properties of BLA principal neurons filled with NB or stained using a modified Golgi–Cox method. We chose to compare quantitative data using both methods, based on previous studies reporting marked quantitative discrepancies for this neuronal type from rats of similar ages. We show that BLA principal neurons filled with NB have significant quantitative differences, compared to Golgi–Cox-stained neurons, including increased apical and basal dendritic length and branching complexity. In addition, we observed marked differences in apical spine density estimates between the two methods.
It is important to note that, even within our current study, the variation in tissue processing and mounting between the two techniques may contribute in part to the observed differences. In particular, Golgi–Cox techniques dehydrate the sample before the sectioning process. Thus, any tissue shrinkage that occurs during processing is consistent in x-
planes. By contrast, with NB-filling, the sectioning takes place before the cell is labelled, and the tissue does not undergo dehydration. During mounting, Golgi–Cox sections are mounted in a solidifying medium, with shrinkage in the z-
plane highly consistent [28
] and minimal stretching/distortion in x-
planes. By contrast, NB-filled techniques require the slice to be mounted in an aqueous mounting media, with higher variability in the z
] and greater potential for stretching/distortion in x
- and y
In recent morphological investigations of BLA principal neurons, total dendritic arbor lengths range from a low of 1338 µm [11
] to a high of 7908 μm [14
] in rats of similar ages (8.5–10-week-old Sprague-Dawley rats). Interestingly, large discrepancies in mean values have been consistently observed in studies that have implemented Golgi–Cox staining or dye-filling methods to quantify BLA principal cell morphology. In line with this, we found that the total dendritic arbor of NB-filled BLA principal neurons was ~2 fold larger than Golgi–Cox-stained neuronal arbors. Our branch order data demonstrated that this was primarily because NB-filling revealed significantly more dendritic branches across second to fifth and greater branch orders, compared to Golgi–Cox staining; this finding was consistent with increased radial Sholl interactions in NB-filled principal neurons. This demonstrates that the method used to delineate dendritic structures significantly influences quantitative measurements of dendrites.
In addition to these methodological differences in BLA principal neuron dendritic morphology, there are also significant disparities between studies using similar dye-filling techniques. Imaging fidelity remains the major limitation in fluorescent analysis, with the finer, more filamentous, dendritic processes captured at higher magnification. Using similar imaging acquisition methods, Ryan et al., 2016 reported a total dendrite length of 7908 μm for BLA principal cells from 8.5-week-old Sprague-Dawley rats filled with biocytin. Additionally, Faber et al., 2001 reported estimates for total dendrite length of 6299 to 6722 μm for principal neurons in the lateral amygdala from 17 to 20-day-old Wistar rats. A combination of species differences and animal age may, in part, contribute to the difference of these estimates, compared with our estimates, and those reported by others. Importantly, these two studies also applied correction factors to account for post-processing tissue shrinkage. Because we did not apply tissue correction factors to our samples, it is to be expected that our total dendrite estimates will be lower, compared to these previous reports. Further investigations are needed to determine the effect of post-processing and the accuracy of correction factors, which vary widely between individual samples [13
], to account for variability in quantitative dendritic morphology resulting from tissue shrinkage. Comparing our current study to expected results from past studies in the BLA from our lab using Golgi–Cox [24
] and NB-filling [8
], indicates that the Golgi–Cox method is less variable than the NB method. Golgi–Cox-stained neurons in this study had ~10% greater total arbor length, ~10% greater basal arbor length and ~15% greater apical arbor length compared with our previous report [24
]. With NB-filling, we observed a two-fold increase in dendrite lengths compared to our past results [8
]. In the current study, the ability to discriminate signal from remarkably small voxel size, in this case, 0.26 µm in x
, compared to 0.62 µm in our previous report [8
], may underlie this difference.
As mentioned previously, the distortion of the brain slice in aqueous mounting media is known to be highly variable [13
], while less tissue shrinkage is observed with Golgi-impregnation [29
]. This might, in part, contribute to the high variation in reports of BLA principal neuron arbor lengths using NB/dye-filling methods by us and others. Of note, there was low variability in the measurements of individual basal tree lengths and maximum terminal reach of apical dendrites between both techniques. Indeed, the ability of dye-filling methods that resolve more filamentous parts of the dendritic tree may aid in the classification of pyramidal cells within different brain regions [8
], including the BLA [13
]. Additionally, it is important to note that these differences may also be predicated on the ability of fluorescent confocal imaging to resolve the NB-filled material with less opacity than brightfield imaging.
We also compared spine densities of NB-filled and Golgi–Cox-stained principal neurons in the BLA. No overall difference in total spine density was found in BLA principal cells from both methods. A more nuanced approach revealed significantly higher spine densities in the apical proximal dendrites of Golgi–Cox-stained neurons compared to NB-filled cells, consistent with branch order analysis findings. A possible explanation for this difference may result from photon detector saturation of brighter proximal processes obliterating the fine spine structures close to the soma and thicker dendrites, which occurs when imaging is optimized for the thinner, less vivid smaller dendritic processes. We also found that the combined distal spine densities (third-order and greater) of NB-filled BLA principal cell apical dendrites were higher, compared to Golgi–Cox-stained cells. This reduction combined with reduced dendritic complexity in high order branch ramifications suggests that NB-filling provides a greater labeling efficiency of distal branch orders of BLA principal neurons compared to Golgi–Cox impregnation. This result might contribute to the consistent reporting of larger dendritic arbors of BLA principal neurons from dye-filling methods compared to Golgi–Cox studies [8
In conclusion, our study has provided a detailed quantitative comparison of BLA principal neuron morphology using NB-filling and modified Golgi–Cox staining. Our morphological analyses have highlighted several quantitative differences with respect to general morphology and branch order characteristics of NB-filled and Golgi–Cox-stained BLA principal cells. Taken together, our findings indicate that NB-labeling provides a higher recovery of filamentous basal trees and the more distal accessory apical branches in BLA principal cells, leading to increased total dendrite arbor lengths and greater dendritic complexity, compared to that seen for Golgi–Cox impregnation. The increased efficacy of dye-filling compared to Golgi–Cox impregnation in distal branches is mirrored in the increased distal spine density of neurons assessed with NB. Importantly, the mean basal tree length and major central projections of the apical tree are preserved using both techniques, suggesting that mean basal tree length and maximal apical terminal lengths are particularly robust measures of BLA principal cell morphology. These results provide insights into differences between NB-filling and Golgi–Cox staining, and highlight methodological considerations contributing to quantitative and regional differences in BLA principal cell morphology reported in the literature. Furthermore, these results, combined with recent advancements in fluorescent microscopy, demonstrate a continued evolution and improvement in identifying and quantifying finer dendritic processes, allowing a more complete quantitative analysis of neuronal morphology.