Research Article: Ultrahigh Voltage Electron Microscopy Links Neuroanatomy and Neuroscience/Neuroendocrinology

Date Published: December 8, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Hirotaka Sakamoto, Mitsuhiro Kawata.


The three-dimensional (3D) analysis of anatomical ultrastructures is extremely important in most fields of biological research. Although it is very difficult to perform 3D image analysis on exact serial sets of ultrathin sections, 3D reconstruction from serial ultrathin sections can generally be used to obtain 3D information. However, this technique can only be applied to small areas of a specimen because of technical and physical difficulties. We used ultrahigh voltage electron microscopy (UHVEM) to overcome these difficulties and to study the chemical neuroanatomy of 3D ultrastructures. This methodology, which links UHVEM and light microscopy, is a useful and powerful tool for studying molecular and/or chemical neuroanatomy at the ultrastructural level.

Partial Text

The three-dimensional (3D) analysis of anatomical ultrastructures is extremely important in most fields of biological research. However, it is considerably difficult to perform a 3D image analysis of exact serial sets of ultrathin sections. Although 3D reconstruction from ultrathin sections (~100 nm thickness) has been generally used to obtain 3D information, this technique is applicable only for small specimen areas because of the technical and physical difficulties under the transmission electron microscopy, restricted to approximately 1 mm2 area. On the other hand, due to tremendous development of various techniques in molecular biology (e.g., green fluorescent proteins and their color variants), as well as the development of live imaging techniques, the structure of biological molecules and their functional changes are calculated and visualized in 3D at subnanometer resolution [1, 2]. With the aid of confocal laser scanning microscopy, it is now possible to image and quantify the 3D organization of these cell processes; however, the detailed morphology of the complicated terminal processes of these cells remains obscure because of the insufficient spatial resolution of light microscopy and visualization methods that depend on fluorescence [3–6]. In addition, unstained domains are very difficult to recognize [3]. In contrast, conventional transmission electron microscopy provides extremely detailed and fine structural information, but the images obtained are mostly 2D due to the physical properties of this imaging technique (use of ultrathin sections). Consequently, it is too difficult to relate electron micrographs to the 3D structures of cells.

The gross morphology of neurons and glial cells have been described in detail using light microscopy in combination with various metal impregnation techniques such as Golgi silver staining [10]. The Golgi method is very useful and has been utilized by many neuroanatomists over the past century. Subsequently, the rapid Golgi impregnation procedure, a newly developed method, is also applied in qualitative and quantitative characterization of neuronal morphology analyses both at light and electron microscopic levels [11–14]. We used rapid Golgi impregnation, in combination with UHVEM to visualize neuronal structures at the electron microscopic levels.

Onuf’s nucleus, located in the ventral horn of the sacral spinal cord of many mammals, including humans, is a sexually dimorphic nucleus that innervates the perineal muscles that are involved in sexual behavior. In humans, it is a distinct group of neurons located in the ventral part of the anterior horn of the sacral region of the spinal cord involved in the maintenance of micturition and defecatory continence, as well as muscular contraction during orgasm [18]. The number of neurons in Onuf’s nucleus is greater in males than in females [18–21]. On the other hand, the spinal nucleus of the bulbocavernosus (SNB) of rats, located in the lower lumbar and upper sacral spinal segments, is homologous to Onuf’s nucleus in that it innervates the striated perineal muscles that are attached to the base of the penis [21–23]. The distribution of serotonergic fibers and terminals in this nucleus in rats is also different between the sexes (male dominant) [24–26]. SNB also plays a significant role in male sexual functions in the rat [22, 23, 27, 28]. Male rats have a larger and a greater number of SNB motoneurons than females; this dimorphism results from differences in perinatal androgen signaling through a mechanism mediated by the androgen receptor [22]. On the other hand, we recently reported that a collection of neurons within the upper lumbar spinal cord (L3-L4 level) project axons with gastrin-releasing peptide (GRP) to the lower lumbar spinal cord, controlling male reproductive functions in rats [9, 29, 30]. It has also been reported that the sexually dimorphic distribution of GRP-immunoreactive fibers in the lower lumbar spinal cord is profoundly regulated by circulating androgen levels [31], mirroring changes in SNB motoneuron arborizations and other synaptic populations [23]. However, due to methodological difficulties, no direct evidence has been reported regarding GRP synaptic inputs to the SNB motoneurons. The aim of the current study was to determine the axodendritic synaptic inputs of GRP neurons that project into perineal SNB motoneurons and bulbocavernosus muscles. Immunoelectron microscopy, in combination with a retrograde tracing technique using UHVEM, was employed to visualize the 3D ultrastructures of the central nervous system [8].

Here, we summarize the 3D analysis of neuroanatomy at the ultrastructure level using UHVEM. Both UHVEM stereoscopy and morphometry are useful for elucidating the functions of living matter. These techniques can easily be combined with Golgi impregnation, conventional neurotracing, and/or immunoelectron microscopic methods to reveal the fine details of 3D neuroanatomy. In conclusion, we believe that this mixed methodology, which links UHVEM and light microscopy, is a useful and powerful tool for studying molecular and chemical neuroanatomy at the ultrastructural level.