Application of stereology to the central nervous system: estimation of numerical densities of neurons and synapses or neuron number

ABSTRACT Two unbiased stereological techniques, the disector and the fractionator have been introduced. Synapse-to-neuron ratios were calculated from estimates of the numerical densities of neurons and synapses in the rat cerebral cortex. These estimates were made using the disector method at the light and electron microscopical levels. Neuronal nuclei were as the counting unit for neurons and paramembranous densities for synapses. The fractionator method was employed to estimate the total number of cerebellar Purkinje cells in rats. We employed a five-stage scheme to count the nucleoli of Purkinje cells. The cerebellum was first divided into uniform random slices, which were cut, in turn, into strips. The chosen strips were cut into pieces. A systematic selection of tissues was taken, and embedded together in the same mold. Subsequently the sections sampled were examined with a light microscope. All Purkinje cell nucleoli visible in these sections were counted.     

突触数密度Disector测量法的建立与计算机辅助分析的实现

目的 建立基于Ｄｉｓｅｃｔｏｒ技术的突触数密度定量研究方法;利用Ｖｉｓｕａｌ Ｂａｓｉｃ语言计算机辅助的突触数密度定量分析。方法 应用连续超薄切片技术和航向电镜技术观察并记录位于同一纵轴上的神经毡,选取已知距离的相邻平行电镜照片构成Ｐｈｙｓｉｃａｌ Ｄｉｓｅｃｔｏｒ,依据Ｄｉｓｅｃｔｏｒ原理计算突触的数密度;以Ｖｉｓｕａｌ Ｂａ－ｓｉｃ４．０为开发工具,在Ｗｉｎｄｏｗｓ９５平台上开发基于Ｄｉｓｅｃ     

Synaptic correlates of memory and menopause in the hippocampal dentate gyrus in rhesus monkeys

Aged rhesus monkeys exhibit deficits in hippocampus-dependent memory, similar to aging humans. Here we explored the basis of cognitive decline by first testing young adult and aged monkeys on a standard recognition memory test (delayed nonmatching-to-sample test; DNMS). Next we quantified synaptic density and morphology in the hippocampal dentate gyrus (DG) outer (OML) and inner molecular layer (IML). Consistent with previous findings, aged monkeys were slow to learn DNMS initially, and they performed significantly worse than young subjects when challenged with longer retention intervals. Although OML and IML synaptic parameters failed to differ across the young and aged groups, the density of perforated synapses in the OML was coupled with recognition memory accuracy. Independent of chronological age, monkeys classified on the basis of menses data as peri- or post-menopausal scored worse on DNMS, and displayed lower OML perforated synapse density, than premenopausal monkeys. These results suggest that naturally occurring reproductive senescence potently influences synaptic connectivity in the DG OML, contributing to individual differences in the course of normal cognitive aging.     

雌激素对大鼠红核神经元保护作用的体视学定量

目的 观察雌激素（E₂）替代治疗对去卵巢SD大鼠脊髓损伤后红核神经元逆行性损伤的影响。方法 成年雌性SD大鼠80只, 随机分为正常组、红核脊髓束(RST)损伤组、E2替代治疗组、雌激素受体拮抗剂（ICI）治疗组和E₂+ICI联合治疗组。SD大鼠去势后1周采用选择性切断脊髓C3～C4左侧背外侧索制作单侧红核脊髓束（RST）横断损伤模型,给予不同条件治疗后1周、2周和4周对各组大鼠采用前肢支撑探测实验进行行为学评价,用红色荧光金（FR）逆行荧光示踪及体视学定量分析法,观察红核神经元的形态及数目变化。结果 前肢支撑探测实验结果显示,各时间点E2替代治疗组的左前肢使用率均高于除正常组外其他各组,但无统计学意义（P >0.05）;形态学检测结果可见各组大鼠中脑右侧FR阳性红核神经元数目除正常组外均有不同程度减少,尤以4周时最为明显。E2替代治疗组右侧中脑FR阳性红核神经元与RST损伤组、ICI治疗组和E₂+ICI 联合治疗组相比胞体饱满、轮廓清晰、突起长,体视框计数结果显示,E₂替代治疗组红核FR阳性神经元数目明显多于其余各组（P<0.05）,但RST损伤组、ICI治疗组和E₂+ICI联合治疗组之间右侧中脑FR阳性红核神经元数目未见明显差异（P>0.05）。结论 大鼠脊髓横断损伤后中脑红核神经元发生逆行性损伤,E₂替代治疗可减轻脊髓损伤引发的继发性红核神经元损伤。     

Efficient quantification of afferent cochlear ultrastructure using design-based stereology

The afferent synapse between the auditory nerve fiber and the inner hair cell (IHC) represents a critical junction for hearing. Elucidation of the structure at this site will help establish the substrate for normal sound encoding as well as pathologic processes associated with hearing dysfunction. Previous applications of unbiased (design-based) stereological principles have expanded our knowledge of neuro-morphological changes evident with the light microscope. Applying these principles at the level of the synapse is a promising morphometric approach for the efficient sampling of large reference spaces with electron microscopy. This study tests the accuracy of using ultra-thin sections at a fixed interval, known as disector pairs, to quantify afferent innervation density. We analyzed the total numbers of afferent terminals, synaptic thickenings, and synaptic bodies associated with each IHC in the C57BL/6J mouse cochlea, and confirmed the accuracy of the stereological approach in comparison to three-dimensional reconstructions of serial alternate sections. The higher sampling efficiency of the disector pair method rapidly increases precision while also reducing the largest source of variability, inter-animal differences. We conclude that ultrastructural quantification of afferent innervation can be accomplished in the cochlea using efficient design-based stereology.     

A quantitative analysis of the synaptic development of the lobus parolfactorius of the chick (Gallus domesticus)

Summary The lobus parolfactorius (LPO) of the chick has been shown to undergo an increase in the mean synaptic numerical density (Nv _syn) in response to one-trial passive avoidance learning (Stewart et al. 1987). The present study was undertaken in order to describe the pattern of normal development of synapses in the LPO, to further investigate the significance of this plastic response. The LPO's from each hemisphere of pre-hatch (16 days), and post-hatch (1 day, 9 day and 22 day old) chicks were processed for electron microscopy. Synapses were classified into asymmetric spine, asymmetric shaft, symmetric spine, and symmetric shaft synapses, on the basis of the density of the post-synaptic thickening and the nature of the post-synaptic target. A 3-dimensional stereological probe was used (the disector) to calculate Nv _syp, and mean projected height (H _syp) of the post-synaptic density (PSD). Mean values for each age and hemisphere were compared with a 2-way analysis of variance test using paired samples. A six-fold increase in Nv _syp was seen between 16 days in ovo, and 9 days post-hatch. There was a hemispheric asymmetry at 9 days post-hatch, with the left hemisphere LPO containing 1.6 times as many synapses per m^-3. There was a subsequent period of reduction in synaptic density in the left hemisphere LPO between 9 and 22 days post-hatch. The Nv of all classes of synapse increased with age, but the proportions of the symmetrical synapses with respect to the total number of synapses, decreased with age. This decrease was of a similar magnitude for each hemisphere. A hemispheric difference was seen in post-hatch asymmetric synapses, with a greater proportion of asymmetric spine synapses in the left hemisphere. The magnitude of the hemispheric asymmetry was constant throughout the 3 week period of post-hatch development, but was not present in pre-hatch chicks. The PSD increased in length in each hemisphere by approximately 40% between post-hatch day 1 and post-hatch day 9. These data show that the LPO contains a synaptic population which undergoes substantial modification during the first week post-hatch. An asymmetry exists at post-hatch day 9 which is not present at the earlier ages investigated, nor indeed after 22 days post-hatch. This may have significance with regard to studies of passive avoidance learning in the one-day old chick, where an increase in both the size and number of synapses in the LPO has been demonstrated (Stewart et al. 1987). It is possible that training, in this situation, may simply enhance the timing of synaptic events that result as a consequence of normal development.     

Estimate of size and total number of neurons in superior cervical ganglion of rat, capybara and horse

The superior (cranial) cervical ganglion was investigated by light microscopy in adult rats, capybaras (Hydrochaeris hydrochaeris) and horses. The ganglia were vascularly perfused, embedded in resin and cut into semi-thin sections. An unbiased stereological procedure (disector method) was used to estimate ganglion neuron size, total number of ganglion neurons, neuronal density. The volume of the ganglion was 0.5 mm³ in rats, 226 mm³ in capybaras and 412 mm³ in horses. The total number of neurons per ganglion was 18,800, 1,520,000 and 3,390,000 and the number of neurons per cubic millimetre was 36,700, 7,000 and 8,250 in rats, capybaras and horses, respectively. The average neuronal size (area of the largest sectional profile of a neuron) was 358, 982 and 800 µm², and the percentage of volume occupied by neurons was 33, 21 and 17% in rats, capybaras and horses, respectively. When comparing the three species (average body weight: 200 g, 40 kg and 200 kg), most of the neuronal quantitative parameters change in line with the variation of body weight. However, the average neuronal size in the capybara deviates from this pattern in being larger than that of in the horse. The rat presented great interindividual variability in all the neuronal parameters. From the data in the literature and our new findings in the capybara and horse, we conclude that some correlations exist between average size of neurons and body size and between total number of neurons and body size. However, these correlations are only approximate and are based on averaged parameters for large populations of neurons: they are less likely to be valid if one considers a single quantitative parameter. Several quantitative features of the nervous tissue have to be taken into account together, rather than individually, when evolutionary trends related to size are considered.     

Nerve cell loss in the thalamic mediodorsal nucleus in Huntington’s disease. II. Optimization of a stereological estimation procedure

This study provides the theoretical background of the decision to count approximately 750–1,300 neurons per individual in the preceding study of Heinsen et al. [6] finding a significant (P < 0.05) nerve cell loss in the thalamic mediodorsal nucleus in Huntington’s disease with the so-called V_Ref× N_V method. Using a computer simulation of the study of Heinsen et al., it was shown that the legitimation for counting only 100–200 neurons per individual in previous studies comparable to that carried out by Heinsen et al. was based on incorrect assumptions. In this context it was of particular importance to confirm the theoretical prediction in the literature that the random error of total neuron number estimates obtained with the V_Ref× N_V method is actually greater than assumed in current stereological studies. In summary, this study revives the question of how many individuals need to be investigated and how many neurons (or other cell types, respectively) need to be counted per individual in studies comparable to that carried out by Heinsen et al. Received: 9 September 1997 / Revised: 24 August 1998 / Accepted: 24 October 1998     

大鼠海马生后早期突触定量研究

为研究大鼠海马ＣＡ１ 区生后发育早期突触的变化,对发育过程中突触的可塑性进行分析,采用连续电镜切片观察与体视学技术——Ｄｉｓｅｃｔｏｒ法测量突触总体、穿孔突触及非穿孔突触的数密度并计算穿孔突触的比例。结果显示生后０～３０ ｄ 大鼠海马ＣＡ１ 区中,上述测量项目均随年龄增长而增长,提示在海马ＣＡ１ 区存在生后突触发育阶段     

Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Quantification of the number of myelinated fibers in peripheral nerves is a common requirement in quantitative morphology. This parameter provides important information on the consequences of various physiological, pathological and experimental conditions on the nerve structure and is one of the main indicators of success of peripheral nerve repair. In this paper, the theoretical rationale for the application of stereological principles to obtain unbiased estimates of the density and total number of myelinated fibers in peripheral nerves is discussed and a simple stereological method is described. The method is applied together with a systematic random sampling scheme, that was optimized for the purposes of the present study, and with sampling scheme analysis by calculating the coefficient of error (CE). The stereological method, which consists of a two-dimensional variation of the classical disector procedure (two-dimensional disector), and the sampling scheme are verified by comparing estimates with the true density and total number of myelinated fibers in peripheral nerve trunks where true values have been accurately determined by extensive counting. The verification of the 2-D disector method, both of normal and regenerated nerves, showed that estimates of density and total number of myelinated nerve fibers are unbiased. The method also proved to be efficient (time-saving): Estimation of density and total number of myelinated fibers in a single nerve takes about 2-3 hours.