Estimates Of Neuron Number Research Articles

Peeking Inside the Lizard Brain: Neuron Numbers in Anolis and Its Implications for Cognitive Performance and Vertebrate Brain Evolution.

Studies of vertebrate brain evolution have mainly focused on measures of brain size, particularly relative mass and its allometric scaling across lineages, commonly with the goal of identifying the substrates that underly differences in cognition. However, recent studies on birds and mammals have demonstrated that brain size is an imperfect proxy for neuronal parameters that underly function, such as the number of neurons that make up a given brain region. Here we present estimates of neuron numbers and density in two species of lizard, Anolis cristatellus and A. evermanni, representing the first such data from squamate species, and explore its implications for differences in cognitive performance and vertebrate brain evolution. The isotropic fractionator protocol outlined in this article is optimized for the unique challenges that arise when using this technique with lineages having nucleated erythrocytes and relatively small brains. The number and density of neurons and other cells we find in Anolis for the telencephalon, cerebellum, and the rest of the brain (ROB) follow similar patterns as published data from other vertebrate species. Anolis cristatellus and A. evermanni exhibited differences in their performance in a motor task frequently used to evaluate behavioral flexibility, which was not mirrored by differences in the number, density, or proportion of neurons in either the cerebellum, telencephalon, or ROB. However, the brain of A. evermanni had a significantly higher number of nonneurons and a higher nonneuron to neuron ratio across the whole brain, which could contribute to the observed differences in problem solving between A. cristatellus and A. evermanni. Although limited to two species, our findings suggest that neuron number and density in lizard brains scale similarly to endothermic vertebrates in contrast to the differences observed in brain to body mass relationships. Data from a wider range of species are necessary before we can fully understand vertebrate brain evolution at the neuronal level.

Postnatal development of the entorhinal cortex: A stereological study in macaque monkeys.

The entorhinal cortex is the main gateway for interactions between the neocortex and the hippocampus. Distinct regions, layers, and cells of the hippocampal formation exhibit different profiles of structural and molecular maturation during postnatal development. Here, we provide estimates of neuron number, neuronal soma size, and volume of the different layers and subdivisions of the monkey entorhinal cortex (Eo, Er, Elr, Ei, Elc, Ec, Ecl) during postnatal development. We found different developmental changes in neuronal soma size and volume of distinct layers in different subdivisions, but no changes in neuron number. Layers I and II developed early in most subdivisions. Layer III exhibited early maturation in Ec and Ecl, a two-step/early maturation in Ei and a late maturation in Er. Layers V and VI exhibited an early maturation in Ec and Ecl, a two-step and early maturation in Ei, and a late maturation in Er. Neuronal soma size increased transiently at 6 months of age and decreased thereafter to reach adult size, except in Layer II of Ei, and Layers II and III of Ec and Ecl. These findings support the theory that different hippocampal circuits exhibit distinct developmental profiles, which may subserve the emergence of different hippocampus-dependent memory processes. We discuss how the early maturation of the caudal entorhinal cortex may contribute to path integration and basic allocentric spatial processing, whereas the late maturation of the rostral entorhinal cortex may contribute to the increased precision of allocentric spatial representations and the temporal integration of individual items into episodic memories.

Implementation of deep neural networks to count dopamine neurons in substantia nigra.

Unbiased estimates of neuron numbers within substantia nigra are crucial for experimental Parkinson's disease models and gene‐function studies. Unbiased stereological counting techniques with optical fractionation are successfully implemented, but are extremely laborious and time‐consuming. The development of neural networks and deep learning has opened a new way to teach computers to count neurons. Implementation of a programming paradigm enables a computer to learn from the data and development of an automated cell counting method. The advantages of computerized counting are reproducibility, elimination of human error and fast high‐capacity analysis. We implemented whole‐slide digital imaging and deep convolutional neural networks (CNN) to count substantia nigra dopamine neurons. We compared the results of the developed method against independent manual counting by human observers and validated the CNN algorithm against previously published data in rats and mice, where tyrosine hydroxylase (TH)‐immunoreactive neurons were counted using unbiased stereology. The developed CNN algorithm and fully cloud‐embedded Aiforia™ platform provide robust and fast analysis of dopamine neurons in rat and mouse substantia nigra.

Stereological analysis of the rhesus monkey entorhinal cortex.

The entorhinal cortex is a prominent structure of the medial temporal lobe, which plays a pivotal role in the interaction between the neocortex and the hippocampal formation in support of declarative and spatial memory functions. We implemented design-based stereological techniques to provide estimates of neuron numbers, neuronal soma size, and volume of different layers and subdivisions of the entorhinal cortex in adult rhesus monkeys (Macaca mulatta; 5-9 years of age). These data corroborate the structural differences between different subdivisions of the entorhinal cortex, which were shown in previous connectional and cytoarchitectonic studies. In particular, differences in the number of neurons contributing to distinct afferent and efferent hippocampal pathways suggest not only that different types of information may be more or less segregated between caudal and rostral subdivisions, but also, and perhaps most importantly, that the nature of the interaction between the entorhinal cortex and the rest of the hippocampal formation may vary between different subdivisions. We compare our quantitative data in monkeys with previously published stereological data for the rat and human, in order to provide a perspective on the relative development and structural organization of the main subdivisions of the entorhinal cortex in two model organisms widely used to decipher the basic functional principles of the human medial temporal lobe memory system. Altogether, these data provide fundamental information on the number of functional units that comprise the entorhinal-hippocampal circuits and should be considered in order to build realistic models of the medial temporal lobe memory system.

Comparison of estimates of neuronal number obtained using the isotropic fractionator method and unbiased stereology in day old chicks (Gallus domesticus)

BackgroundThe relative size and neuronal density of brain regions are important metrics in both comparative and experimental studies in neuroscience. Consequently, it is imperative to have accurate, reliable and reproducible methods of quantifying cell number. New methodThe isotropic fractionator (IF) method estimates the number of neurons and non-neurons in the central nervous system by homogenizing tissue into discrete nuclei and determining the proportion of neurons from non-neurons using immunohistochemistry (Herculano- Herculano-Houzel and Lent, 2005). Comparison with existing methodOne of the advantages of IF is that it is considerably faster than stereology. However, as the method is relatively new, concerns about its accuracy remain, particularly whether homogenization results in underestimation of cell number. In this study, we compared estimates of neuronal number in the telencephalon and ‘rest of brain’ (i.e. the diencephalon and brainstem excluding the optic lobes) of day old chicks using the IF method and stereology. ResultsIn the telencephalon, there was a significant difference in estimates of neuronal number between the 2 methods, but not estimates of neuronal density (neurons/mg of tissue). Whereas in the ‘rest of brain’, there was a significant difference in estimates of neuronal density, but not neuronal number. In all cases, stereological estimates were lower than those obtained using the IF method. ConclusionDespite the statistically significant differences, there was considerable overlap (all estimates were within 16% of one another) between estimates obtained using the two methods suggesting that the two methods provide comparable estimates of neuronal number in birds.

Reduced neuronal density in the CA1 anterodorsal hippocampus of the mdx mouse

Duchenne muscular dystrophy (DMD) is associated with non-progressive cognitive dysfunction including hippocampal-dependent memory deficits. Loss of the cytoskeleton-associated dystrophin protein in central inhibitory synapses, associated with consequent alterations in GABAergic function and synaptic plasticity, has been proposed as a primary mechanism responsible for cognitive impairments. However, several lines of evidence suggest a multifactorial etiology involving alternative signaling pathways, some of which could affect neuronal survival. To determine whether changes in neuronal density in the hippocampus could contribute to the emergence of memory deficits, we undertook an unbiased stereological estimation of neuron number in the anterodorsal CA1 region of the hippocampus of the dystrophin-deficient mdx mouse model of DMD. We found a significant reduction (~34%) in the number of pyramidal neurons, with a heterogeneous magnitude of genotype differences along the hippocampal antero-posterior axis. This extends previous knowledge of brain morphofunctional alterations induced by dystrophin loss and suggests that putative mechanisms involved in neurogenesis and/or neuron survival might contribute to the emergence of hippocampal-dependent learning and memory deficits in DMD.

Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods

BackgroundNeuN and Nissl staining (toluidine blue, cresyl violet staining) are routinely used methods in unbiased stereological estimation of the total number of hippocampal neurons. New methodIn the present study, we stained serial frozen coronal sections from 5 normal adult male Sprague-Dawley rat brains with different methods, measured the deformation of hippocampal area in brain sections and estimated the total number of hippocampal neurons using the optical fractionator. ResultsThe deformation in x, y-axis was not obviously different with different staining protocols, but shrinkage in z-axis was significant after staining (p<0.001). NeuN staining produced significant higher estimate number than cresyl violet staining by 24% (p=0.002), however, NeuN and Cresyl Violet staining showed a high degree of correlation in quantification of total neuronal numbers and both methods are suitable for unbiased stereological estimation. Comparison with existing method (s)NeuN is more reliable but if time is limited or the number of animals used in experiments is high, cresyl violet staining may be a feasible method. ConclusionsCompared with previous estimates of the neurons number in rat hippocampus, our present data is reliable and the stereological analysis based on our system is a cost-effective unbiased method for estimation of neuron number.

Stereological estimation of neuron number and plaque load in the hippocampal region of a transgenic rat model of Alzheimer's disease.

The main hallmarks of Alzheimer's disease (AD) are senile plaques, neurofibrillary tangles and neuronal death. The McGill-R-Thy1-APP rat is one of the few transgenic rat models of AD that displays progressive amyloid pathology. This study aimed to further characterise this rat model, focusing on the pathological changes in the hippocampal formation and the parahippocampal region. These structures, that are important for episodic memory and spatial navigation, are affected in the early stages of the disease. This study used unbiased stereology to investigate possible neuronal loss in the CA1, subiculum and entorhinal cortex of 18-month-old homozygous McGill-R-Thy1-APP rats, and also quantified the plaque load in all the areas of the hippocampal formation and parahippocampal region from 9 to 18 months old. A significant reduction of neurons at 18 months was only seen in the subiculum. The first plaque pathology was seen at 9 months in the subiculum. Although the quantified plaque load was variable between animals, the pattern of spatiotemporal progression was similar for all animals. The spread of plaque pathology mainly affected anatomically connected regions. Overall, the plaque pathology observed in the transgenic rats was similar to the early phases of amyloid beta (Aβ)-deposition described in human patients. The findings here thus indicate that the McGill-R-Thy1-APP rat could be a good model of the Aβ pathology in AD, but less so with respect to neuron loss.

Cytoarchitecture of the human superior olivary complex: Nuclei of the trapezoid body and posterior tier

The superior olivary complex (SOC) is a cluster of nuclei situated in the caudal brainstem tegmentum that forms an essential component of the auditory pathway. The SOC includes two principal nuclei, the medial and lateral superior olives (MSO and LSO respectively), that have clear roles in sound source localization. Surrounding the principal nuclei are a number of periolivary nuclei (PON) that vary significantly between mammalian species but function in multiple aspects of hearing. Although the PON have been studied in numerous laboratory animals, these nuclei have not been delineated in human. The major goal of this study is to, based on myeloarchitecture, location, neuronal morphology and cytoarchitecture, define the PON within the human SOC and provide estimates of neuronal number within these nuclei. Results from the study of twelve human brainstems provide evidence for six morphologically distinct cell groups: three within the trapezoid body and three along the posterior aspect of the SOC. Based on the analysis of human tissue stained for myelin, Nissl substance, or impregnated with silver, the human PON appear largely homologous to the PON described in other low-frequency hearing animals.

Design-based estimation of neuronal number and individual neuronal volume in the rat hippocampus

Tools recently developed in stereology were employed for unbiased estimation of the neuronal number and volume in three major subdivisions of rat hippocampus (dentate granular, CA1 and CA3 pyramidal layers).The optical fractionator is used extensively in quantitative studies of the hippocampus; however, the classical optical fractionator design may be affected by tissue deformation in the z-axis of the section. In this study, we applied an improved optical fractionator design to estimate total number of neurons on 100μm thick vibratome sections that had been deformed, in the z-dimension, during histological processing.For estimating cell number, it is necessary to randomize only the location of section planes. But, in the local stereological methods, like cell volume estimation, the orientation of sections must also be randomized. We present a detailed application of a method for making vertical sections from the hippocampus. The volume of hippocampal neurons was estimated using the rotator principle on 40μm thick plastic vertical uniform random sections and corrected for tissue shrinkage. Application of the proposed new design should result in more accurate estimates of neuron number and neuronal volumes in tissue sections affected by homogenous non-uniform shrinkage

Cytoarchitecture of the human superior olivary complex: Medial and lateral superior olive

The superior olivary complex is a group of brainstem nuclei involved in hearing and includes the medial superior olive (MSO) and the lateral superior olive (LSO), surrounded by periolivary cell groups. The structure and functional roles of the MSO and LSO have been the subject of many investigations in laboratory animals and it has largely been assumed that these findings are directly transferable to humans. However, little is known conclusively regarding the detailed organization of the human superior olivary complex. The goal of this study is to provide a detailed analysis of the cytoarchitecture of the human MSO and LSO. Results from the examination of eight human brainstems confirm the existence of a conserved MSO and provide evidence of a prominent and highly ordered LSO. Unbiased stereological estimates of neuronal number indicate approximately 15,500 neurons in the MSO and 5600 neurons in the LSO. Additionally, a three-dimensional model of the MSO and LSO was constructed and provides evidence that the human LSO is composed of medial and lateral segments. Finally, an analysis of neuronal morphology, in Nissl stained and Golgi impregnated tissue, provides evidence of multiple neuronal classes within each nucleus and further that these neurons demonstrate a precise geometric arrangement (depending on the nucleus) that is suggestive of isofrequency laminae.

Late differentiation contributes to the apparent increase in sensory neuron number in juvenile rat

Using both profile counts and unbiased stereological methods, estimates of neuron number in the lumbar dorsal root ganglia of the rat have been shown to increases approximately 35% during postnatal life [J. Comp. Neurol. 386 (1997) 8–15; J. Comp. Neurol. 449 (2002) 158–165]. The mechanism underlying this addition of neurons was investigated. No evidence of incorporation of (BrdU), a mitotic marker, was found. Similarly, counts of myelinated and unmyelinated axons in the sural nerve were the same in neonates and adults. These results are not consistent with the possibility that neurogenesis accounts for neuron addition. A population of neurons that stains with TuJ1, an antibody against neuronal class III β tubulin, but not with an antibody against the phosphorylated and non-phosphorylated forms of heavy chain neurofilament protein (NF-H) was found in neonates, but not adults. These less-differentiated (type-L) neurons are not detected by either profile counts or unbiased stereology and do not transport HRP retrogradely. Maturation of this pool of incompletely differentiated neurons appears to be one mechanism whereby neuron number is augmented during postnatal life.

Unbiased stereological estimates of neuron number in subcortical auditory nuclei of the rat

The mammalian auditory system consists of a large number of cell groups, each containing its own complement of neuronal cell types. In recent years, much effort has been devoted to the quantitation of auditory neurons with common morphological, connectional, pharmacological or functional features. However, it is difficult to place these data into the proper quantitative perspective due to our lack of knowledge of the number of neurons contained within each auditory nucleus. To this end, we have employed unbiased stereological methods to estimate neuron number in the cochlear nuclei, superior olivary complex, lateral lemniscus, inferior colliculus and medial geniculate body. Additionally, we generated a three-dimensional model of the superior olivary complex. The utility of unbiased stereological estimates of auditory nuclei is discussed in the context of various experimental paradigms.

Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry

A rapid counting protocol is described which combines the optical disector and Cavalieri methods on non-embedded slices of fixed tissue viewed in the confocal laser scanning microscope. By eliminating the embedding stage and avoiding the need to align adjacent sections in the z plane for counting, considerable time savings are gained over physical disector methods. It also allows the remaining non-embedded sections to be used for other purposes, such as in situ hybridisation and immunocytochemistry. Starting with fixed brainstem, it was possible in less than 2 h to determine the total number of motoneurons in both facial nuclei of an adult Sprague–Dawley rat. This method revealed that the normal facial nucleus contained approximately 3200 motoneurons ( n=12 rats). One month following facial nerve avulsion ( n=4 rats), mean numbers of motoneurons were reduced by 75%. Using intervening sections, changes in neuronal number were compared with changes in in situ hybridisation signal and immunostaining.

A-fiber sensory input induces neuronal cell death in the dorsal horn of the adult rat spinal cord.

Excitotoxicity due to excessive synaptic glutamate release is featured in many neurological conditions in which neuronal death occurs. Whether activation of primary sensory pathways can ever produce sufficient over-activity in secondary sensory neurons in the dorsal horn of the spinal cord to induce cell death, however, has not been determined. In this study, we asked whether activity in myelinated afferents (A fibers), which use glutamate as a transmitter, can induce cell death in the dorsal horn. Using stereological estimates of neuron numbers from electron microscopic sections, we found that stimulation of A-fibers in an intact sciatic nerve at 10 Hz, 20 Hz, and 50 Hz in 10-minute intervals at a stimulus strength that activates both Abeta and Adelta fibers resulted in the loss of 25% of neurons in lamina III, the major site of termination of large Abeta fibers, but not in lamina I, where Adelta fibers terminate. Furthermore, sciatic nerve lesions did not result in detectable neuron loss, but activation of A fibers in a previously sectioned sciatic nerve did cause substantial cell death not only in lamina III but also in laminae I and II. The expansion of the territory of A-fiber afferent-evoked cell death is likely to reflect the sprouting of the fibers into these laminae after peripheral nerve injury. The data show, therefore, that primary afferent A-fiber activity can cause neuronal cell death in the dorsal horn with an anatomical distribution that depends on whether intact or injured fibers are activated. Stimulation-induced cell death potentially may contribute to the development of persistent pain.

Use of cryostat sections from snap-frozen nervous tissue for combining stereological estimates with histological, cellular, or molecular analyses on adjacent sections

Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal histological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochemistry may be problematic with regard to adequate stereological design. In this study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimates of neuron number obtained using the optical disector. Neuronal nuclei and nucleoli were clearly present in thin sections of snap-frozen rat (3 μm) and human (6 μm) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. In order to quantify the potential impact of any nuclear loss, optical fractionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cerebellar granule and Purkinje cells were made using minimal (1 μm) upper guard zones. Estimates did not differ from data reported previously in the literature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers using optical disectors.

Requirements for obtaining unbiased estimates of neuronal numbers in frozen sections

The use of frozen sectioning is a convenient and rapid means of observing the results obtained using fluorescent retrograde tracers. Quantitation of these results using the biased stereological methods currently available can be associated with large errors. A recently developed stereological tool, the optical dissector, provides unbiased and efficient results, however, the requirements for its use in frozen sections has not previously been established. In this study, a comparison was made of neuron numbers, estimated using the optical dissector method, in the motoneuron pool retrogradely labelled from the rat sciatic nerve with either Fast Blue or Tetramethylrhodamine dextran (fluoro-ruby) in methacrylate embedded and frozen spinal cord specimens. Despite over 50% shrinkage in the frozen sections, compared with virtually no shrinkage in the methacrylate sections, no significant difference in labelled motoneuron numbers was observed, provided this shrinkage was taken into account. Correction for section shrinkage is therefore essential in order to use the optical dissector with confidence to count fluorescent labelled neurons in frozen tissue.

Effect of nerve crush on perikaryal number and volume of neurons in adult rat dorsal root ganglion.

Assumption-free stereological methods were applied to assess the effect of nerve crush on perikaryal number and mean volume of neuronal subpopulations in adult rat dorsal root ganglion (DRG). The L5 spinal nerve of 20 Wistar rats was crushed approximately 7 mm distal to the DRG, and the contralateral spinal nerve and DRG were left intact and used as controls. After four, 15, 45, and 120 days, the rats were killed, and the tissue was fixed and processed for subsequent preparation of 30-microm-thick sections. Estimates of neuron number were obtained with the optical fractionator technique and estimates of the mean perikaryal volume with the vertical planar rotator principle. Perikaryal loss was progressive during the early study period but stabilized 45 days after nerve injury. The mean number (n) of all neurons in intact L5 DRG was 16,400 (S.D. = 2,000). The loss of perikarya was 16% (P < 0.05) after four days, 15% (P < 0.05) after 15 days, 30% (P = 0.059) after 45 days, and 34% (P < 0. 05) after 120 days. B cells were lost at an earlier time than were A cells, and the B cell loss was more pronounced (39% vs. 22%, respectively, after 120 days). For A cells, the mean perikaryal volume was initially reduced but was normalized at the end of the study. Distributions of perikaryal volume showed that the curves of both A and B cells were uniformly displaced toward smaller values 15 and 45 days after injury. Neuronal loss caused by crush seems similar to that seen in rats exposed to permanent axotomy (Vestergaard et al. [1997] J Comp Neurol 388:307-312) at the same location, indicating that survival of perikarya is not dependent on possibility for fiber growth.

Transient effects of nerve injury on estimates of sensory neuron number in juvenile bullfrog

The effect of lumbar spinal nerve (SN) transection on estimates of neuron number was investigated in the dorsal root ganglia (DRGs) of juvenile bullfrogs (Rana catesbeiana). SN8 and SN10 were transected on one side, and SN9 was left intact. Two weeks after nerve injury, estimates of neuron number in DRG8 and DRG10 on both the operated and unoperated sides were more than twice the estimates obtained from control animals. Neuron number in the uninjured DRG9 was also elevated relative to that of control animals. Eight weeks after axotomy, differences in neuron number were less apparent. The mean cross-sectional area of DRG neurons was reduced 2 weeks after nerve injury in all DRGs. The decrease in mean area was the result of the addition of neurons to the smallest size classes. These data are discussed in the context of previous results showing that neurons are added as juvenile frogs grow to adult size (St. Wecker and Farel [1994] J. Comp. Neurol. 342:430-438). This addition results from the maturation of a population of incompletely differentiated neurons (Meeker and Farel [1997] J. Comp. Neurol. 389:569-576). The present results suggest that axotomy precipitates differentiation of these incompletely differentiated neurons, perhaps as a compensatory response to nerve injury.

Analysis of cell death in the trochlear nucleus of the chick embryo: Calibration of the optical disector counting method reveals systematic bias

Detection of changes in numbers of neurons is essential for an understanding of neuronal development, function, and death. Optical disector counting is claimed to be the most efficient technique to estimate accurate numbers of neurons in microscopic sections. We calibrated the optical disector by comparison with three-dimensional reconstructions from serial sections and determined how accurate this technique is relative to conventional profile counting methods. The calibration was performed on the trochlear nucleus in developing chicks. Optical disector estimates, when obtained as generally recommended, were about 25% lower than the actual number of neurons. This underestimate was caused by a nonuniform (bimodal) distribution of neuronal nuclei in paraffin and plastic (glycolmethacrylate) sections, but not in cryosections. The density of neurons in the core of the paraffin and plastic sections was substantially lower than in the upper and lower margins of these sections. Accurate estimates of neuronal numbers were obtained with a modified optical disector method that sampled the entire extent of tissue sections. Previous estimates of numbers of trochlear neurons in the developing chick have been controversial. The modified (calibrated) optical disector method revealed that the number of trochlear neurons decreased from about 1,600 at day 8.5 of incubation (embryonic day, [E]8.5) to about 900 at the time of hatching. Numbers of pyknotic nuclei peaked at E6 and at E9, revealing an additional early, but postproliferative, period of cell death. Taken together, these data emphasize the need for calibration of stereological counting techniques and the need to examine sampling strategies for potential bias. J. Comp. Neurol. 409:169–186, 1999. © 1999 Wiley-Liss, Inc.