RESUMO
Isotropic fractionation is a quantitative technique that allows reliable estimates of absolute numbers of neuronal and non-neuronal brain cells. However, being fast for single small brains, it requires a long time for processing large brains or many small ones, if done manually. To solve this problem, we developed a machine to automate the method, and tested its efficiency, consistency, and reliability as compared with manual processing. The machine consists of a set of electronically controlled rotation and translation motors coupled to tissue grinders, which automatically transform fixed tissue into homogeneous nuclei suspensions. Speed and torque of the motors can be independently regulated by electronic circuits, according to the volume of tissue being processed and its mechanical resistance to fractionation. To test the machine, twelve paraformaldehyde-fixed rat brains and eight human cerebella were separated into two groups, respectively: one processed automatically and the other, manually. Both pairs of groups (rat and human tissue) followed the same, published protocol of the method. We compared the groups according to nuclei morphology, degree of clustering and number of cells. The machine proved superior for yielding faster results due to simultaneous processing in multiple grinders. Quantitative analysis of machine-processed tissue resulted in similar average numbers of total brain cells, neurons, and non-neuronal cells, statistically similar to the manually processed tissue and equivalent to previously published data. We concluded that the machine is more efficient because it utilizes many homogenizers simultaneously, equally consistent in producing high quality material for counting, and quantitatively reliable as compared to manual processing.
Assuntos
Encéfalo/citologia , Contagem de Células/instrumentação , Contagem de Células/métodos , Processamento Eletrônico de Dados/métodos , Neurônios/fisiologia , Análise de Variância , Animais , Núcleo Celular/fisiologia , Processamento Eletrônico de Dados/instrumentação , Humanos , Técnicas In Vitro , Indóis , Neuroglia/citologia , Neurônios/citologia , Fosfopiruvato Hidratase/metabolismo , RatosRESUMO
Owing to methodological shortcomings and a certain conservatism that consolidates wrong assumptions in the literature, some dogmas have become established and reproduced in papers and textbooks, derived from quantitative features of the brain. The first dogma states that the cerebral cortex is the pinnacle of brain evolution - based on the observations that its volume is greater in more 'intelligent' species, and that cortical surface area grows more than any other brain region, to reach the largest proportion in higher primates and humans. The second dogma claims that the human brain contains 100 billion neurons, plus 10-fold more glial cells. These round numbers have become widely adopted, although data provided by different authors have led to a broad range of 75-125 billion neurons in the whole brain. The third dogma derives from the second, and states that our brain is structurally special, an outlier as compared with other primates. Being so large and convoluted, it is a special construct of nature, unrelated to evolutionary scaling. Finally, the fourth dogma appeared as a tentative explanation for the considerable growth of the brain throughout development and evolution - being modular in structure, the brain (and particularly the cerebral cortex) grows by tangential addition of modules that are uniform in neuronal composition. In this review, we sought to examine and challenge these four dogmas, and propose other interpretations or simply their replacement with alternative views.
Assuntos
Neurônios/citologia , Neurociências , Animais , Evolução Biológica , Cerebelo/citologia , Córtex Cerebral/citologia , Humanos , Neuroglia/citologia , PrimatasRESUMO
The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells ("neurons") and 84.6 +/- 9.8 billion NeuN-negative ("nonneuronal") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain.