ALTMAN CHALLENGES THE DOGMA AND IS IGNORED
An important advance in the study of neurogenesis came in the late 1950s with the introduction of [3H]-thymidine autoradiography. [3H]-thymidine is incorporated into the DNA of dividing cells. Therefore, the progeny of cells that had just divided could be labeled, and their time and place of birth determined. Initially, this new method was applied exclusively to the study of developing rodents.7 The emphasis on using this method to study pre-and perinatal development, rather than looking across the life span of the animal, reflected the persistence of the belief that neurogenesis did not occur in the adult mammal.
Starting in the early 1960s, Joseph Altman (b. 1925) challenged this idea of “no new neurons in the adult brain.” He published a series of papers reporting thymidine autoradiographic evidence for new neurons in the dentate gyrus of the hippocampus, the olfactory bulb, and the cerebral cortex of the adult rat.8 He also reported new neurons in the neocortex and elsewhere in the adult cat.9 (See figure 10.1.) Most of the new neurons were small, and Altman suggested that they were crucial for learning and memory. 10 Although published in the most prestigious journals of the time, such as the Journal of Comparative Neurology, Science, and Nature, these findings were totally ignored or dismissed as unimportant for over two decades. As late as 1970, an authoritative textbook of developmental neuroscience stated that “there is no convincing evidence of neuron production in the brains of adult mammals.”11
Figure 10.1
Autoradiograms of apparently labeled neocortical (lateral gyrus) neuron nuclei in an adult cat that was injected intraventricularly with [3H]-thymidine cresyl violet stain (Altman, 1963).
Altman was not granted tenure at MIT and moved to Purdue University where he eventually turned to more conventional developmental questions, perhaps because of the lack of recognition of his work on adult neurogenesis. Unable to get grants, he supported his work by producing magnificent brain atlases.12
KAPLAN CONFIRMS ALTMAN AND IS ALSO DISMISSED
Fifteen years after Altman’s first report, direct support for his claim of adult neurogenesis came from a series of electron microscopy studies by Michael Kaplan and his coauthors. First, they showed that [3H]-thymidine-labeled cells in the dentate gyrus and olfactory bulb of adult rats have the ultrastructural characteristics of neurons, such as dendrites and synapses, but not of glia (astrocytes or oligodendrocytes).13 Then Kaplan reported autoradiographic and ultrastructural evidence for new neurons in the cerebral cortex of adult rats, again confirming the earlier claims of Altman.14 Finally, he showed mitosis in the subventricular zone of adult macaque monkeys by again combining [3H]-thymidine labeling and electron microscopy.15 During this period, Kaplan was, successively, a graduate student at Boston University, a postdoctoral fellow at Florida State University, and an assistant professor at the University of New Mexico. Attacked for his iconoclastic claims, Kaplan left the field, became a medical student, and now works in rehabilitation medicine.16 In spite of his evidence for adult neurogenesis, Kaplan’s work had little effect at the time, as measured by citations or follow-up studies. Again, as in Altman’s case, publication in prestigious and rigorously reviewed journals, such as Science, the Journal of Comparative Neurology, and the Journal of Neuroscience by an unknown figure was not sufficient to make any marked dent in the dogma.
An important reason for the small impact of Kaplan’s work may have been a study presented at a meeting in 1984 and published the following year. Pasko Rakic, the author of the study, was (and still is) professor at Yale Medical School and arguably the leading student of primate brain development. He carried out a [3H]-thymidine study of adult rhesus monkeys in which he examined “all major structures and subdivisions of the brain including the visual, motor, and association neocortex, hippocampus, [and] olfactory bulb.” Rakic found “not a single heavily labelled cell with the morphological characteristics of a neuron in any brain of any adult animal” and concluded that “all neurons of the rhesus monkey brain are generated during prenatal and early postnatal life.”17
Rakic’s papers had a profound influence on the development of the field.18 Subsequent work in adult rhesus monkeys by Eckenhoff and Rakic, using a combination of thymidine autoradiography and electron microscopy, also failed to find new neurons in the adult.19 Furthermore, the authors questioned the reports of adult neurogenesis in rats with the suggestion that rats never stop growing and so never become adults. (In fact, there are strains of rats that do stop growing and also show adult neurogenesis,20 but this was not known at the time.) For Eckenhoff and Rakic, the supposed lack of adult neurogenesis in primates made sense, because “a stable population of neurons may be a biological necessity in an organism whose survival relies on learned behavior acquired over a long period of time.”21 Furthermore, Rakic suggested that the “social and cognitive behavior” of primates may require the absence of adult neurogenesis.22
Humans often show a basic need to distinguish themselves from other animals, and their order—primates—from other orders on cognitive grounds.23 Although neuroscientists have often tried to make these distinctions in terms of brain structure or function, Rakic’s suggestion may be the only time that the social and cognitive differences between primates and nonprimates was attributed to the presence or absence of adult neurogenesis and, more generally, to the structural stability of the brain.
There were three developments that led to a vindication of Altman’s pioneering work and to general acceptance that new neurons are added to the adult mammalian brain and that this was probably an interesting and important phenomenon. The first development was the demonstration of neurogenesis in adult birds. The second was the introduction of new methods for labeling new cells and for distinguishing neurons from glia. Finally, demonstrations that neurogenesis could be up-and downregulated by important psychological variables such as stress, environmental complexity, and learning raised the possibility that adult hippocampal neurogenesis might be important for cognition in higher animals.
AVIAN NEUROGENESIS
Starting in the late 1980s, Nottebohm and his colleagues at Rockefeller University began a systematic analysis of the neural basis of song learning in birds. They discovered a set of brain mechanisms that are crucial for bird song and showed how the volume of two nuclei were a function of variables such as sex, sexual maturity, song complexity, species, testosterone level, and season.24 The seasonal and hormonal changes in the volume of these song-related nuclei were so great in some species that Nottebohm set out to examine the possibility that these changes were due to fluctuations in the actual number of neurons in the adult avian brain.
In a series of elegant experiments, Nottebohm and his colleagues showed that, indeed, thousands of new neurons are added every day to the avian brain. They did so by, first, showing the production of new cells with thymidine labeling;25 second, producing ultrastructural evidence that the new cells were neurons receiving synapses;26 and last, in a technical tour de force, showing that the putative neurons responded to sound with action potentials.27 In subsequent studies, they showed that the axons of new neurons extended over long distances, that neuronal birth and death proceeded in parallel, that in both singing and nonsinging species neurogenesis was widespread throughout the avian forebrain—including the hippocampus—and that in the latter structure it was modulated by environmental complexity and learning experience.28
In spite of this unassailable evidence of neurogenesis in parts of the adult bird brain known to be homologous to primate cerebral cortex and primate hippocampus, these studies tended to be viewed as irrelevant to the primate or even the mammalian brain. Rather, the evidence for avian neurogenesis was viewed as an exotic specialization related to the necessity for flying creatures to have light cerebrums and to their seasonal cycles of singing.
NEW TECHNIQUES FOR DETECTING NEUROGENESIS
Beginning around the 1990s, there were several developments that finally established the reality of neurogenesis in th
e dentate gyrus of the adult rat. One was the demonstration that the new cells in the rat dentate gyrus extend axons into the mossy fiber pathway.29
The second important development was the introduction of the synthetic thymidine analog BrdU (5-bromo-3’-deoxyuridine). Like thymidine, BrdU is taken up by cells during the S-phase of mitosis and is a marker of proliferating cells and their progeny. BrdU labeling can be visualized with immunocytochemical techniques and does not require autoradiography.30 More recently, an endogenous marker for cell proliferation, Ki-67, was introduced. Ki-67 is a protein that is a cellular marker for cell proliferation. It is present during mitosis but is absent in the resting cell.31
Perhaps the most important advance was the use of cell-type-specific markers enabling the immunohistochemical distinction of the newly generated neurons from glia cells. Among the markers for mature neurons are NSE, MAP-2, TuJ1, and NeuN. Although some of these markers have been shown to stain nonneuronal cells under certain conditions and others do not stain all neuronal types,32 the expression of several of these antigens in a population of adult-generated cells is considered good evidence that new neurons have been produced. There are also markers for immature neurons and for glia (oligodendrocytes and astrocytes). The combination of BrdU for detecting new cells with immunochemical markers for neurons now allowed the identification of new neurons. Other markers for new neurons are now available.33
REGULATION OF NEUROGENESIS
The advent of these new techniques meant that by the 1990s, Altman’s claim that new neurons were added to the adult dentate gyrus had been confirmed several times, and by now it is well established for a variety of mammals including humans and other primates.34
But was this phenomenon more than some ontogenetic lag or phylogenetic vestige? At least in rats, the finding that the number of new hippocampal cells is so large, over 9,000 cells per day, most of which are neurons, makes this very unlikely.35 Furthermore, dentate gyrus neurogenesis in the rodent can be modulated by a number of experiential variables and so might be important for cognitive function.36 For example, acute and chronic stress decreases hippocampal neurogenesis. Adrenal steroids probably underlie this effect as stress increases adrenal steroid levels and glucocorticoids decrease the rate of neurogenesis. By contrast, there are several conditions that increase the number of adult-generated dentate gyrus cells, environmental complexity and wheel-running being particularly well studied enhancers of adult neurogenesis.
In Altman’s earliest studies he speculated that adult neurogenesis might play a crucial role in learning and memory. 37 In recent years this idea has been subjected to an increasing amount of experimental examination. Although there are conflicting results, the preponderance of evidence supports Altman’s speculation: the number of new neurons often positively correlates with learning of hippocampal-dependent tasks: learning such tasks tends to increase the number of new neurons and the depletion of new hippocampal neurons is reported to impair hippocampal-dependent learning.38
ADULT NEUROGENESIS IN THE OLFACTORY BULB AND CEREBRAL CORTEX
At about the time that Altman’s finding of neurogenesis in the dentate gyrus was confirmed, his report of neurogenesis in the adult olfactory bulb was also replicated.39 Neurogenesis in the adult olfactory bulb has now been shown for a variety of mammals, including humans,40 and, at least in rats, it is modulated by olfactory experience and learning.41
The status of Altman’s report of adult neurogenesis in the cerebral cortex is less clear. Beyond Altman and Kaplan’s work, a number of investigators have reported cortical neurogenesis in the hamster, rat, marmoset, and macaque cortex. However, others have failed to find cortical neurogenesis. Gould and Cameron and Dayer have reviewed the positive and negative studies and suggested that the negative results were due to insufficient sensitivity of the methods used and the small number and size of the new cortical neurons.42 (See this chapter’s postscript).
WHY WERE ALTMAN’S DISCOVERIES IGNORED FOR ALMOST 30 YEARS?
There appear to be several reasons why Altman’s discovery of neurogenesis in the hippocampus and the olfactory bulb was ignored. First, there were not accessible and reliable techniques for the objective differentiation of small neurons from glia, particularly astrocytes. Until the 1990s this distinction could be made only by “an expert eye,” and almost by definition, “experts knew” that adult neurogenesis did not occur in mammals. Another reason was that Altman, although in a leading university (MIT), was at the time of his early adult neurogenesis papers a junior faculty member in a psychology department and had not been trained in a distinguished, or indeed any, developmental laboratory or one using autoradiographic techniques. Finally, the dogma of “no new neurons” was universally held and vigorously defended by the most powerful and leading primate developmental anatomists of his time.
The continued resistance to acceptance of neurogenesis in the adult cerebral cortex may be due, in part, to the much lower incidence of cortical neurogenesis than hippocampal neurogenesis and therefore the greater importance of sensitive methods for detecting new neurons there. It may also reflect the continued investment of more traditional members of the community in denying neuronal plasticity.
POSTSCRIPT
COMPARISON OF FOUR WHO WERE “ BEFORE THEIR TIME ”
In my previous Tales in the History of Neuroscience I described a more extreme case of someone “before his time”—Emmanuel Swedenborg (1688–1772), who anticipated sensory and motor function of the cortex and, arguably, even the neuron theory by over 100 years.43 Yet his writings on brain function remained unknown until the twentieth century, by which time many of his ideas had been confirmed. There are both common elements and ones that were very different in the neglect by their contemporaries of Swedenborg’s ideas on the cortex, Bernard’s dictum on the constancy of the internal environment (chapter 8), Panizza’s discovery of visual cortex (chapter 9), and Altman’s discovery of neurogenesis.
Swedenborg, Panizza, and Altman faced impregnable ideological resistance: for Swedenborg, the dogma that the cortex was a functionless rind, for Panizza the dogma that the cortex had “higher functions” but neither sensory nor motor ones, and for Altman the dogma of no new neurons.
Swedenborg’s publications never reached the scientific community until they were long outdated. Panizza’s paper was published in a local journal but one that circulated to major scientific societies (exactly as was the case for Gregor Mendel). By contrast both Bernard’s and Altman’s were very widely available.
Bernard was the most famous French scientist of his time (and arguably, all time); Panizza was very highly regarded in his university, Altman was at a distinguished research institution whereas Swedenborg was not even recognized as a biologist.
All four had ideas that were difficult or impossible to test in their own time.
It took over 150 years for Swedenborg to be rediscovered. Bernard’s ideas on the internal milieu took about 50 years to be understood. Panizza was cited only after his discovery was rediscovered, and then was returned to his previous totally obscure status. Altman’s adult neurogenesis findings were ignored for about 30 years and are now ubiquitous in the opening paragraphs of papers on adult neurogenesis.
They were all iconoclasts and their icons were resilient.
ADULT NEUROGENESIS IN THE CEREBRAL CORTEX TODAY
By 2008 Altman’s reports of adult neurogenesis in the dentate gyrus of the hippocampus and in the olfactory bulb had been repeatedly confirmed with a variety of techniques and in many mammals including humans. By contrast his claim for adult neurogenesis in the cerebral cortex has remained a subject for debate and controversy. Since 1999 there have been at least five groups of investigators finding positive evidence for adult cortical neurogenesis in rodents and primates and about the same number failing to do so.44
There are several possible reasons for the difficulty in replicating the positive findings of cortical neurogenesis. First, the new cortical neurons that have been r
eported are much harder to detect than those in the olfactory bulb and hippocampus because they are much rarer and distributed unevenly over a much larger volume of tissue. Second, the new cortical neurons are interneurons much smaller than the typical cortical pyramidal cells among which they are sparsely scattered.45 Third, in many of the negative studies, histological protocols were used that have been shown to tend to reduce tissue quality and diminish the signal-to-noise ratio for detecting new neurons. Finally, and perhaps most important, the studies that failed to find cortical neurogenesis used techniques that also failed to show adequate evidence for new neurons in the dentate gyrus, presumably because of their poor sensitivity or tissue destructiveness. For inferences to be made about failure to find cortical neurogenesis it is necessary to have the positive control of good evidence for hippocampal neurogenesis with the same techniques, and this has not yet been the case.46 New, more sensitive techniques and the use of positive controls are needed to resolve this issue.
It should be noted that the very small reported incidence of new cortical neurons, namely 1–2 cells per mm3, should not belie the possible importance of these cells. At least in sensory systems, the activity of a single or small number of neurons has been shown to influence behavior.47
In recent years adult neurogenesis has also been reported in the striatum, amygdala, hypothalamus, substantia nigra, and brain stem in rodents or primates or both.48 Some of these observations have been confirmed but others have not, perhaps because of the sensitivity issues discussed for cortex.
Altman’s speculation that the new neurons in the hippocampus might play a role in learning and memory has belatedly resulted in a burgeoning literature on the subject.49 Since my paper was published there has been considerable additional evidence of the involvement of new hippocampal neurons in learning and memory tasks that require the hippocampus, such as spatial learning and eye-blink conditioning.50 However, the task and timing parameters for the involvement of new hippocampal neurons are not well understood and seeming contradictions in the literature abound. The role of the new neurons in the processes underlying learning and memory remains totally obscure.
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