Fernando Nottebohm - US grants

Autoradiographic studies using 3H-thymidine indicate that new neurons are added to hyperstriatum ventralis, pars caudalis (HVc) of the adult canary brain. HVc is in the forebrain and is part of the song control system used in song learning. The new neurons are formed by division of ventricular zone cells, then migrate, differentiate and become connected to existing circuitry. Research planned for the next 5 years will describe the dynamics of neuronal recruitment in this system, the factors that control it, and the possibility that the new neurons replace older neurons. 3H-thymidine and autoradiography will continue to be used for this work, as well as silastic implants of steroid hormones, deafening by removal of both cochleas, and kainic acid lesions. The significance of neurogenesis and neuronal replacement in adult HVc will be studied by searching for temporal correlations between these phenomena and times of year during which adult canaries are particularly prone to learn new song syllables and forget old ones. The hypothesis tested here is that there is a correlation between the temporal occurrence of song learning, song forgetting and replacement of HVc neurons. The behavioral studies will involve song recording and sound-spectrographic analysis. Within this scenario our specific aims will be: 1. To measure the half-life of song perceptual and motor memories in adult male canaries, and the extent to which they are hormone dependent. 2. To determine the seasonal occurrence of neurogenesis and neuronal replacement in adult canaries, and their temporal relation to periods of song instability, forgetting and learning. 3. To determine to what extent, if any, hormones and experience influence neurogenesis and neuronal replacement in HVc. 4. To determine the survival curves of new HVc neurons, and the extent to which they may vary between different neuronal classes. 5. To determine how new HVc neurons orient during migration and find a place to work. 6. To determine to what extent neuronal recruitment occurs during the period from hatching to sexual maturity, so that patterns of recruitment (and replacement?) occurring at that time can be compared with those occurring in adulthood. 7. To interfere with neuronal recruitment and neuronal replacement in adult HVc to see how this affects memory retention and learning of new songs. An appreciaton of the occurrence and significance of neurogenesis and neuronal replacement in adult brain could have profound effects on neurological practice.

The work proposed will increase our understanding of brain pathways used for the acquisition of a complex skill and the way they are connected and altered by experience. The subjects to be used are two species of songbird, the canary and zebra finch, that learn their song by imitating sounds they hear. Anterograde and retrograde tracers will be used to study the connectivity -- inputs and outputs -- of parts of the brain involved in the control of this learned behavior. Extracellular and intracellular electrodes will be used to characterize the electrical activity of neurons during song production and at the time the animal is presented with various sounds. Among the questions asked are, what is the relation between sound input and vocal output?, and how are pathways that mediate this relation affected by experience, sex, season and hormonal variables? Another question asked is how do anatomically symmetrical pathways for singing function so as to generate an asymmetry in manifest control? The health relatedness of this work comes by way of the information it might contribute towards an understanding of the mechanisms that determine the "when", "what", "how much" and "how" of learning, so that malfunction of these higher functions of the vertebrate brain can be approached in a more scientific manner than at present. An added expectation, based on preliminary observations, is that an understanding of the kinds of brain plasticity used in learning in adulthood may tell us about the brain's potential for self-repair and the factors that control it.

Work to be supported by this grant will continue to investigate the anatomy and physiology of the song control system of birds. Specific aims are to better understand, 1) the relation between production and perception of learned song; 2) brain asymmetries and sexual dimorphism that may occur in the perception of learned song; 3) the rules that govern the recognition of conspecific song and the preference that females show for the song of some individual birds; 4) the anatomical and physiological regulation of song learning; 5) the relation between pathways used for song learning and pathways governing other reproductive behaviors. Information on these matters should uncover interesting principles of brain function that can then be related to learning. The P.I. expects that these insights will be applicable to other systems, including vocal learning and brain function in humans. Information derived from these various related projects might also help understand how the newly discovered phenomenon of neuronal replacement in adult avian brain relates to how song learning circuits are organized. Health relatedness: The P.I. believes that the kinds of circuit plasticity that birds seem to use for learning -- neurite growth and retraction, formation and culling of synapses, neurogenesis - - will, once understood at the molecular level form the basis of inducible and controlled brain self-repair. In addition, a better understanding of the factors that restrict or encourage learning could be of great benefit to humans. The song learning system of birds is good material to study these matters.

Autoradiographic studies using 3H-thymidine indicate that new neurons are added to hyperstriatum ventralis, pars caudalis (HVc) of the adult canary brain. HVc is in the forebrain and is part of the song control system used in song learning. The new neurons are formed by division of ventricular zone cells, then migrate, differentiate and become connected to existing circuitry. Research planned for the next 5 years will describe the dynamics of neuronal recruitment in this system, the factors that control it, and the possibility that the new neurons replace older neurons. 3H-thymidine and autoradiography will continue to be used for this work, as well as silastic implants of steroid hormones, deafening by removal of both cochleas, and kainic acid lesions. The significance of neurogenesis and neuronal replacement in adult HVc will be studied by searching for temporal correlations between these phenomena and times of year during which adult canaries are particularly prone to learn new song syllables and forget old ones. The hypothesis tested here is that there is a correlation between the temporal occurrence of song learning, song forgetting and replacement of HVc neurons. The behavioral studies will involve song recording and sound-spectrographic analysis. Within this scenario our specific aims will be: 1. To measure the half-life of song perceptual and motor memories in adult male canaries, and the extent to which they are hormone dependent. 2. To determine the seasonal occurrence of neurogenesis and neuronal replacement in adult canaries, and their temporal relation to periods of song instability, forgetting and learning. 3. To determine to what extent, if any, hormones and experience influence neurogenesis and neuronal replacement in HVc. 4. To determine the survival curves of new HVc neurons, and the extent to which they may vary between different neuronal classes. 5. To determine how new HVc neurons orient during migration and find a place to work. 6. To determine to what extent neuronal recruitment occurs during the period from hatching to sexual maturity, so that patterns of recruitment (and replacement?) occurring at that time can be compared with those occurring in adulthood. 7. To interfere with neuronal recruitment and neuronal replacement in adult HVc to see how this affects memory retention and learning of new songs. An appreciaton of the occurrence and significance of neurogenesis and neuronal replacement in adult brain could have profound effects on neurological practice.

9319638 Nottenbohm Macklis Kirn Many brain disorders, result from the death of brain cells. We do not know how to replace these cells, or even if this is possible. However, death and replacement of brain cells (neurons) occurs spontaneously in the brain of adult birds. The new neurons are born in the walls of the lateral ventricle of the brain. Some of these cells are part of the vocal control system, and are known to be involved in controlling the learning and remembering of song. Since this constant replacement is not preceded or accompanied by overt pathology, it can be thought of as a form of brain rejuvenation. In collaborative research proposed here by Drs. Nottenbohm, Macklis and Kirn, a new technique for inducing selective cell death in the brain will be used and resulting effects on the generation and function of new brain cells will be tested. The questions that will be answered are: 1) Is cell replacement restricted to a few kinds of cells, or will any type of brain cell that dies be replaced by another one of the same kind? 2) Do signals emitted by dying cells play a role in the birth, migration, and survival of the new cells? 3) Does cell replacement affect memory and the acquisition of new memories? The answers to these questions will help reveal the cellular dynamics underlying memory formation, and may help in the development of new approaches to repairing brains and controlling the decrement of function that accompanies aging.***

The long range goal of work described in this proposal is the correction of handicaps, as found in Alzheimer's, Parkinson's and stroke patients, that result from the selective and partial loss of brain cells. It is suggested that a possible solution to these handicaps is to tap the brain's own potential for neuronal replacement. This potential is widely expressed in oscine songbirds, such as the canary and zebra finch. The work described here uses their song system, which is necessary for the acquisition and production of learned song. We will study the factors that regulate neuronal replacement in this system, including the role of experience, learning, hormones and trophic factors. An advantage of this system is that it allows us to test for possible relations between neuronal replacement and the acquisition and production of a learned behavior. Specific hypotheses that we will test are: 1) that experience affects the survival of new cells; 2) that experience affects the place of insertion of new cells; 3) that acquisition of long-term memory alters gene expression in a long-lasting, possibly irreversible manner, as occurs during tissue differentiation. Behavioral, anatomical, cellular and molecular tools will be used to test these hypotheses. Understanding the processes of neuronal turnover in adult songbirds may suggest approaches for making this happen, under controlled conditions, in the human brain when this seems clinically desirable.

DESCRIPTION:( from applicant's abstract) Neuron production, migration, and differentiation are mostly restricted to early development in warm-blooded vertebrates; however, neurogenesis also persists into adulthood in a broad range of species, among them songbirds, who incorporate new neurons into a brain region (HVC) that controls song production. Increases in spontaneous neuronal replacement in HVC correlate with song changes and with cell death. Therefore, songbirds are an excellent model for studies on functional significance of spontaneous adult neuronal replacement and adult brain repair. Most new HVC neurons become projection neurons in the motor pathway that controls the production of learned song. A second type of HVC neuron is not produced in adulthood. The factors governing the recruitment of one cell type but not the other are not known. In experiments to address this question, we recently demonstrated that targeted photolytic neuronal death of the projection neuron type that normally turns over results in compensatory replacement of the same type. Induced death of the normally not replaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. This suggests that neuronal death regulates the recruitment of neurons but only of the replaceable kind. After elimination of replaceable neurons, song deteriorated in some birds; behavioral deficits were transient and followed by variable degrees of recovery, raising the possibility that induced neuronal replacement can restore a learned behavior. In this application we propose to further investigate the relationship between the experimentally induced brain injury, the ensuing brain repair and song behavior. Specifically, we will determine how induced neuronal death causes song deterioration and how subsequent recovery occurs. We will address (1) What variables govern the type and severity of song deterioration and what is the time course of deterioration? (2) Does behavioral recovery depend on the same neural substrates that guide vocal learning during development? (3) Does behavioral recovery after induction of neuronal death depend on neuronal replacement?

DESCRIPTION (provided by applicant): The work proposed here is designed to build on what we have accomplished thus far toward establishing an efficient procedure for producing transgenic zebra finches. Zebra finches are songbirds that learn their song by imitating that of an adult. The brain regions that mediate this behavior are highly modular and well defined anatomically;they are collectively referred to as the "song system". Within this system a key population of neurons encoding the song is replaced continuously in adulthood. The tight relationship between anatomy and behavior, together with the rare ability to continuously replace those cells that produce the behavior, have made the song system a very attractive model for studying the basic biology of learning and neuronal replacement. Several key resources have been and are presently being generated to facilitate the study of this system at ever more reductionistic levels: the genome of this species has been sequenced, cDNA libraries have been generated, sequenced and published, and a BAC library has been created and made available. These resources have the potential to help elucidate the molecular mechanisms and cellular properties that mediate song behavior. However, to take full advantage of these resources it has to be possible to manipulate gene expression in vivo, which we cannot yet do in zebra finches. Over the last year this laboratory has devoted its resources to the development of such a tool, and the methods we have employed have allowed us to overcome key obstacles and make significant progress. That progress is outlined here as well as the steps we will take to produce an efficient protocol for making transgenic songbirds. PUBLIC HEALTH RELEVANCE: Neurogenesis and neuronal replacement occur in the context of song learning in adult zebra finches. This project is about the production of transgenic zebra finches that will help us understand the mechanisms, function and therapeutic potential of neuronal replacement in adult brain.