How do neurons and their networks learn from experience? How does the organ governing motor activity and sensation also learn and remember? We are studying neural plasticity, the manner in which nerve cell structure and function changes with alteration in the external and internal milieu. Our studies indicate that environmental influences alter brain function at the most fundamental level, that of gene action. These conclusions derive from multiple related projects, two of which are detailed here, the molecular basis of learning and memory, and stem cell plasticity.

Approximately fifteen years ago, we and others discovered that the brain contains multiple growth factors that regulate long term development and survival of brain neurons over days to weeks. Recently, we found that one of the factors, BDNF (brain derived neurotrophic factor), also acutely increases neuronal synaptic communication within minutes. This increased neural transmission occurs in hippocampal neurons that subserve memory. Detailed analysis in a number of laboratories indicated that BDNF enhanced transmission through actions both on the signal sending and signal receiving neurons. Focusing initially on the receiving neurons in cell culture, we found that BDNF increased the activity of the receptor lock for the neurotransmitter signal key that transmits nerve impulses. In sum, BDNF increases responsiveness of the receiving memory neuron to neurotransmitter signals. We were able to identify the biochemical changes that increased receptor activity: Specific subunits of the receptor were phosphorylated indirectly by BDNF, increasing responsiveness. In parallel studies, BDNF also increased the release of transmitter signal by the transmitting neuron. In summary, the growth factor potentially increases memory function by acting both on the sending and receiving neurons.

To define the genomic basis of the growth factor action, we combined electrophysiological and molecular biological investigation at the single neuron level. Exposure to BDNF markedly increased synaptic transmission, as expected, and transcriptional analysis identified increases in 11 genes. We examined one gene, Rab3A, in detail as a prototype. The Rab3A gene product is a small GTP-binding protein that plays a critical role in transmitter release. To analyze function, we examined hippocampal neurons from mutant mice lacking Rab3A. Although basal synaptic transmission was normal, the neurons failed to respond to the growth factor with enhanced transmission, indicating that Rab3A is required for BDNF-induced synaptic plasticity associated with learning and memory. More generally, our studies suggest that gene expression governing mental function can be approached at the single cell level. Since BDNF is regulated by a variety of environmental stimuli from stress to seizures, our findings integrate experience, gene expression and brain function.

The striking synaptic plasticity uncovered by BDNF raised a host of general questions concerning cellular plasticity and cellular identity. What are the limits of plasticity? We approached this question by studying stem cells, the paradigm of plasticity. These undifferentiated elements can differentiate into a variety of specialized cell types and also retain the ability to self-renew.

We recently differentiated adult human and rat bone marrow stromal stem cells (BMSCs) into neurons. Our ultimate goal is to use these cells in Alzheimer's and Parkinson's diseases and in spinal cord injury. BMSCs normally differentiate only into mesenchymal cells, including bone, cartilage, muscle, tendon and fat. Differentiation into non-mesenchymal fates had not been demonstrated. A relatively simple treatment protocol induced the stromal cells to differentiate into neurons, exhibiting neuronal morphological traits, and expressing a variety of neuron-specific genes. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Our observations suggest that intrinsic genomic mechanisms of commitment, lineage restriction and cell fate are mutable. Environmental signals apparently can elicit the expression of pluripotentiality that extends well beyond the accepted fate restrictions of cells originating in classical embryonic germ layers.

We have transplanted these neurons into various regions of the rat brain and spinal cord. The neurons survive and the rats exhibit no untoward effects. We are now transplanting the cells into rats with experimental diseases. Our approach, using adult stem cell-derived neurons confers several potential future advantages: (1) Use of the patient's own cells for autologous transplantation eliminates the danger of immunorejection and the need for toxic immunosuppressive agents; (2) The self-renewing BMSCs and the neurons grow vigorously in culture providing a vast reservoir of source material; (3 ) Neuronal differentiation is achieved by environmental manipulation only, without altering the genome, eliminating the need for immortalization and minimizing the probability of neoplastic transformation; (4) The use of adult cells circumvents the ethical concerns associated with the use of embryos.

We are presently examining the molecular mechanisms governing differentiation and self-renewal in BMSCs and in stem cells from other sources. In parallel we are transplanting stem cells and derived neurons in a variety of experimental diseases.

Key References

Levine, E., Dreyfus, C.F., Black, I.B. and Plummer, M.: Brain Derived Neurotrophic Factor Rapidly Enhances Synaptic Transmission in Hippocampal Neurons via Postsynaptic Tyrosine Kinase Receptors. Proc. Natl. Acad. Sci., USA, 92(17), 8074-8077, 1995.

Black, I. B.:Trophic Interactions and Brain Plasticity. In: The Cognitive Neurosciences (Gazzaniga, M. S., ed.) MIT Press, MA, 1995.

Suen, P-C, Wu, K., Levine, E.S., Mount, H.T.J., Xu, J-L, Lin, S-Y, Black, I.B.: Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit 1. Proc. Natl. Acad. Sci. USA, 94:8191-8195, 1997.

Levine, E.S., Black, I.B.: Trophic factors, synaptic plasticity, and memory. Ann. NY Acad Sci., 835:12-18, 1997.

Lin, S-Y., Wu, K., Levine, E.S., Mount, H.T.J., Suen, P-C., Black, I.B.: BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities. Mol. Br. Res., 55, 20-27, 1998.

Levine, E.S., Crozier, R.A., Black, I.B., Plummer, M.R.: Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing NMDA receptor activity. Proc. Natl. Acad. Sci., USA, 95, 10235-10239, 1998.

Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B.: Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res., 61, 364-370, 2000.
The Dying of Enoch Wallace: LIFE, DEATH, and the CHANGING BRAIN, McGraw-Hill, October, 2000.