Long-term enhancement of synaptic efficacy in the hippocampus is an important model for studying the cellular mechanisms of neuronal plasticity, circuit reorganization, and even learning and memory. Although these long-lasting functional changes are easy to induce, it has been very difficult to demonstrate that they are accompanied or even caused by morphological changes on the subcellular level. Here we combined a local superfusion technique with two-photon imaging, which allowed us to scrutinize specific regions of the postsynaptic dendrite where we knew that the synaptic changes had to occur. We show that after induction of long-lasting (but not short-lasting) functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite, whereas in control regions on the same dendrite or in slices where long-term potentiation was blocked, no significant spine growth occurred.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo-and heterozygous mutant mice showed significantly reduced LTP in the CAl region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.Neurotrophic factors, in particular the members of the nerve growth factor (NGF) gene family, have so far been considered predominantly with regard to their function in regulating survival and differentiation of specific neuronal populations during embryonic development and the maintenance of characteristic neuronal function in adulthood (1-3). There is, however, evidence that neurotrophins might also be involved in neuronal plasticity (4-10). Long-term potentiation (LTP) is the most widely used paradigm to study cellular and molecular events underlying neuronal plasticity (11). We therefore used this paradigm in slices of the hippocampus from mice with targeted deletion of the brain-derived neurotrophic factor (BDNF) gene to test whether BDNF has a role in this important phenomenon of synaptic plasticity. MATERIALS AND METHODSIn the gene-targeting construct, a 560-bp fragment from the BDNF protein-coding exon was replaced by the selection marker-a neomycin-resistance gene flanked by a glycerate kinase gene promoter and a polyadenylylation signal-thus deleting most of the mature BDNF coding sequence (Fig. 1). Embryonic stem cells (D3, 129Sv) containing the disrupted BDNF gene were injected into BALB/c mouse blastocysts for subsequent generation of chimeric mice. Chimeric males were crossed with NMRI females to produce heterozygotes. In keeping with previously published reports (12, 13), homozygous BDNF (-/-) mutant mice were retarded in growth and had reduced weight (down to only 25% of the wild type) from postnatal day 3 (P3) on. They displayed aberrant limb coordination and balance, showed a loss of neurons in the dorsal root ganglia, and usually died between 2 and 4 weeks after birth. Such abnormalities were never observed in heterozygous BDNF (+/ -) mice.Transverse hippocampal slices (400 ,um thick) were prepared and maintained by standard procedures (medium, 124 mM NaCl/3 mM KCl/1.25 mM...

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (∼0.8–12 mm2). The surgical procedure can be completed within ∼1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

Dendritic spines are morphological specializations that receive synaptic inputs and compartmentalize calcium. In spite of a long history of research, the specific function of spines is still not well understood. Here we review the current status of the relation between morphological changes in spines and synaptic plasticity. Since Cajal and Tanzi proposed that changes in the structure of the brain might occur as a consequence of experience, the search for the morphological correlates of learning has constituted one of the central questions in neuroscience. Although there are scores of studies that encompass this wide field in many species, in this review we focus on experimental work that has analyzed the morphological consequences of hippocampal long-term potentiation (LTP) in rodents. Over the past two decades many studies have demonstrated changes in the morphology of spines after LTP, such as enlargements of the spine head and shortenings of the spine neck. Biophysically, these changes translate into an increase in the synaptic current injected at the spine, as well as shortening of the time constant for calcium compartmentalization. In addition, recent online studies using time-lapse imaging have reported increased spinogenesis. The currently available data show a strong correlation between synaptic plasticity and morphological changes in spines, although at the same time, there is no evidence that these morphological changes are necessary or sufficient for the induction or maintenance of LTP. Still, they highlight once more how form and function go hand in hand in the central nervous system.