The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments.

Brain-derived neurotrophic factor (BDNF) is a member of the NGF gene family, which 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 like 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 is affected by this mutation. Mutant mice showed significantly weaker LTP in the CA1 region. The magnitude of the potentiation as well as the percentage of cases in which LTP could be induced successfully was clearly reduced whereas important pharmacological and morphological control parameters in the hippocampus of these animals were unaffected. Adenoviral vectors were used to re-express BDNF in acute slices of BDNF-knock-out mice. In most cases LTP could be rescued with this approach. These results suggest that BDNF has an important functional role in the expression of LTP in the hippocampus.

Optical recording with single cell resolution from monolayered slice cultures of rat hippocampus.

Voltage-sensitive dyes were used to optically record the membrane potentials from neurons in hippocampal slice cultures. Multi-channel recordings from these monolayered but otherwise 'organotypic' slice cultures had very good spatial as well as good temporal resolution (15 x 15 micron, 0.5 ms respectively). We show that in this preparation action potentials elicited by intracellular current pulses can be recorded optically and that single spikes are readily detectable without averaging. Furthermore, a new procedure which significantly reduces photodynamic damage is described. Our study demonstrates the feasibility of optical recording with single cell resolution in an organotypic mammalian CNS preparation.

Formation of specific afferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus.

The development of the cerebral cortex involves the specification of intrinsic circuitry and extrinsic connections, the pattern of inputs and outputs. To investigate the development of a major afferent input to the cortex, we studied the formation of thalamocortical connections in an organotypic culture system. Slices from the lateral thalamus of young rats were cocultured with slices from the visual cortex. Thalamocortical projections in vitro were examined anatomically with fluorescent dyes and physiologically with electrophysiological and optical recording techniques. Axons emerged from thalamic explants radially in all directions. The outgrowth of thalamic fibers and the course of the axonal trajectories were not influenced by the presence of the cocultured cortex. Only those thalamic axons that happened to grow toward the cortical slices invaded their target tissue. Thalamocortical projection cell in vitro had the characteristic morphology of thalamic relay neurons. Cells with the morphology of interneurons were present in thalamic explants, but these neurons did not project to the cocultured cortex. Thalamocortical axons in vitro terminated in their appropriate cortical target layer, formed axonal arbors, and made functional synaptic contacts. Such specific connections between thalamic neurons and their cortical target cells were established regardless of whether thalamocortical axons invaded the cortex from the white matter side or from the pial surface. These results suggest that thalamic projection neurons have an innate mechanism that allows them to recognize their cortical target cells. Thus, intrinsic factors play a significant role in the laminar specification of cortical connections during development.

Synaptic plasticity in rat hippocampal slice cultures: local "Hebbian" conjunction of pre- and postsynaptic stimulation leads to distributed synaptic enhancement.

A central theme in neurobiology is the search for the mechanisms underlying learning and memory. Since the seminal work, first of Cajal and later of Hebb, the synapse is thought to be the basic "storing unit." Hebb proposed that information is stored by correlation: synapses between neurons, which are often coactive, are enhanced. Several recent findings suggest that such a mechanism is indeed operative in the central nervous system. Pairing of activity on presynaptic fibers with strong postsynaptic depolarization results in synaptic enhancement. While there is substantial evidence in favor of a postsynaptic locus for detection of the synchronous pre- and postsynaptic event and subsequent initiation of synaptic enhancement, the locus of this enhancement and its ensuing persistence is still disputed: both pre- and postsynaptic contributions have been suggested. In all previous studies, the enhancement was presumed to be specific to the synapses where synchronous pre- and postsynaptic stimulation was applied. We report here that two recording techniques--optical recording, using voltage-sensitive dyes, and double intracellular recordings--reveal that synaptic enhancement is not restricted to the stimulated cell. Although we paired single afferent volleys with intracellular stimulation confined to one postsynaptic cell, we found that strengthening also occurred on synapses between the stimulated presynaptic fibers and neighboring cells. This suggests that synaptic enhancement by the "paired-stimulation paradigm" is not local on the presynaptic axons and that, in fact, the synapses of many neighboring postsynaptic cells are enhanced.

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Brain-derived neurotrophic factor promotes the differentiation of various hippocampal nonpyramidal neurons, including Cajal-Retzius cells, in organotypic slice cultures.

Brain-derived neurotrophic factor (BDNF) is widely expressed in the central nervous system, where its function is poorly understood. The aim of this study was to investigate the effects of BDNF on the differentiation of hippocampal nonpyramidal neurons using organotypic slice cultures prepared from postnatal rats. The application of BDNF induced an increase in immunostaining for the microtubule-associated protein (MAP)-2 in non-pyramidal neurons of the stratum oriens. BDNF promotes the elongation of the dendrites of these neurons, as demonstrated by analysis after biocytin labeling. Calbindin-D- and calretinin-containing subgroups of nonpyramidal cells in the stratum oriens were responsive to BDNF but not to nerve growth factor, as shown by an increase in the number of neurons immunostained for these proteins. BDNF also induced an increase in neuropeptide Y immunostaining of stratum oriens neurons. In contrast, BDNF had no effect on parvalbumin immunostaining, despite the fact that these cells express the BDNF receptor trkB. In addition, BDNF increased calretinin immunoreactivity in Cajal-Retzius cells situated around the hippocampal fissure. The Cajal-Retzius neurons persisted in slices beyond the time at which they degenerate in vivo. However, BDNF is not required for the survival of these cells, because they also persisted in slices from BDNF knock-out mice. The present results indicate that BDNF exerts an effect on the morphology of stratum oriens nonpyramidal cells and their calcium-binding protein levels. BDNF also regulates the calretinin content of Cajal-Retzius cells but is not necessary for their survival.