Calcium regulation of neuronal gene expression.

Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activity dependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

EphB receptors interact with NMDA receptors and regulate excitatory synapse formation.

EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.

Long-range inhibition within the zebra finch song nucleus RA can coordinate the firing of multiple projection neurons.

The zebra finch forebrain song control nucleus RA (robust nucleus of the archistriatum) generates a phasic and temporally precise neural signal that drives vocal and respiratory motoneurons during singing. RA's output during singing predicts individual notes, even though afferent drive to RA from the song nucleus HVc is more tonic, and predicts song syllables, independent of the particular notes that comprise the syllable. Therefore RA's intrinsic circuitry transforms neural activity from HVc into a highly precise premotor output. To understand how RA's intrinsic circuitry effects this transformation, we characterized RA interneurons and projection neurons using intracellular recordings in brain slices. RA interneurons fired fast action potentials with steep current-frequency relationships and had small somata with thin aspinous processes that extended throughout large portions of the nucleus; the similarity of their fine processes to those labeled with a glutamic acid decarboxylase (GAD) antibody strongly suggests that these interneurons are GABAergic. Electrical stimulation revealed that RA interneurons receive excitatory inputs from RA's afferents, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and HVc, and from local axon collaterals of RA projection neurons. To map the functional connections that RA interneurons make onto RA projection neurons, we focally uncaged glutamate, revealing long-range inhibitory connections in RA. Thus these interneurons provide fast feed-forward and feedback inhibition to RA projection neurons and could help create the phasic pattern of bursts and pauses that characterizes RA output during singing. Furthermore, selectively activating the inhibitory network phase locks the firing of otherwise unconnected pairs of projection neurons, suggesting that local inhibition could coordinate RA output during singing.

To fear or not to fear: what was the question? A potential role for Ras-GRF in memory.

Learning, making memories, and forgetting are thought to require changes in the strengths of connections between neurons. Such changes in synaptic strength occur in two phases: an early phase that is likely mediated by covalent modifications to existing proteins, and a delayed phase that depends on new gene expression and protein synthesis. However, the biochemical mechanisms by which neuronal activity leads to changes in synaptic strength are poorly understood. Recently, it has been shown that animals that lack Ras guanine nucleotide releasing factor (Ras-GRF), a Ca(2+)-dependent activator of the small GTP-binding protein, Ras, do not learn fear responses normally, although other types of learning appear normal. These animals show defects in the delayed phase of memory formation within the neuronal circuit that mediates fear conditioning. This paper suggests that Ras-GRF couples synaptic activity to the molecular mechanisms that consolidate changes in synaptic strength within specific neuronal circuits.

Relationships between local synaptic connections and orientation domains in primary visual cortex.

Combined optical imaging of ferret primary visual cortex in vivo and scanning laser photostimulation in brain slices were used to determine the spatial relationships between synaptic inputs onto individual neurons and the pattern of orientation columns. In the upper cortical layers, both excitatory and inhibitory inputs originated primarily from regions with orientation tuning similar to that of the recorded neurons; the shapes of the input tuning curves were indistinguishable. The orientation distributions of both types of inputs centered around the orientation of the recorded neurons, and no evidence for preferential cross-orientation inputs, either excitatory or inhibitory, was observed. These patterns of synaptic connectivity are most consistent with feedforward models for generation of orientation selectivity and are inconsistent with the patterns required by models based on cross-orientation inhibition.

Scanning laser photostimulation: a new approach for analyzing brain circuits.

A new technique for understanding the organization of complex circuits in the vertebrate brain, scanning laser photostimulation, is described. This approach is based on the photolysis of a caged form of the excitatory neurotransmitter glutamate. Computer-controlled photostimulation and whole cell recording in brain slices allow the construction of detailed maps of the position, strength, sign and number of inputs converging on a single postsynaptic neuron. Scanning laser photostimulation offers many advantages over current techniques: spatial resolution is superb, fibers of passage are not activated, and thousands of presynaptic locations can be stimulated. This review describes the technique of photostimulation, outlines the instrumentation, necessary to implement it, and discusses the interpretation of photostimulation-derived data. Several examples of applications, ranging from mapping circuits in the mammalian visual cortex to determining receptor distributions on single neurons are considered. Although still in its early stages, scanning laser photostimulation offers neuroscientists a powerful tool for determining the organization and function of local brain circuits.

Rearrangements of synaptic connections in visual cortex revealed by laser photostimulation.

Assessing patterns of synaptic connections in the developing mammalian neocortex has relied primarily on anatomical studies. In a physiological approach described here, the patterns of synaptic connections in slices of developing ferret visual cortex were determined with scanning laser photostimulation. Functional synaptic inputs to pyramidal cells in cortical layers 2 and 3 originating from sites close to the neuronal cell body appeared at least 2 weeks before eye opening, prior to the formation of long-range horizontal connections. Extensive long-range horizontal connections appeared in the next 10 days of development. The number of local connections peaked at the time of eye opening; the number of these connections subsequently declined to the level found in the adult while the specificity of long-distance connections increased. Thus, the relative influence of local connections on the activity of layer 2 and layer 3 neurons declines as the cortex matures while the influence of longer range connections increases substantially.

Independent control of dendritic and axonal form in the developing lateral geniculate nucleus.

To identify mechanisms that regulate neuronal form in the mammalian CNS, we have examined dendritic development in the lateral geniculate nucleus (LGN) during the period of segregation of retinal ganglion cell axons. The tracer Dil was used to label retrogradely LGN neurons that send their axons to primary visual cortex at different ages between embryonic day 36 (E36) and E60 in the cat. LGN neurons grow extensively during this period, in concert with the progressive restriction of ganglion cell axons from the two eyes to their appropriate eye-specific layers. At E36 neurons have simple bipolar morphology; by E60 all have acquired complex multipolar dendritic trees. During this period, soma size increases by 190% and total dendritic length increases 240%. Dendritic complexity, as measured by dendritic branch points, also increases. As dendrites grow, the number of spines increases, but their density remains constant at 0.015/micron throughout this period. Since it is known that blockade of action potential activity significantly alters the branching pattern and extent of retinal ganglion cell axonal arbors within the LGN, we also investigated whether the dendritic development of the postsynaptic LGN neurons is similarly susceptible. Following 2 weeks of the intracranial minipump infusion of TTX between E42 and E56, the morphology of LGN neurons was examined. Surprisingly in view of the striking effect of the treatment on the morphology of retinal ganglion cell axons, dendritic growth and development were essentially normal. However, the density of dendritic spines increased almost threefold, suggesting that this specific feature of dendritic morphology is highly regulated by action potential activity. These observations indicate that normally during this period of development, the previously described changes that occur in the morphology of the presynaptic inputs to LGN neurons are accompanied by a progressive growth of post-synaptic dendrites. Because the intracranial TTX infusions have almost certainly blocked all sodium action potentials, our results suggest that the basic dendritic framework of LGN neurons can be achieved even in the absence of this form of neural activity. Moreover, since the same treatment causes a profound change in the morphology of the presynaptic axons, at least some aspects of axonal and dendritic form must be controlled independently during this prenatal period of development.