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Recently, there has been increasing interest in the use of in vivo imaging approaches in the study of the way that synaptic circuits become established and the degree to which they stabilize in mature brains. We review progress since the first efforts, two decades ago, at in vivo synaptic imaging and highlight the more recent advances in molecular biology, optics and neurobiological imaging that have fueled a mini-renaissance in this line of inquiry. Many of the technical problems that limited early efforts still remain, but the rapid pace of molecular and optical innovation might soon transform this specialized field into one that is more 'mainstream'.

In developing mammalian muscle, axon branches of several motor neurons co-innervate the same muscle fibre. Competition among them results in the strengthening of one and the withdrawal of the rest. It is not known why one particular axon branch survives or why some competitions resolve sooner than others. Here we show that the fate of axonal branches is strictly related to the identity of the axons with which they compete. When two neurons co-innervate multiple target cells, the losing axon branches in each contest belong to the same neuron and are at nearly the same stage of withdrawal. The axonal arbor of one neuron engages in multiple sets of competitions simultaneously. Each set proceeds at a different rate and heads towards a common outcome based on the identity of the competitor. Competitive vigour at each of these sets of local competitions depends on a globally distributed resource: neurons with larger arborizations are at a competitive disadvantage when confronting neurons with smaller arborizations. An accompanying paper tests the idea that the amount of neurotransmitter released is this global resource.

The structural dynamics of synapses probably has a crucial role in the development and plasticity of the nervous system. In the mammalian brain, the vast majority of excitatory axo-dendritic synapses occur on dendritic specializations called 'spines'. However, little is known about their long-term changes in the intact developing or adult animal. To address this question we developed a transcranial two-photon imaging technique to follow identified spines of layer-5 pyramidal neurons in the primary visual cortex of living transgenic mice expressing yellow fluorescent protein. Here we show that filopodia-like dendritic protrusions, extending and retracting over hours, are abundant in young animals but virtually absent from the adult. In young mice, within the 'critical period' for visual cortex development, approximately 73% of spines remain stable over a one-month interval; most changes are associated with spine elimination. In contrast, in adult mice, the overwhelming majority of spines (approximately 96%) remain stable over the same interval with a half-life greater than 13 months. These results indicate that spines, initially plastic during development, become remarkably stable in the adult, providing a potential structural basis for long-term information storage.

The use of fluorescence-based calcium indicators has, over the years, unraveled important calcium-dependent mechanisms underlying neuronal function and development. However, difficulties associated with the loading of calcium indicators have limited their widespread use, particularly for the study of neuronal processing in the adult nervous system. Here, we show that in the central and peripheral nervous systems, populations of neurons and their processes, including dendritic spines and filopodia, can be labeled rapidly and efficiently by delivering calcium indicator-coated particles using a 'gene gun'. Importantly, neuronal labeling occurred both in vitro and in vivo, and across a wide range of ages and preparations. The labeled cells demonstrate spontaneous and evoked calcium transients, indicating that particle-mediated delivery is not deleterious to neuronal function. Furthermore, unlike loading with patch pipettes, cytoplasmic content is preserved following ballistic loading. This enables the study of calcium-dependent second messenger pathways without loss of signaling components. The ballistic delivery of calcium indicators thus opens up many new avenues for further exploration of the structure and function of the nervous system from single spines to neuronal networks.

1. Coherent network oscillations in several distinct frequency bands are seen in the hippocampus of behaving animals. To investigate how different neuronal types within this network respond to oscillatory inputs we made whole-cell current clamp recordings from three different types of neurones in the CA1 region of rat hippocampal slices: pyramidal cells, fast-spiking interneurones and horizontal interneurones, and recorded their response to sinusoidal inputs at physiologically relevant frequencies (1-100 Hz). 2. Pyramidal neurones showed firing preference to inputs at theta frequencies (range 2-7 Hz; n = 30). They showed subthreshold resonance in the same frequency range (2-7 Hz; mean 4.1 +/- 0.4 Hz; n = 19). 3. Interneurones differed in their firing properties. Horizontal interneurones in the stratum oriens showed firing preference to inputs at theta frequencies (range 1.5-10 Hz; n = 10). These interneurones also showed resonance at low frequencies (range 1-5 Hz; mean 2.4 +/- 0.5 Hz; n = 7). In contrast, fast-spiking interneurones with cell bodies in the pyramidal cell layer fired preferentially at input frequencies in the gamma band (range 30-50 Hz; n = 10/12). These interneurones showed resonance at beta-gamma frequencies (10-50 Hz; mean 26 +/- 5 Hz; n = 7/8). 4. Thus, in the hippocampus, different types of neurones have distinct frequency preferences. Therefore, in the CA1 layer of the hippocampal network, a compound oscillatory input may be segregated into distinct frequency components which are processed locally by distinct types of neurones.