Near-infrared (NIR) radiation has been employed using one- and two-photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR-low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

We found that rat central auditory neurons fire action potentials in a precise sequence of mini-bursts prior to hearing onset. This stereotyped pattern is initiated by hair cells within the cochlea, which trigger brief bursts of action potentials in auditory neurons each time they fire a Ca2+ spike. By generating theta-like activity, hair cells may limit the influence of synaptic depression in developing auditory circuits and promote consolidation of synapses.

The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held.auditory system ͉ medial nucleus of the trapezoid body ͉ two-photon imaging ͉ structural plasticity ͉ axon collateral S tudying the formation of individual, identified synapses in the CNS presents a formidable challenge because of their small size, their incredibly high density, and their protracted formation period (1). Imaging studies in living animals have provided insights into the structural changes that presynaptic axons undergo during development, which complements our understanding of how specific brain connections form (2). An emerging view from these studies is that axonal dynamics are age-and cell type-dependent (3) and strongly correlated with the formation of synaptic contacts, which may ultimately guide the growth of the axonal arbor (2, 4-6).Here, we study the development of a CNS synapse that can be identified relatively easily because it is probably the largest synaptic contact in the mammalian brain (7). The calyx of Held connects the globular bushy cells of the anteroventral cochlear nucleus and the principal cells of the medial nucleus of the trapezoid body (MNTB) in the brainstem. Studies in rodents have shown that shortly before birth, the principal cells of the MNTB are innervated by small glutamatergic boutons (8). Morphological and functional identification of nascent calyces is possible between postnatal days 3 and 5 (8-10), which suggests that the characteristic one-to-one innervation observed in mature animals is achieved very rapidly (10). However, previous studies have not been able to study the dynamic aspects of the calyx of Held development.As a first step toward elucidating the cellular mechanisms that ensure that each MNTB principal cell is always innervated by only a single calyx of Held, we sought an imaging approach. Because it has not been possible to study this unique synapse in culture, we carried out studies in vivo. We developed a surgical procedure to label brainstem axons in anesthetized rat pups and imaged them with a two-photon microscope. Using this approach, we provide evidence of structural dynamics r...