Presynaptic supervision of cortical spine dynamics in motor learning.

In mammalian neocortex, learning triggers the formation and turnover of new postsynaptic spines on pyramidal cell dendrites. However, the biological principles of spine reorganization during learning remain elusive because the identity of their presynaptic neuronal partners is unknown. Here, we show that two presynaptic neural circuits supervise distinct programs of spine dynamics to execute learning. We imaged spine dynamics in motor cortex during learning and performed post hoc identification of their afferent presynaptic neurons. New spines that appeared during learning formed small transient contacts with corticocortical neurons that were eliminated on skill acquisition. In contrast, persistent spines with axons from thalamic neurons were formed and enlarged. These results suggest that pyramidal cell dendrites in motor cortex use a neural circuit division of labor during skill learning, with dynamic teaching contacts from top-down intracortical axons followed by synaptic memory formation driven by thalamic axons. Dual spine supervision may govern diverse skill learning in the neocortex.

A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure.

Automated tape-collecting ultramicrotomy in conjunction with scanning electron microscopy (SEM) is a powerful approach for volume electron microscopy and three-dimensional neuronal circuit analysis. Current tapes are limited by section wrinkle formation, surface scratches and sample charging during imaging. Here we show that a plasma-hydrophilized carbon nanotube (CNT)-coated polyethylene terephthalate (PET) tape effectively resolves these issues and produces SEM images of comparable quality to those from transmission electron microscopy. CNT tape can withstand multiple rounds of imaging, offer low surface resistance across the entire tape length and generate no wrinkles during the collection of ultrathin sections. When combined with an enhanced en bloc staining protocol, CNT tape-processed brain sections reveal detailed synaptic ultrastructure. In addition, CNT tape is compatible with post-embedding immunostaining for light and electron microscopy. We conclude that CNT tape can enable high-resolution volume electron microscopy for brain ultrastructure analysis.

Selective Thalamic Innervation of Rat Frontal Cortical Neurons.

Most glutamatergic inputs in the neocortex originate from the thalamus or neocortical pyramidal cells. To test whether thalamocortical afferents selectively innervate specific cortical cell subtypes and surface domains, we investigated the distribution patterns of thalamocortical and corticocortical excitatory synaptic inputs in identified postsynaptic cortical cell subtypes using intracellular and immunohistochemical staining combined with confocal laser scanning and electron microscopic observations in 2 thalamorecipient sublayers, lower layer 2/3 (L2/3b) and lower layer 5 (L5b) of rat frontal cortex. The dendrites of GABAergic parvalbumin (PV) cells preferentially received corticocortical inputs in both sublayers. The somata of L2/3b PV cells received thalamic inputs in similar proportions to the basal dendritic spines of L2/3b pyramidal cells, whereas L5b PV somata were mostly innervated by cortical inputs. The basal dendrites of L2/3b pyramidal and L5b corticopontine pyramidal cells received cortical and thalamic glutamatergic inputs in proportion to their local abundance, whereas crossed-corticostriatal pyramidal cells in L5b exhibited a preference for thalamic inputs, particularly in their distal dendrites. Our data demonstrate an exquisite selectivity among thalamocortical afferents in which synaptic connectivity is dependent on the postsynaptic neuron subtype, cortical sublayer, and cell surface domain.

Functional effects of distinct innervation styles of pyramidal cells by fast spiking cortical interneurons.

Inhibitory interneurons target precise membrane regions on pyramidal cells, but differences in their functional effects on somata, dendrites and spines remain unclear. We analyzed inhibitory synaptic events induced by cortical, fast-spiking (FS) basket cells which innervate dendritic shafts and spines as well as pyramidal cell somata. Serial electron micrograph (EMg) reconstructions showed that somatic synapses were larger than dendritic contacts. Simulations with precise anatomical and physiological data reveal functional differences between different innervation styles. FS cell soma-targeting synapses initiate a strong, global inhibition, those on shafts inhibit more restricted dendritic zones, while synapses on spines may mediate a strictly local veto. Thus, FS cell synapses of different sizes and sites provide functionally diverse forms of pyramidal cell inhibition.

Important factors for the three-dimensional reconstruction of neuronal structures from serial ultrathin sections.

Quantitative analysis of anatomical synaptic connectivity in microcircuits depends upon accurate three-dimensional (3D) reconstructions of synaptic ultrastructure using electron microscopy of serial ultrathin sections. Here we address two pitfalls in current methodology that lead to inaccurate reconstructions and compromise conclusions drawn from the data. The first pitfall is inaccurate determination of ultrathin section thickness, which negatively affects the 3D shape of reconstructions and therefore impairs quantitative measurement of synaptic structures. Secondly, current methodology significantly underestimates the number of synaptic junctions, with only two-thirds or less of genuine synaptic contacts being identified in dendrites that radiate within the plane of section. Here we propose a new methodology utilizing precise optical measurements of section thickness and successive observations of synaptic elements across serial ultrathin sections that corrects for these limitations to allow accurate 3D reconstruction of synaptic ultrastructure. We use this methodology to reveal that parvalbumin-expressing cortical interneurons have a much higher synaptic density than previously shown. This result suggests that this technique will be useful for re-examining synaptic connectivity of other cell types.

Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents.

Fast inhibition in the cortex is gated primarily at GABAergic synapses formed by local interneurons onto postsynaptic targets. Although GABAergic inputs to the somata and axon initial segments of neocortical pyramidal neurons are associated with direct inhibition of action potential generation, the role of GABAergic inputs to distal dendritic segments, including spines, is less well characterized. Because a significant proportion of inhibitory input occurs on distal dendrites and spines, it will be important to determine whether these GABAergic synapses are formed selectively by certain classes of presynaptic cells onto specific postsynaptic elements. By electron microscopic observations of synapses formed by different subtypes of nonpyramidal cells, we found that a surprisingly large fraction (33.4 +/- 9.3%) of terminals formed symmetrical synaptic junctions onto a subset of cortical spines that were mostly coinnervated by an asymmetrical terminal. Using VGLUT1 and VGLUT2 isoform of the glutamate vesicular transporter immunohistochemistry, we found that the double-innervated spines selectively received thalamocortical afferents expressing the VGLUT2 but almost never intracortical inputs expressing the VGLUT1. When comparing the volumes of differentially innervated spines and their synaptic junction areas, we found that spines innervated by VGLUT2-positive terminal were significantly larger than spines innervated by VGLUT1-positive terminal and that these spines had larger, and more often perforated, synapses than those of spines innervated by VGLUT1-positive afferent. These results demonstrate that inhibitory inputs to pyramidal cell spines may preferentially reduce thalamocortical rather than intracortical synaptic transmission and are therefore positioned to selectively gate extracortical information.

Further mapping of quantitative trait loci for postnatal growth in an inter-sub-specific backcross of wild Mus musculus castaneus and C57BL/6J mice.

We performed a quantitative trait locus (QTL) analysis of eight body weights recorded weekly from 3 weeks to 10 weeks after birth and two weight gains recorded between 3 weeks and 6 weeks, and between 6 weeks and 10 weeks in an inter-sub-specific backcross population of wild Mus musculus castaneus mice captured in the Philippines and the common inbred strain C57BL/6J ( M. musculus domesticus ), to elucidate the complex genetic architecture of body weight and growth. Interval mapping identified 17 significant QTLs with main effects on 11 chromosomes. In particular, the main effect of the most potent QTL on proximal chromosome 2 increased linearly with age, whereas other QTLs exerted effects on either the early or late growth period. Surprisingly, although wild mice displayed 60% of the body size of their C57BL/6J counterparts, the wild-derived allele enhanced growth at two QTLs. Interestingly, five of the 17 main-effect QTLs identified had significant epistatic interaction effects. Five new epistatic QTLs with no main effects were identified on different chromosomes or regions. For one pair of epistatic QTLs, mice that were heterozygous for the wild-derived allele at one QTL and homozygous for that allele at another QTL exhibited the most rapid growth in all four possible genotypic combinations. Out of the identified QTLs, several showed significant sex-specific effects.