In vivo intracellular recordings of hippocampal neurons reveal the occurrence of fast events of small amplitude called spikelets or fast prepotentials. Because intracellular recordings have been restricted to anesthetized or head-fixed animals, it is not known how spikelet activity contributes to hippocampal spatial representations. We addressed this question in CA1 pyramidal cells by using in vivo whole-cell recording in freely moving rats. We observed a high incidence of spikelets that occurred either in isolation or in bursts and could drive spiking as fast prepotentials of action potentials. Spikelets strongly contributed to spiking activity, driving approximately 30% of all action potentials. CA1 pyramidal cell firing and spikelet activity were comodulated as a function of the animal’s location in the environment. We conclude that spikelets have a major impact on hippocampal activity during spatial exploration.

Dopaminergic neurons in mammals respond to rewards and reward-predicting cues, and are thought to play an important role in learning actions or sensory cues that lead to reward. The anatomical sources of input that drive or modulate such responses are not well understood; these ultimately define the range of behavior to which dopaminergic neurons contribute. Primary rewards are not the immediate objective of all goal-directed behavior. For example, a goal of vocal learning is to imitate vocal-communication signals. Here, we demonstrate activation of dopaminergic neurons in songbirds driven by a basal ganglia region required for vocal learning, area X. Dopaminergic neurons in anesthetized zebra finches respond more strongly to the bird’s own song (BOS) than to other sounds, and area X is critical for these responses. Direct pharmacological modulation of area X output, in the absence of auditory stimulation, is sufficient to bidirectionally modulate the firing rate of dopaminergic neurons. The only known pathway from song control regions to dopaminergic neurons involves a projection from area X to the ventral pallidum (VP), which in turn projects to dopaminergic regions. We show that VP neurons are spontaneously active and inhibited preferentially by BOS, suggesting that area X disinhibits dopaminergic neurons by inhibiting VP. Supporting this model, auditory-response latencies are shorter in area X than VP, and shorter in VP than dopaminergic neurons. Thus, dopaminergic neurons can be disinhibited selectively by complex sensory stimuli via input from the basal ganglia. The functional pathway we identify may allow dopaminergic neurons to contribute to vocal learning.

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued, discrete, alternate phenotypes (polyphenisms) during its life cycle. In the case of the reproductive polyphenism, differences in day length determine whether mothers will produce daughters that reproduce either sexually by laying fertilized eggs (oviparous sexual reproduction), or asexually by allowing oocytes to complete embryogenesis within the mother without fertilization (viviparous parthenogenesis). Oocytes and embryos that are produced asexually and develop within the mother develop more rapidly, are yolk-free, and much smaller than oocytes and embryos that are produced sexually. These overt differences suggest that there may be underlying differences in the molecular mechanisms of pattern formation. Indeed, our preliminary comparative gene expression work suggests that there are important differences in the terminal patterning system, involving the Torso pathway, between viviparous and oviparous development. We have so far examined the expression of homologs of torso-like and capicua, members of the Drosophila Torso pathway. We have detected clear differential expression of torso-like and possible differential expression of capicua. Establishing such differences in the expression of patterning genes between these developmental modes is a first step toward understanding how a single genome manages to direct patterning events in such different embryological contexts.

Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set.

Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Ca(v)2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Ca(v)2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.

Data acquisition of cryo-electron tomography (CET) samples described in previous chapters involves relatively imprecise mechanical motions: the tilt series has shifts, rotations, and several other distortions between projections. Alignment is the procedure of correcting for these effects in each image and requires the estimation of a projection model that describes how points from the sample in three-dimensions are projected to generate two-dimensional images. This estimation is enabled by finding corresponding common features between images. This chapter reviews several software packages that perform alignment and reconstruction tasks completely automatically (or with minimal user intervention) in two main scenarios: using gold fiducial markers as high contrast features or using relevant biological structures present in the image (marker-free). In particular, we emphasize the key decision points in the process that users should focus on in order to obtain high-resolution reconstructions.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Mitochondria are organelles that have been primarily known as the powerhouse of the cell. However, recent advances in the field have revealed that mitochondria are also involved in many other cellular activities like lipid modifications, redox balance, calcium balance, and even controlled cell death. These multifunctional organelles are motile and highly dynamic in shapes and forms; the dynamism is brought about by the mitochondria's ability to undergo fission and fusion with each other. Therefore, it is very important to be able to image mitochondrial shape changes to relate to the variety of cellular functions these organelles have to accomplish. The protocols described here will enable researchers to perform steady-state and time-lapse imaging of mitochondria in live cells by using confocal microscopy. High-resolution three-dimensional imaging of mitochondria will not only be helpful in understanding mitochondrial structure in detail but it also could be used to analyze their structural relationships with other organelles in the cell. FRAP (fluorescence recovery after photobleaching) studies can be performed to understand mitochondrial dynamics or dynamics of any mitochondrial molecule within the organelle. The microirradiation assay can be performed to study functional continuity between mitochondria. A protocol for measuring mitochondrial potential has also been included in this unit. In conclusion, the protocols described here will aid the understanding of mitochondrial structure-function relationship.