Integration of signals from different cortical areas in higher order thalamic neurons

Higher order thalamic neurons receive driving inputs from cortical layer 5 and project back to the cortex, reflecting a transthalamic route for corticocortical communication. To determine whether or not individual neurons integrate signals from different cortical populations, we combined electron microscopy “connectomics” in mice with genetic labeling to disambiguate layer 5 synapses from somatosensory and motor cortices to the higher order thalamic posterior medial nucleus. A significant convergence of these inputs was found on 19 of 33 reconstructed thalamic cells, and as a population, the layer 5 synapses were larger and located more proximally on dendrites than were unlabeled synapses. Thus, many or most of these thalamic neurons do not simply relay afferent information but instead integrate signals as disparate in this case as those emanating from sensory and motor cortices. These findings add further depth and complexity to the role of the higher order thalamus in overall cortical functioning.

Genetic Labeling of Cells Allows Identification and Tracking of Transgenic Platelets in Mice

Background: The use of knock-out mouse models is crucial to understand platelet activation and aggregation. Methods: Analysis of the global double fluorescent Cre reporter mouse mT/mG that has been crossbred with the megakaryocyte/platelet specific PF4-Cre mouse. Results: Platelets show bright mT (PF4-Cre negative) and mG (PF4-Cre positive) fluorescence. However, a small proportion of leukocytes was positive for mG fluorescence in PF4-Cre positive mice. In mT/mG;PF4-Cre mice, platelets, and megakaryocytes can be tracked by their specific fluorescence in blood smear, hematopoietic organs and upon thrombus formation. No differences in platelet activation and thrombus formation was observed between mT/mG;PF4-Cre positive and negative mice. Furthermore, hemostasis and in vivo thrombus formation was comparable between genotypes as analyzed by intravital microscopy. Transplantation studies revealed that bone marrow of mT/mG;PF4-Cre mice can be transferred to C57BL/6 mice. Conclusions: The mT/mG Cre reporter mouse is an appropriate model for real-time visualization of platelets, the analysis of cell morphology and the identification of non-recombined platelets. Thus, mT/mG;PF4-Cre mice are important for the analysis of platelet-specific knockout mice. However, a small proportion of leukocytes exhibit mG fluorescence. Therefore, the analysis of platelets beyond hemostasis and thrombosis should be critically evaluated when recombination of immune cells is increased.

Pyramidal Neurons of the Zebrafish Tectum Receive Highly Convergent Input From Torus Longitudinalis

The torus longitudinalis (TL) is a midbrain structure unique to ray finned fish. Although previously implicated in orienting behaviors elicited by changes in ambient lighting, the role of TL in visual processing is not well-understood. TL is reciprocally connected to tectum and is the only known source of synaptic input to the stratum marginalis (SM) layer of tectal neuropil. Conversely, tectal pyramidal neurons (PyrNs) are the only identified tectal neuron population that forms a dendrite in SM. In this study we describe a zebrafish gal4 transgenic that labels TL neurons that project to SM. We demonstrate that the axonal TL projection to SM in zebrafish is glutamatergic. Consistent with these axons synapsing directly onto PyrNs, SM-targeted dendrites of PyrNs contain punctate enrichments of the glutamatergic post-synaptic marker protein PSD95. Sparse genetic labeling of individual TL axons and PyrN dendrites enabled quantitative morphometric analysis that revealed (1) large, sparsely branched TL axons in SM and (2) small, densely innervated PyrN dendrites in SM. Together this unique combination of morphologies support a wiring diagram in which TL inputs to PyrNs exhibit a high degree of convergence. We propose that this convergence functions to generate large, compound visual receptive fields in PyrNs. This quantitative anatomical data will instruct future functional studies aimed at identifying the precise contribution of TL-PyrN circuitry to visual behavior.

Cocaine causes rapid remodeling of mesoaccumbens dopaminergic axons, synapses, and mitochondria

Detailing the ways drugs of abuse physically alter dopaminergic circuits would provide new mechanisms for explaining addictive behaviors, future targets for therapeutic intervention, and insights into the nature of synaptic plasticity. We combine recent advances in genetic labeling with large volume serial electron microscopy to detail how normal dopaminergic (DA) axons interact with putative targets in the Nucleus Accumbens (NAc) and how those interactions change in mice briefly exposed to cocaine. We find that while most DA axonal boutons are devoid of obvious signs of synapses (i.e. synaptic vesicles or synaptic densities), many DA boutons physically interdigitate with dendrites or excitatory and inhibitory axons. A brief exposure to cocaine results in large-scale remodeling: extensive DA axonal branching and frequent occurrences of axonal blind-ended “bulbs”, filled with mitochondria and reminiscent of axonal retraction in the developing and damaged brain. The number of physical interdigitations and vesicle filled boutons in DA axons scales linearly with the length of axon in both controls and cocaine exposed animals and the size or the type of interaction (i.e. axo-axonic or axo-dendritic) do not change. Finally, we find in cocaine exposed animals, mitochondrial lengths are increased ~2.5 times relative to control. Mitochondrial elongation is cell type specific: primarily in DA neurons and downstream spiny dendrites, and localized to DA axons and not DA soma or dendrites. We show for the first time the effects of cocaine on remodeling of dopamine axon morphology and mitochondria and reveal new details on how dopamine neurons physically associate with downstream targets.

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Although several sleep-regulating neuronal populations have been identified, little is known about how they interact with each other to control sleep/wake states. We previously identified neuropeptide VF (NPVF) and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). Here we show using zebrafish that npvf-expressing neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that is critical for sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, we show that npvf-expressing neurons innervate and can activate serotonergic RN neurons. We also demonstrate that chemogenetic or optogenetic stimulation of npvf-expressing neurons induces sleep in a manner that requires NPVF and serotonin in the RN. Finally, we provide genetic evidence that NPVF acts upstream of serotonin in the RN to maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain neuronal circuit for sleep/wake control.

Molecular and genetic approaches for assaying human cell type synaptic connectivity

ABSTRACTProspective and post-hoc molecular identification of specific neuron types is essential for functional studies of cellular and synaptic properties. We demonstrate a thick brain slice mFISH technique applied to multi-patch-clamp recordings in human cortical slices obtained from neurosurgical-excised tissue to reveal the molecular and morpho-electric properties of synaptically connected neurons, both with and without prospective AAV based genetic labeling. This “quadruple modality” methodology should be extensible to other local brain circuits in many organisms.