Reproducible protocol to obtain and measure first-order relay human thalamic white-matter tracts

Available online 13 August 2022The “primary ”or “first-order relay ”nuclei of the thalamus feed the cerebral cortex with information about on- going activity in the environment or the subcortical motor systems. Because of the small size of these nuclei and the high specificity of their input and output pathways, new imaging protocols are required to investigate thala- mocortical interactions in human perception, cognition and language. The goal of the present study was twofold: I) to develop a reconstruction protocol based on in vivo diffusion MRI to extract and measure the axonal fiber tracts that originate or terminate specifically in individual first-order relay nuclei; and, II) to test the reliability of this reconstruction protocol. In left and right hemispheres, we investigated the thalamocortical/corticothalamic axon bundles linking each of the first-order relay nuclei and their main cortical target areas, namely, the lateral geniculate nucleus (optic radiation), the medial geniculate nucleus (acoustic radiation), the ventral posterior nu- cleus (somatosensory radiation) and the ventral lateral nucleus (motor radiation). In addition, we examined the main subcortical input pathway to the ventral lateral posterior nucleus, which originates in the dentate nucleus of the cerebellum. Our protocol comprised three components: defining regions-of-interest; preprocessing diffu- sion data; and modeling white-matter tracts and tractometry. We then used computation and test-retest methods to check whether our protocol could reliably reconstruct these tracts of interest and their profiles. Our results demonstrated that the protocol had nearly perfect computational reproducibility and good-to-excellent test-retest reproducibility. This new protocol may be of interest for both basic human brain neuroscience and clinical studies and has been made publicly available to the scientific community.This work was supported by grants from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (grant agreement No. 713673 ), and from “la Caixa ”Foundation (grant No. 11660016 ) to M.L.; grants from the Span- ish Ministerio de Ciencia e Innovación ( IJC2020-042887-I ; PID2021- 123577NA-I00 ) to G.L.-U.; grants from the European Union ’s Horizon 2020 Research and Innovation Program, European Commission (grant agreement No. 945539 - HBP SGA3 ) and from the Ministerio de Ciencia e Innovación FLAG-ERA grant NeuronsReunited ( MICINN-AEI PCI2019-111900-2 ) to F.C.; and grants from the Ministerio de Ciencia e Innovación ( PGC2018-093408-B-I00 ; PID2021-123574NB-I00 ), Neuro- science projects from the Fundación Tatiana Pérez de Guzmán el Bueno , Basque Government ( PIBA-2021-1-0003 ), and a grant from “la Caixa ”Banking Foundation under the project code LCF/PR/HR19/52160002 to P.M.P.-A. BCBL acknowledges support by the Basque Government through the BERC 2022-2025 program and by the S panish State Re- search Agency through BCBL Severo Ochoa excellence accreditation CEX2020-001010-S

High-Resolution Tractography Protocol to Investigate the Pathways between Human Mediodorsal Thalamic Nucleus and Prefrontal Cortex

Published: November 15, 2023Animal studies have established that the mediodorsal nucleus (MD) of the thalamus is heavily and reciprocally connected with all areas of the prefrontal cortex (PFC). In humans, however, these connections are difficult to investigate. High-resolution imaging protocols capable of reliably tracing the axonal tracts linking the human MD with each of the PFC areas may thus be key to advance our understanding of the variation, development, and plastic changes of these important circuits, in health and disease. Here, we tested in adult female and male humans the reliability of a new reconstruction protocol based on in vivo diffusion MRI to trace, measure, and characterize the fiber tracts interconnecting the MD with 39 human PFC areas per hemisphere. Our protocol comprised the following three components: (1) defining regions of interest; (2) preprocessing diffusion data; and, (3) modeling white matter tracts and tractometry. This analysis revealed largely separate PFC territories of reciprocal MD–PFC tracts bearing striking resemblance with the topographic layout observed in macaque connection-tracing studies. We then examined whether our protocol could reliably reconstruct each of these MD–PFC tracts and their profiles across test and retest sessions. Results revealed that this protocol was able to trace and measure, in both left and right hemispheres, the trajectories of these 39 area-specific axon bundles with good-to-excellent test-retest reproducibility. This protocol, which has been made publicly available, may be relevant for cognitive neuroscience and clinical studies of normal and abnormal PFC function, development, and plasticity.L.M. was supported by Horizon 2020 the European Union’s research and innovation program under Marie Skłodowska-Curie Grant 713673 and from “la Caixa” Foundation (Grants 11660016 and 100010434 under Agreement HR18-00178-DYSTHAL). G.L-U. was supported by the Spanish Ministry of Science and Innovation (Grants IJC2020-042887-I and PID2021-123577NA-I00) and the Basque Government (Grant PIBA-2022-1- 0014). F.C. was supported by the Spanish Ministry of Science and Innovation (Grants MICINN-AEI PCI2019- 111900-2 and PID2020-115780GB-I00). P.M.P-A. was supported by the Spanish Ministry of Science and Innovation (Grant PID2021-123574NB-I00), the Basque Government (Grant PIBA-2021-1-0003), and the Red guipuzcoana de Ciencia, Tecnología e Innovación of the Diputación Foral de Gipuzkoa (Grant FA/OF 422/2022), and “la Caixa” Foundation (Grant 100010434 under Agreement HR18-00178-DYSTHAL). The Basque Center on Cognition, Brain and Language (BCBL) acknowledges funding from the Basque Government through the BERC 2022-2025 program and by the Spanish State Research Agency through BCBL Severo Ochoa Excellence Accreditation CEX2020-001010-S

Posterior thalamic nucleus axon terminals have different structure and functional impact in the motor and somatosensory vibrissal cortices

Rodents extract information about nearby objects from the movement of their whiskers through dynamic computations that are carried out by a network of forebrain structures that includes the thalamus and the primary sensory (S1BF) and motor (M1wk) whisker cortices. The posterior nucleus (Po), a higher order thalamic nucleus, is a key hub of this network, receiving cortical and brainstem sensory inputs and innervating both motor and sensory whisker-related cortical areas. In a recent study in rats, we showed that Po inputs differently impact sensory processing in S1BF and M1wk. Here, in C57BL/6 mice, we measured Po synaptic bouton layer distribution and size, compared cortical unit response latencies to “in vivo” Po activation, and pharmacologically examined the glutamatergic receptor mechanisms involved. We found that, in S1BF, a large majority (56%) of Po axon varicosities are located in layer (L)5a and only 12% in L2–L4, whereas in M1wk this proportion is inverted to 18% and 55%, respectively. Light and electron microscopic measurements showed that Po synaptic boutons in M1wk layers 3–4 are significantly larger (~ 50%) than those in S1BF L5a. Electrical Po stimulation elicits different area-specific response patterns. In S1BF, responses show weak or no facilitation, and involve both ionotropic and metabotropic glutamate receptors, whereas in M1wk, unit responses exhibit facilitation to repetitive stimulation and involve ionotropic NMDA glutamate receptors. Because of the different laminar distribution of axon terminals, synaptic bouton size and receptor mechanisms, the impact of Po signals on M1wk and S1BF, although simultaneous, is likely to be markedly different.This study was supported by European Union’s Horizon 2020 (Grant Agreement no. 785907 HBP SGA2) and Ministerio de Economía y Competitividad/Fondo Europeo para el Desarrollo Regional (MINECO/FEDER) Grant BFU2017-88549 to F.C., and MINECO/FEDER Grant BFU2012-36107 to A.N

Cerebellar and basal ganglia inputs define three main nuclei in the mouse ventral motor thalamus

The thalamus is a central link between cortical and subcortical brain motor systems. Axons from the deep nuclei of the cerebellum (DCN), or the output nuclei of the basal ganglia system (substantia nigra reticulata, SNr; and internal pallidum GPi/ENT) monosynaptically innervate the thalamus, prominently some nuclei of the ventral nuclear group. In turn, axons from these ventral nuclei innervate the motor and premotor areas of the cortex, where their input is critical for planning, execution and learning of rapid and precise movements. Mice have in recent years become a widely used model in motor system research. However, information on the distribution of cerebellar and basal ganglia inputs in the rodent thalamus remains poorly defined. Here, we mapped the distribution of inputs from DCN, SNr, and GPi/ENT to the ventral nuclei of the mouse thalamus. Immunolabeling for glutamatergic and GABAergic neurotransmission markers delineated two distinct main territories, characterized each by the presence of large vesicular glutamate transporter type 2 (vGLUT2) puncta or vesicular GABA transporter (vGAT) puncta. Anterograde labeling of axons from DCN revealed that they reach virtually all parts of the ventral nuclei, albeit its axonal varicosities (putative boutons) in the vGAT-rich sector are consistently smaller than those in the vGLUT2-rich sector. In contrast, the SNr axons innervate the whole vGAT-rich sector, but not the vGLUT2-rich sector. The GPi/ENT axons were found to innervate only a small zone of the vGAT-rich sector which is also targeted by the other two input systems. Because inputs fundamentally define thalamic cell functioning, we propose a new delineation of the mouse ventral motor nuclei that is consistent with the distribution of DCN, SNr and GPi/ENT inputs and resembles the general layout of the ventral motor nuclei in primates

Benchmarking of tools for axon length measurement in individually-labeled projection neurons

Projection neurons are the commonest neuronal type in the mammalian forebrain and their individual characterization is a crucial step to understand how neural circuitry operates. These cells have an axon whose arborizations extend over long distances, branching in complex patterns and/or in multiple brain regions. Axon length is a principal estimate of the functional impact of the neuron, as it directly correlates with the number of synapses formed by the axon in its target regions; however, its measurement by direct 3D axonal tracing is a slow and labor-intensive method. On the contrary, axon length estimations have been recently proposed as an effective and accessible alternative, allowing a fast approach to the functional significance of the single neuron. Here, we analyze the accuracy and efficiency of the most used length estimation tools—design-based stereology by virtual planes or spheres, and mathematical correction of the 2D projected-axon length—in contrast with direct measurement, to quantify individual axon length. To this end, we computationally simulated each tool, applied them over a dataset of 951 3D-reconstructed axons (from NeuroMorpho.org), and compared the generated length values with their 3D reconstruction counterparts. The evaluated reliability of each axon length estimation method was then balanced with the required human effort, experience and know-how, and economic affordability. Subsequently, computational results were contrasted with measurements performed on actual brain tissue sections. We show that the plane-based stereological method balances acceptable errors (~5%) with robustness to biases, whereas the projection-based method, despite its accuracy, is prone to inherent biases when implemented in the laboratory. This work, therefore, aims to provide a constructive benchmark to help guide the selection of the most efficient method for measuring specific axonal morphologies according to the particular circumstances of the conducted research

Cyto- and Myelo-Architecture of the Amygdaloid Complex of the Common Marmoset Monkey (Callithrix jacchus)

The amygdaloid complex (AC) is a heterogeneous aggregate of nuclei located in the rostromedial region of the temporal lobe. In addition to being partly connected among themselves, the AC nuclei are strongly interconnected with the cerebral cortex, striatum, basal forebrain, hypothalamus and brainstem. Animal and human functional studies have established that the AC is a central hub of the neuronal networks supporting emotional responsivity, particularly its negative/aversive components. Dysfunction of AC circuits in humans has been implicated in anxiety, depression, schizophrenia and bipolar disorder. The small New-World marmoset monkey (Callithrix jacchus) has recently become a key model for neuroscience research. However, the nuclear and fiber tract organization of marmoset AC has not been examined in detail. Thus, the extent to which it can be compared to the AC of Old-World (human and macaque) primates is yet unclear. Here, using Nissl and acetylcholinesterase (AChE) histochemical stains as a reference, we analyzed the cytoarchitecture and nuclear parcellation of the marmoset AC. In addition, given the increasing relevance of tractographic localization for high-resolution in vivo imaging studies in non-human primates, we also identified the myelin fiber tracts present within and around the AC as revealed by the Gallyas method. The present study provides a detailed atlas of marmoset AC. Moreover, it reveals that, despite phylogenetic distance and brain size differences, every nucleus and myelinated axon bundle described in human and macaque studies can be confidently recognized in marmosets

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Unveiling the diversity of thalamocortical neuron subtypes

Our current understanding of thalamocortical (TC) circuits is largely based on studies investigating so-called 'specific' thalamic nuclei, which receive and transmit sensory-triggered input to specific cortical target areas. TC neurons in these nuclei have a striking point-to-point topography and a stereotyped laminar pattern of termination in the cortex, which has made them ideal models to study the organization, plasticity, and development of TC circuits. However, despite their experimental importance, neurons within these nuclei only represent a fraction of all thalamic neurons and do not reflect the diversity of the TC neuron population. Here we review the distinct subtypes of projection neurons that populate the thalamus, both within and across anatomically-defined nuclei, with regard to differences in their morphology, input/output connectivity and target specificity, as well as more recent findings on their neuron type-specific gene expression and development. We argue that a detailed understanding of the biology of TC neurons is critical to understand the role of the thalamus in normal and pathological perception, voluntary movement, cognition and attention

Thalamocortical Projection Neuron and Interneuron Numbers in the Visual Thalamic Nuclei of the Adult C57BL/6 Mouse

A key parameter to constrain predictive, bottom-up circuit models of a given brain domain is the number and position of the neuronal populations involved. These include not only the neurons whose bodies reside within the domain, but also the neurons in distant regions that innervate the domain. The mouse visual cortex receives its main subcortical input from the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior (LP) complex of the thalamus. The latter consists of three different nuclei: lateral posterior lateral (LPL), lateral posterior medial rostral (LPMR), and lateral posterior medial caudal (LPMC), each exhibiting specific patterns of connections with the various visual cortical areas. Here, we have determined the number of thalamocortical projection neurons and interneurons in the LP complex and dLGN of the adult C57BL/6 male mouse. We combined Nissl staining and histochemical and immunolabeling methods for consistently delineating nuclei borders, and applied unbiased stereological cell counting methods. Thalamic interneurons were identified using GABA immunolabeling. The C57BL/6 dLGN contains ∼21,200 neurons, while LP complex contains ∼31,000 total neurons. The dLGN and LP are the only nuclei of the mouse dorsal thalamus containing substantial numbers GABA-immunoreactive interneurons. These interneurons, however, are scarcer than previously estimated; they are 5.6% of dLGN neurons and just 1.9% of the LP neurons. It can be thus inferred that the dLGN contains ∼20,000 and the LP complex ∼30,400 thalamocortical projection neurons (∼12,000 in LPL, 15,200 in LPMR, and 4,200 in LPMC). The present dataset is relevant for constraining models of mouse visual thalamocortical circuits, as well as for quantitative comparisons between genetically modified mouse strains, or across species

A simple and Efficient in Vivo non-viral RNA transfection method for labeling the whole axonal tree of individual adult long-range projection neurons

We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “in vivo” transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al. 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs