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

[This corrects the article DOI: 10.3389/fnana.2023.1242839.].

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.

A Combinatorial Input Landscape in the "Higher-Order Relay" Posterior Thalamic Nucleus.

All pathways targeting the thalamus terminate directly onto the thalamic projection cells. As these cells lack local excitatory interconnections, their computations are fundamentally defined by the type and local convergence patterns of the extrinsic inputs. These two key variables, however, remain poorly defined for the "higher-order relay" (HO) nuclei that constitute most of the thalamus in large-brained mammals, including humans. Here, we systematically analyzed the input landscape of a representative HO nucleus of the mouse thalamus, the posterior nucleus (Po). We examined in adult male and female mice the neuropil distribution of terminals immunopositive for markers of excitatory or inhibitory neurotransmission, mapped input sources across the brain and spinal cord and compared the intranuclear distribution and varicosity size of axons originated from each input source. Our findings reveal a complex landscape of partly overlapping input-specific microdomains. Cortical layer (L)5 afferents from somatosensory and motor areas predominate in central and ventral Po but are relatively less abundant in dorsal and lateral portions of the nucleus. Excitatory inputs from the trigeminal complex, dorsal column nuclei (DCN), spinal cord and superior colliculus as well as inhibitory terminals from anterior pretectal nucleus and zona incerta (ZI) are each abundant in specific Po regions and absent from others. Cortical L6 and reticular thalamic nucleus terminals are evenly distributed across Po. Integration of specific input motifs by particular cell subpopulations may be commonplace within HO nuclei and favor the emergence of multiple, functionally diverse input-output subnetworks.SIGNIFICANCE STATEMENT Because thalamic projection neurons lack local interconnections, their output is essentially determined by the kind and convergence of the long-range inputs that they receive. Fragmentary evidence suggests that these parameters may vary within the "higher-order relay" (HO) nuclei that constitute much of the thalamus, but such variation has not been systematically analyzed. Here, we mapped the origin and local convergence of all the extrinsic inputs reaching the posterior nucleus (Po), a typical HO nucleus of the mouse thalamus by combining multiple neuropil labeling and axon tracing methods. We report a complex mosaic of partly overlapping input-specific domains within Po. Integration of different input motifs by specific cell subpopulations in HO nuclei may favor the emergence of multiple, computationally specialized thalamocortical subnetworks.

Implication of type 4 NADPH oxidase (NOX4) in tauopathy.

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.

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.

Variation of connectivity across exemplar sensory and associative thalamocortical loops in the mouse.

The thalamus engages in sensation, action, and cognition, but the structure underlying these functions is poorly understood. Thalamic innervation of associative cortex targets several interneuron types, modulating dynamics and influencing plasticity. Is this structure-function relationship distinct from that of sensory thalamocortical systems? Here, we systematically compared function and structure across a sensory and an associative thalamocortical loop in the mouse. Enhancing excitability of mediodorsal thalamus, an associative structure, resulted in prefrontal activity dominated by inhibition. Equivalent enhancement of medial geniculate excitability robustly drove auditory cortical excitation. Structurally, geniculate axons innervated excitatory cortical targets in a preferential manner and with larger synaptic terminals, providing a putative explanation for functional divergence. The two thalamic circuits also had distinct input patterns, with mediodorsal thalamus receiving innervation from a diverse set of cortical areas. Altogether, our findings contribute to the emerging view of functional diversity across thalamic microcircuits and its structural basis.

Area-Specific Synapse Structure in Branched Posterior Nucleus Axons Reveals a New Level of Complexity in Thalamocortical Networks.

Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Here, using fine-scale high-end 3D electron microscopy, we demonstrate in adult male C57BL/6 wild-type mice marked differences between MC versus S1 Po synapses in (1) bouton and active zone size, (2) neurotransmitter vesicle pool size, (3) distribution of mitochondria around synapses, and (4) proportion of synapses established on dendritic spines and dendritic shafts. These differences are as large, or even more pronounced, than those between Po and ventro-posterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we demonstrate that the above differences actually occur on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Along with recently-discovered divergences in efficacy and plasticity, the synaptic structure differences reported here thus reveal a new subcellular level of complexity. This is a finding that upends current models of thalamocortical circuitry, and that might as well illuminate the functional logic of other branched projection axon systems.SIGNIFICANCE STATEMENT Many long-distance brain connections depend on neurons whose branched axons target separate regions. Using 3D electron microscopy and single-cell transfection, we investigated the mouse Posterior thalamic nucleus (Po) cell axons that simultaneously innervate motor and sensory areas of the cerebral cortex involved in whisker movement control. We demonstrate significant differences in the size of the boutons made in each area by individual Po axons, as well as in functionally-relevant parameters in the composition of their synapses. In addition, we found similarly large differences between the synapses of Po versus ventral posteromedial thalamic nucleus axons in the whisker sensory cortex. Area-specific synapse structure in individual axons implies a new, unsuspected level of complexity in long-distance brain connections.

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.

A highly collateralized thalamic cell type with arousal-predicting activity serves as a key hub for graded state transitions in the forebrain.

Sleep cycles consist of rapid alterations between arousal states, including transient perturbation of sleep rhythms, microarousals, and full-blown awake states. Here we demonstrate that the calretinin (CR)-containing neurons in the dorsal medial thalamus (DMT) constitute a key diencephalic node that mediates distinct levels of forebrain arousal. Cell-type-specific activation of DMT/CR+ cells elicited active locomotion lasting for minutes, stereotyped microarousals, or transient disruption of sleep rhythms, depending on the parameters of the stimulation. State transitions could be induced in both slow-wave and rapid eye-movement sleep. The DMT/CR+ cells displayed elevated activity before arousal, received selective subcortical inputs, and innervated several forebrain sites via highly branched axons. Together, these features enable DMT/CR+ cells to summate subcortical arousal information and effectively transfer it as a rapid, synchronous signal to several forebrain regions to modulate the level of arousal.

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.

Specific activation of the paralemniscal pathway during nociception.

Two main neuronal pathways connect facial whiskers to the somatosensory cortex in rodents: (i) the lemniscal pathway, which originates in the brainstem principal trigeminal nucleus and is relayed in the ventroposterior thalamic nucleus and (ii) the paralemniscal pathway, originating in the spinal trigeminal nucleus and relayed in the posterior thalamic nucleus. While lemniscal neurons are readily activated by whisker contacts, the contribution of paralemniscal neurons to perception is less clear. Here, we functionally investigated these pathways by manipulating input from the whisker pad in freely moving mice. We report that while lemniscal neurons readily respond to neonatal infraorbital nerve sectioning or whisker contacts in vivo, paralemniscal neurons do not detectably respond to these environmental changes. However, the paralemniscal pathway is specifically activated upon noxious stimulation of the whisker pad. These findings reveal a nociceptive function for paralemniscal neurons in vivo that may critically inform context-specific behaviour during environmental exploration.

Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice.

Transgenic mouse lines in which a fluorescent protein is constitutively expressed under the Thy1 gene promoter have become important models in cell biology and pathology studies of specific neuronal populations. As a result of positional insertion and/or copy number effects on the transgene, the populations expressing the fluorescent protein (eYFP+) vary markedly among the different mice lines. However, identification of the eYFP+ subpopulations has remained sketchy and fragmentary even for the most widely used lines such as Thy1-eYFP-H mice (Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman and J.W., Sanes. J.R. 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41-51). Here, we provide a comprehensive mapping of labeled cell types throughout the central nervous system in adult and postnatal (P0-P30) Thy1-eYFP-H mice. Cell type identification was based on somatodendritic morphology, axon trajectories, and, for cortical cells, retrograde labeling with Fast Blue to distinguish between subpopulations with different axonal targets. In the neocortex, eYFP+ cells are layers 5 and 6 pyramidal neurons, whose abundance and sublaminar distribution varies markedly between areas. Labeling is particularly prevalent in the corticospinal cells; as a result, the pyramidal pathway axons are conspicuously labeled down to the spinal cord. Large populations of hippocampal, subicular and amygdaloid projection neurons are eYFP+ as well. Additional eYFP+ cell groups are located in specific brainstem nuclei. Present results provide a comprehensive reference dataset for adult and developmental studies using the Thy1-eYFP-H mice strain, and show that this animal model may be particularly suitable for studies on the cell biology of corticospinal neurons.

Thalamic input to distal apical dendrites in neocortical layer 1 is massive and highly convergent.

Input to apical dendritic tufts is now deemed crucial for associative learning, attention, and similar "feedback" interactions in the cerebral cortex. Excitatory input to apical tufts in neocortical layer 1 has been traditionally assumed to be predominantly cortical, as thalamic pathways directed to this layer were regarded relatively scant and diffuse. However, the sensitive tracing methods used in the present study show that, throughout the rat neocortex, large numbers (mean approximately 4500/mm(2)) of thalamocortical neurons converge in layer 1 and that this convergence gives rise to a very high local density of thalamic terminals. Moreover, we show that the layer 1-projecting neurons are present in large numbers in most, but not all, motor, association, limbic, and sensory nuclei of the rodent thalamus. Some layer 1-projecting axons branch to innervate large swaths of the cerebral hemisphere, whereas others arborize within only a single cortical area. Present data imply that realistic modeling of cortical circuitry should factor in a dense axonal canopy carrying highly convergent thalamocortical input to pyramidal cell apical tufts. In addition, they are consistent with the notion that layer 1-projecting axons may be a robust anatomical substrate for extensive "feedback" interactions between cortical areas via the thalamus.

[Diversity in thalamic relay neurons: evidence for "bottom-up" and "top-down" information flow in thalamocortical pathways]. / Nuevas perspectivas anatomofuncionales sobre las redes talamocorticales.

Thalamocortical (TC) pathways are still mainly understood as the gateway for ascending sensory-motor information into the cortex. However, it is now clear that a great many TC cells are involved in interactions between cortical areas via the thalamus. We review recent data, including our own, which demonstrate the generalized presence in rodent thalamus of two major TC cell types characterized, among other features, by their axon development, arborization and laminar targeting in the cortex. Such duality may allow inputs from thalamus to access cortical circuits via "bottom-up"-wired axon arbors or via "top-down"-wired axon arbors.

Embryonic and postnatal development of the layer I-directed ("matrix") thalamocortical system in the rat.

Inputs to the layer I apical dendritic tufts of pyramidal cells are crucial in "top-down" interactions in the cerebral cortex. A large population of thalamocortical cells, the "matrix" (M-type) cells, provides a direct robust input to layer I that is anatomically and functionally different from the thalamocortical input to layer VI. The developmental timecourse of M-type axons is examined here in rats aged E (embryonic day) 16 to P (postnatal day) 30. Anterograde techniques were used to label axons arising from 2 thalamic nuclei mainly made up of M-type cells, the Posterior and the Ventromedial. The primary growth cones of M-type axons rapidly reached the subplate of dorsally situated cortical areas. After this, interstitial branches would sprout from these axons under more lateral cortical regions to invade the overlying cortical plate forming secondary arbors. Moreover, retrograde labeling of M-type cell somata in the thalamus after tracer deposits confined to layer I revealed that large numbers of axons from multiple thalamic nuclei had already converged in a given spot of layer I by P3. Because of early ingrowth in such large numbers, interactions of M-type axons may significantly influence the early development of cortical circuits.

Extracellular matrix molecules and synaptic plasticity: immunomapping of intracellular and secreted Reelin in the adult rat brain.

Reelin, a large extracellular matrix glycoprotein, is secreted by several neuron populations in the developing and adult rodent brain. Secreted Reelin triggers a complex signaling pathway by binding lipoprotein and integrin membrane receptors in target cells. Reelin signaling regulates migration and dendritic growth in developing neurons, while it can modulate synaptic plasticity in adult neurons. To identify which adult neural circuits can be modulated by Reelin-mediated signaling, we systematically mapped the distribution of Reelin in adult rat brain using sensitive immunolabeling techniques. Results show that the distribution of intracellular and secreted Reelin is both very widespread and specific. Some interneuron and projection neuron populations in the cerebral cortex contain Reelin. Numerous striatal neurons are weakly immunoreactive for Reelin and these cells are preferentially located in striosomes. Some thalamic nuclei contain Reelin-immunoreactive cells. Double-immunolabeling for GABA and Reelin reveals that the Reelin-immunoreactive cells in the visual thalamus are the intrinsic thalamic interneurons. High local concentrations of extracellular Reelin selectively outline several dendrite spine-rich neuropils. Together with previous mRNA data, our observations suggest abundant axoplasmic transport and secretion in pathways such as the retino-collicular tract, the entorhino-hippocampal ('perforant') path, the lateral olfactory tract or the parallel fiber system of the cerebellum. A preferential secretion of Reelin in these neuropils is consistent with reports of rapid, activity-induced structural changes in adult brain circuits.