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•The thalamus contains a diversity set of cortical connections that are associated with differential genetic expression.•We used genetic expression of calcium binding proteins to relate thalamus and cortex functional connectivity.•We observed a unique relationships between the thalamus and cortical gradients, timescales and dynamics.•The patterns were replicated in independent 7T and 3T resting-state fMRI datasets. Recent neuroimaging experiments have defined low-dimensional gradients of functional connectivity in the cerebral cortex that subserve a spectrum of capacities that span from sensation to cognition. Despite well-known anatomical connections to the cortex, the subcortical areas that support cortical functional organization have been relatively overlooked. One such structure is the thalamus, which maintains extensive anatomical and functional connections with the cerebral cortex across the cortical mantle. The thalamus has a heterogeneous cytoarchitecture, with at least two distinct cell classes that send differential projections to the cortex: granular-projecting ‘Core’ cells and supragranular-projecting ‘Matrix’ cells. Here we use high-resolution 7T resting-state fMRI data and the relative amount of two calcium-binding proteins, parvalbumin and calbindin, to infer the relative distribution of these two cell-types (Core and Matrix, respectively) in the thalamus. First, we demonstrate that thalamocortical connectivity recapitulates large-scale, low-dimensional connectivity gradients within the cerebral cortex. Next, we show that diffusely-projecting Matrix regions preferentially correlate with cortical regions with longer intrinsic fMRI timescales. We then show that the Core–Matrix architecture of the thalamus is important for understanding network topology in a manner that supports dynamic integration of signals distributed across the brain. Finally, we replicate our main results in a distinct 3T resting-state fMRI dataset. Linking molecular and functional neuroimaging data, our findings highlight the importance of the thalamic organization for understanding low-dimensional gradients of cortical connectivity.

Cortico-basal ganglia-thalamocortical loops are largely conceived as parallel circuits that process limbic, associative, and sensorimotor information separately. Whether and how these functionally distinct loops interact remains unclear. Combining genetic and viral approaches, we systemically mapped the limbic and motor cortico-basal ganglia-thalamocortical loops in rodents. Despite largely closed loops within each functional domain, we discovered a unidirectional influence of the limbic over the motor loop via ventral striatum-substantia nigra (SNr)-motor thalamus circuitry. Slice electrophysiology verifies that the projection from ventral striatum functionally inhibits nigro-thalamic SNr neurons. In vivo optogenetic stimulation of ventral or dorsolateral striatum to SNr pathway modulates activity in medial prefrontal cortex (mPFC) and motor cortex (M1), respectively. However, whereas the dorsolateral striatum-SNr pathway exerts little impact on mPFC, activation of the ventral striatum-SNr pathway effectively alters M1 activity. These results demonstrate an open cortico-basal ganglia loop whereby limbic information could modulate motor output through ventral striatum control of M1.

Striatal cholinergic (ChAT) and parvalbumin (PV) interneurons exert powerful influences on striatal function in health and disease, yet little is known about the organization of their inputs. Here using rabies tracing, electrophysiology and genetic tools, we compare the whole-brain inputs to these two types of striatal interneurons and dissect their functional connectivity in mice. ChAT interneurons receive a substantial cortical input from associative regions of cortex, such as the orbitofrontal cortex. Amongst subcortical inputs, a previously unknown inhibitory thalamic reticular nucleus input to striatal PV interneurons is identified. Additionally, the external segment of the globus pallidus targets striatal ChAT interneurons, which is sufficient to inhibit tonic ChAT interneuron firing. Finally, we describe a novel excitatory pathway from the pedunculopontine nucleus that innervates ChAT interneurons. These results establish the brain-wide direct inputs of two major types of striatal interneurons and allude to distinct roles in regulating striatal activity and controlling behavior.

The claustrum (CLA) is a subcortical structure, present only in mammals, whose function remains uncertain. Previously, using resting-state functional magnetic resonance imaging (rs-fMRI) in awake head-fixed rats, we found evidence that the CLA is part of the rodent homolog of the default mode network (DMN; Smith et al., 2017). This network emerged as strong functional connections between the medial prefrontal cortex (mPFC), mediodorsal (MD) thalamus, and CLA in the awake state, which was not present following administration of isoflurane anesthesia. In the present report, we review evidence indicating that the rodent CLA also has connections with structures identified in the rodent homolog of the salience network (SN), a circuit that directs attention towards the most relevant stimuli among a multitude of sensory inputs (Seeley et al., 2007; Menon and Uddin, 2010). In humans, this circuit consists of functional connections between the anterior cingulate cortex (ACC) and a region that encompasses both the CLA and insular cortex. We further go on to review the similarities and differences between the functional and anatomical connections of the CLA and insula in rodents using both rs-fMRI and neuroanatomical tracing, respectively. We analyze in detail the connectivity of the CLA with the cingulate cortex, which is a major node in the SN and has been shown to modulate attention. When considered with other recent behavior and physiology studies, the data reveal a role for the CLA in salience-guided orienting. More specifically, we hypothesize that limbic information from mPFC, MD thalamus, and the basolateral amygdala (BLA) are integrated by the CLA to guide modality-related regions of motor and sensory cortex in directing attention towards relevant (i.e., salient) sensory events.

The basal ganglia and pontocerebellar systems regulate somesthetic-guided motor behaviors and receive prominent inputs from sensorimotor cortex. In addition, the claustrum and thalamus are forebrain subcortical structures that have connections with somatosensory and motor cortices. Our previous studies in rats have shown that primary and secondary somatosensory cortex (S1 and S2) send overlapping projections to the neostriatum and pontine nuclei, whereas, overlap of primary motor cortex (M1) and S1 was much weaker. In addition, we have shown that M1, but not S1, projects to the claustrum in rats. The goal of the current study was to compare these rodent projection patterns with connections in cats, a mammalian species that evolved in a separate phylogenetic superorder. Three different anterograde tracers were injected into the physiologically identified forepaw representations of M1, S1, and S2 in cats. Labeled fibers terminated throughout the ipsilateral striatum (caudate and putamen), claustrum, thalamus, and pontine nuclei. Digital reconstructions of tracer labeling allowed us to quantify both the normalized distribution of labeling in each subcortical area from each tracer injection, as well as the amount of tracer overlap. Surprisingly, in contrast to our previous findings in rodents, we observed M1 and S1 projections converging prominently in striatum and pons, whereas, S1 and S2 overlap was much weaker. Furthermore, whereas, rat S1 does not project to claustrum, we confirmed dense claustral inputs from S1 in cats. These findings suggest that the basal ganglia, claustrum, and pontocerebellar systems in rat and cat have evolved distinct patterns of sensorimotor cortical convergence.

The claustrum is a brain region whose function remains unknown, though many investigators suggest it plays a role in conscious attention. Resting-state functional magnetic resonance imaging (RS-fMRI) has revealed how anesthesia alters many functional connections in the brain, but the functional role of the claustrum with respect to the awake versus anesthetized states remains unknown. Therefore, we employed a combination of seed-based RS-fMRI and neuroanatomical tracing to reveal how the anatomical connections of the claustrum are related to its functional connectivity during quiet wakefulness and the isoflurane-induced anesthetic state. In awake rats, RS-fMRI indicates that the claustrum has interhemispheric functional connections with the mediodorsal thalamus (MD) and medial prefrontal cortex (mPFC), as well as other known connections with cortical areas that correspond to the connections revealed by neuroanatomical tracing. During deep isoflurane anesthesia, the functional connections of the claustrum with mPFC and MD were significantly attenuated, while those with the rest of cortex were not significantly altered. These changes in claustral functional connectivity were also observed when seeds were placed in mPFC or MD during RS-fMRI comparisons of the awake and deeply anesthetized states. Collectively, these data indicate that the claustrum has functional connections with mPFC and MD-thalamus that are significantly lessened by anesthesia.

Rodent whisking is an exploratory behavior that can be modified by sensory feedback. Consistent with this, many whisker-sensitive cortical regions project to agranular motor [motor cortex (MI)] cortex, but the relative topography of these afferent projections has not been established. Intracortical microstimulation (ICMS) evokes whisker movements that are used to map the functional organization of MI, but no study has compared the whisker-related inputs to MI with the ICMS sites that evoke whisker movements. To elucidate this relationship, anterograde tracers were placed in posterior parietal cortex (PPC) and in the primary somatosensory (SI) and secondary somatosensory (SII) cortical areas so that their labeled projections to MI could be analyzed with respect to ICMS sites that evoke whisker movements. Projections from SI and SII terminate in a narrow zone that marks the transition between the medial agranular (AGm) and lateral agranular (AGl) cortical areas, but PPC projects more medially and terminates in AGm proper. Paired recordings of MI neurons indicate that the region between AGm and AGl is highly responsive to whisker deflections, but neurons in AGm display negligible responses to whisker stimulation. By contrast, AGm microstimulation is more effective in evoking whisker movements than microstimulation of the transitional region between AGm and AGl. The AGm region was also found to contain a larger concentration of corticotectal neurons, which could convey whisker-related information to the facial nucleus. These results indicate that rat whisker MI is comprised of at least two functionally distinct subregions: a sensory processing zone in the transitional region between AGm and AGl, and a motor-output region located more medially in AGm proper.

The primary (S1) and secondary (S2) somatosensory cortices project to several trigeminal sensory nuclei. One putative function of these corticofugal projections is the gating of sensory transmission through the trigeminal principal nucleus (Pr5), and some have proposed that S1 and S2 project differentially to the spinal trigeminal subnuclei, which have inhibitory circuits that could inhibit or disinhibit the output projections of Pr5. Very little, however, is known about the origin of sensorimotor corticofugal projections and their patterns of termination in the various trigeminal nuclei. We addressed this issue by injecting anterograde tracers in S1, S2 and primary motor (M1) cortices, and quantitatively characterizing the distribution of labeled terminals within the entire rostro-caudal chain of trigeminal sub-nuclei. We confirmed our anterograde tracing results by injecting retrograde tracers at various rostro-caudal levels within the trigeminal sensory nuclei to determine the position of retrogradely labeled cortical cells with respect to S1 barrel cortex. Our results demonstrate that S1 and S2 projections terminate in largely overlapping regions but show some significant differences. Whereas S1 projection terminals tend to cluster within the principal trigeminal (Pr5), caudal spinal trigeminal interpolaris (Sp5ic), and the dorsal spinal trigeminal caudalis (Sp5c), S2 projection terminals are distributed in a continuum across all trigeminal nuclei. Contrary to the view that sensory gating could be mediated by differential activation of inhibitory interconnections between the spinal trigeminal subnuclei, we observed that projections from S1 and S2 are largely overlapping in these subnuclei despite the differences noted earlier.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain 1 , neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties. Quantitative analysis of the methylation of mouse cortical neurons that project to different cortical and subcortical target regions provides insight into genetic mechanisms that contribute to differences in cell function.

Single-cell analyses parse the brain’s billions of neurons into thousands of ‘cell-type’ clusters residing in different brain structures 1 . Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq 2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis -regulatory elements and transcription-factor-binding motifs, and neurotransmitter use. This study uses epi-retro-seq to link single-cell epigenomes and cell types to long-distance projections for neurons dissected from different regions projecting to different targets across the whole mouse brain.