Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 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.

The Anatomy and Physiology of Claustrum-Cortex Interactions.

The claustrum is one of the most widely connected regions of the forebrain, yet its function has remained obscure, largely due to the experimentally challenging nature of targeting this small, thin, and elongated brain area. However, recent advances in molecular techniques have enabled the anatomy and physiology of the claustrum to be studied with the spatiotemporal and cell type-specific precision required to eventually converge on what this area does. Here we review early anatomical and electrophysiological results from cats and primates, as well as recent work in the rodent, identifying the connectivity, cell types, and physiological circuit mechanisms underlying the communication between the claustrum and the cortex. The emerging picture is one in which the rodent claustrum is closely tied to frontal/limbic regions and plays a role in processes, such as attention, that are associated with these areas.

Epigenomic diversity of cortical projection neurons in the mouse brain.

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 brain1, 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.

Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. METHODS: We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. RESULTS: Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. INTERPRETATION: Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.

Core and matrix thalamic sub-populations relate to spatio-temporal cortical connectivity gradients.

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.

The Zona Incerta Regulates Communication between the Superior Colliculus and the Posteromedial Thalamus: Implications for Thalamic Interactions with the Dorsolateral Striatum.

There is uncertainty concerning the circuit connections by which the superior colliculus interacts with the basal ganglia. To address this issue, anterograde and retrograde tracers were placed, respectively, into the superior colliculus and globus pallidus of Sprague-Dawley rats. In this two-tracer experiment, the projections from the superior colliculus terminated densely in the ventral zona incerta (ZIv), but did not overlap the labeled neurons observed in the subthalamic nucleus. In cases in which anterograde and retrograde tracers were placed, respectively, in sensory-responsive sites in the superior colliculus and posteromedial (POm) thalamus, the labeled projections from superior colliculus innervated the ZIv regions that contained the labeled neurons that project to POm. We also confirmed this colliculo-incertal-POm pathway by depositing a mixture of retrograde and anterograde tracers at focal sites in ZIv to reveal retrogradely labeled neurons in superior colliculus and anterogradely labeled terminals in POm. When combined with retrograde tracer injections in POm, immunohistochemical processing proved that most ZIv projections to POm are GABAergic. Consistent with these findings, direct stimulation of superior colliculus evoked neuronal excitation in ZIv and caused inhibition of spontaneous activity in POm. Collectively, these results indicate that superior colliculus can activate the inhibitory projections from ZIv to the POm. This is significant because it suggests that the superior colliculus could suppress the interactions between POm and the dorsolateral striatum, presumably to halt ongoing behaviors so that more adaptive motor actions are selected in response to unexpected sensory events. SIGNIFICANCE STATEMENT: By demonstrating that the zona incerta regulates communication between the superior colliculus and the posteromedial thalamus, we have uncovered a circuit that partly explains the behavioral changes that occur in response to unexpected sensory stimuli. Furthermore, this circuit could explain why deep brain stimulation of the zona incerta is beneficial to patients who suffer from Parkinson's disease.

Neurovascular coupling and decoupling in the cortex during voluntary locomotion.

Hemodynamic signals are widely used to infer neural activity in the brain. We tested the hypothesis that hemodynamic signals faithfully report neural activity during voluntary behaviors by measuring cerebral blood volume (CBV) and neural activity in the somatosensory cortex and frontal cortex of head-fixed mice during locomotion. Locomotion induced a large and robust increase in firing rate and gamma-band (40-100 Hz) power in the local field potential in the limb representations in somatosensory cortex, and was accompanied by increases in CBV, demonstrating that hemodynamic signals are coupled with neural activity in this region. However, in the frontal cortex, CBV did not change during locomotion, but firing rate and gamma-band power both increased, indicating a decoupling of neural activity from the hemodynamic signal. These results show that hemodynamic signals are not faithful indicators of the mean neural activity in the frontal cortex during locomotion; thus, the results from fMRI and other hemodynamic imaging methodologies for studying neural processes must be interpreted with caution.

Brief anesthesia, but not voluntary locomotion, significantly alters cortical temperature.

Changes in brain temperature can alter electrical properties of neurons and cause changes in behavior. However, it is not well understood how behaviors, like locomotion, or experimental manipulations, like anesthesia, alter brain temperature. We implanted thermocouples in sensorimotor cortex of mice to understand how cortical temperature was affected by locomotion, as well as by brief and prolonged anesthesia. Voluntary locomotion induced small (∼ 0.1 °C) but reliable increases in cortical temperature that could be described using a linear convolution model. In contrast, brief (90-s) exposure to isoflurane anesthesia depressed cortical temperature by ∼ 2 °C, which lasted for up to 30 min after the cessation of anesthesia. Cortical temperature decreases were not accompanied by a concomitant decrease in the γ-band local field potential power, multiunit firing rate, or locomotion behavior, which all returned to baseline within a few minutes after the cessation of anesthesia. In anesthetized animals where core body temperature was kept constant, cortical temperature was still > 1 °C lower than in the awake animal. Thermocouples implanted in the subcortex showed similar temperature changes under anesthesia, suggesting these responses occur throughout the brain. Two-photon microscopy of individual blood vessel dynamics following brief isoflurane exposure revealed a large increase in vessel diameter that ceased before the brain temperature significantly decreased, indicating cerebral heat loss was not due to increased cerebral blood vessel dilation. These data should be considered in experimental designs recording in anesthetized preparations, computational models relating temperature and neural activity, and awake-behaving methods that require brief anesthesia before experimental procedures.

Thalamostriatal projections from the medial posterior and parafascicular nuclei have distinct topographic and physiologic properties.

The dorsolateral striatum (DLS) is critical for executing sensorimotor behaviors that depend on stimulus-response (S-R) associations. In rats, the DLS receives it densest inputs from primary somatosensory (SI) cortex, but it also receives substantial input from the thalamus. Much of rat DLS is devoted to processing whisker-related information, and thalamic projections to these whisker-responsive DLS regions originate from the parafascicular (Pf) and medial posterior (POm) nuclei. To determine which thalamic nucleus is better suited for mediating S-R associations in the DLS, we compared their input-output connections and neuronal responses to repetitive whisker stimulation. Tracing experiments demonstrate that POm projects specifically to the DLS, but the Pf innervates both dorsolateral and dorsomedial parts of the striatum. The Pf nucleus is innervated by whisker-sensitive sites in the superior colliculus, and these sites also send dense projections to the zona incerta, a thalamic region that sends inhibitory projections to the POm. These data suggest that projections from POm to the DLS are suppressed by incertal inputs when the superior colliculus is activated by unexpected sensory stimuli. Simultaneous recordings with two electrodes indicate that POm neurons are more responsive and habituate significantly less than Pf neurons during repetitive whisker stimulation. Response latencies are also shorter in POm than in Pf, which is consistent with the fact that Pf receives its whisker information via synaptic relays in the superior colliculus. These findings indicate that, compared with the Pf nucleus, POm transmits somatosensory information to the DLS with a higher degree of sensory fidelity.

Rat claustrum coordinates but does not integrate somatosensory and motor cortical information.

The function of the claustrum is a fundamental issue in neuroscience. Anatomical data indicate that the rat claustrum is part of an interhemispheric circuit that could be involved in the bilateral coordination of whisker movements. Given that whisking is a somesthetic-guided motor behavior, the goal of the current study was to elucidate the connections of the claustrum with respect to the whisker representations in the primary somatosensory (wSI) and motor (wMI) cortical areas. Anterograde tracer injections showed that wMI projects most densely to the claustrum in the contralateral hemisphere, whereas wSI does not project to the claustrum in either hemisphere. Injections of different retrograde tracers into wMI and wSI of the same animal revealed intermingled populations of labeled neurons in the claustrum, as well as many double-labeled neurons. This indicates that the same part of the claustrum projects to the whisker representations in both SI and MI. Finally, injections of different anterograde tracers in the wMI regions of both hemispheres were combined with a retrograde tracer injection in wSI, and this produced dense terminal labeling around retrogradely labeled neurons in the claustrum of both hemispheres. Although the rodent claustrum is probably involved in the interhemispheric coordination of the MI and SI whisker representations, it does not receive inputs from both of these cortical regions. Hence, the claustrum should not be universally regarded as an integrator of somesthetic and motor information.

In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons.

Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. In silico analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.

Thalamic POm projections to the dorsolateral striatum of rats: potential pathway for mediating stimulus-response associations for sensorimotor habits.

The dorsolateral part of the striatum (DLS) represents the initial stage for processing sensorimotor information in the basal ganglia. Although the DLS receives much of its input from the primary somatosensory (SI) cortex, peripheral somesthetic stimulation activates the DLS at latencies that are shorter than the response latencies recorded in the SI cortex. To identify the subcortical regions that transmit somesthetic information directly to the DLS, we deposited small quantities of retrograde tracers at DLS sites that displayed consistent time-locked responses to controlled whisker stimulation. The neurons that were retrogradely labeled by these injections were located mainly in the sensorimotor cortex and, to a lesser degree, in the amygdala and thalamus. Quantitative analysis of neuronal labeling in the thalamus indicated that the strongest thalamic input to the whisker-sensitive part of the DLS originates from the medial posterior nucleus (POm), a somesthetic-related region that receives inputs from the spinal trigeminal nucleus. Anterograde tracer injections in POm confirmed that this thalamic region projects to the DLS neuropil. In subsequent experiments, simultaneous recordings from POm and the DLS during whisker stimulation showed that POm consistently responds before the DLS. These results suggest that POm could transmit somesthetic information to the DLS, and this modality-specific thalamostriatal pathway may cooperate with the thalamostriatal projections that originate from the intralaminar nuclei.

Convergence of forepaw somatosensory and motor cortical projections in the striatum, claustrum, thalamus, and pontine nuclei of cats.

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.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

Functional specificity of claustrum connections in the rat: interhemispheric communication between specific parts of motor cortex.

Recent evidence indicates that the rat claustrum interconnects the motor cortical areas in both hemispheres. To elucidate the functional specificity of the interhemispheric connections between the claustrum and primary motor (MI) cortex, anterograde tracer injections in specific parts of MI were paired with retrograde tracer injections in homotopic sites of the opposite hemisphere. In addition to injecting the MI forepaw (Fp) region in both hemispheres, we injected the region associated with whisker retractions (Re) and the more caudal rhythmic whisking (RW) region. While the MI-Fp region has few connections with the claustrum of either hemisphere, both whisker regions project to the contralateral claustrum, with those from the MI-RW region being denser and more extensive than those originating from the MI-Re region. Retrograde tracer injections in the MI-RW region produced more labeled neurons in the ipsilateral claustrum than retrograde tracer injections in the MI-Re. Consistent with these patterns, the overlap of labeled terminals and soma in the claustrum was greatest when both tracers were injected into the MI-RW region. When retrograde tracers were injected into the claustrum, the highest density of labeled neurons in MI appeared in the contralateral RW region. Tracer injections in the claustrum also revealed hundreds of labeled neurons throughout its rostrocaudal extent, thereby establishing the presence of long-range intraclaustral connections. These results indicate that the intrinsic and extrinsic connections of the rat claustrum are structured for rapid, interhemispheric transmission of information needed for bilateral coordination of the MI regions that regulate whisker movements.

The claustrum.

The claustrum is a brain region that has been investigated for over 200 years, yet its precise function remains unknown. In the final posthumously released article of Francis Crick, written with Christof Koch, the claustrum was suggested to be critically linked to consciousness. Though the claustrum remained relatively obscure throughout the last half century, it has enjoyed a renewed interest in the last 15 years since Crick and Koch's article. During this time, the claustrum, like many other brain regions, has been studied with the myriad of modern systems neuroscience tools that have been made available by the intersection of genetic and viral technologies. This has uncovered new information about its anatomical connectivity and physiological properties and begun to reveal aspects of its function. From these studies, one clear consensus has emerged which supports Crick and Koch's primary interest in the claustrum: the claustrum has widespread extensive connectivity with the entire cerebral cortex, suggesting a prominent role in 'higher order processes'.

A Role for the Claustrum in Salience Processing?

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 relationship between the claustrum and endopiriform nucleus: A perspective towards consensus on cross-species homology.

With the emergence of interest in studying the claustrum, a recent special issue of the Journal of Comparative Neurology dedicated to the claustrum (Volume 525, Issue 6, pp. 1313-1513) brought to light questions concerning the relationship between the claustrum (CLA) and a region immediately ventral known as the endopiriform nucleus (En). These structures have been identified as separate entities in rodents but appear as a single continuous structure in primates. During the recent Society for Claustrum Research meeting, a panel of experts presented data pertaining to the relationship of these regions and held a discussion on whether the CLA and En should be considered (a) separate unrelated structures, (b) separate nuclei within the same formation, or (c) subregions of a continuous structure. This review article summarizes that discussion, presenting comparisons of the cytoarchitecture, neurochemical profiles, genetic markers, and anatomical connectivity of the CLA and En across several mammalian species. In rodents, we conclude that the CLA and the dorsal endopiriform nucleus (DEn) are subregions of a larger complex, which likely performs analogous computations and exert similar effects on their respective cortical targets (e.g., sensorimotor versus limbic). Moving forward, we recommend that the field retain the nomenclature currently employed for this region but should continue to examine the delineation of these structures across different species. Using thorough descriptions of a variety of anatomical features, this review offers a clear definition of the CLA and En in rodents, which provides a framework for identifying homologous structures in primates.

An open cortico-basal ganglia loop allows limbic control over motor output via the nigrothalamic pathway.

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.