Regional heterogeneity in the membrane properties of mouse striatal neurons.

The cytoarchitecture of the striatum is remarkably homogeneous, in contrast to the regional variation in striatal functions. Whether differences in the intrinsic membrane properties of striatal neurons contribute to regional heterogeneity has not been addressed systematically. We made recordings throughout the young adult mouse striatum under identical conditions, with synaptic input blocked, from four major striatal neuron types, namely, the two subtypes of spiny projection neurons (SPNs), cholinergic interneurons (ChIs), and fast-spiking GABAergic interneurons (FSIs), sampling at least 100 cells per cell type. Regional variation manifested across all cell types. All cell types in the nucleus accumbens (NAc) shell had higher input impedance and increased excitability. Cells in the NAc core were differentiated from the caudate-putamen (CPu) for both SPN subtypes by smaller action potentials and increased excitability. Similarity between the two SPN subtypes showed regional variation, differing more in the NAc than in the CPu. So, in the Str, both the intrinsic properties of interneurons and projection neurons are regionally heterogeneous, with the greatest difference between the NAc and CPu; greater excitability of NAc shell neurons may make the region more susceptible to activity-dependent plasticity.

Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

A spatial-temporal map of glutamatergic neurogenesis in the murine embryonic cerebellar nuclei uncovers a high degree of cellular heterogeneity.

The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.

Striosomes Target Nigral Dopamine-Containing Neurons via Direct-D1 and Indirect-D2 Pathways Paralleling Classic Direct-Indirect Basal Ganglia Systems.

Balanced activity of canonical direct D1 and indirect D2 basal ganglia pathways is considered a core requirement for normal movement, and their imbalance is an etiologic factor in movement and neuropsychiatric disorders. We present evidence for a conceptually equivalent pair of direct-D1 and indirect-D2 pathways that arise from striatal projection neurons (SPNs) of the striosome compartment rather than from SPNs of the matrix, as do the canonical pathways. These S-D1 and S-D2 striosomal pathways target substantia nigra dopamine-containing neurons instead of basal ganglia motor output nuclei. They modulate movement oppositely to the modulation by the canonical pathways: S-D1 is inhibitory and S-D2 is excitatory. The S-D1 and S-D2 circuits likely influence motivation for learning and action, complementing and reorienting canonical pathway modulation. A major conceptual reformulation of the classic direct-indirect pathway model of basal ganglia function is needed, as well as reconsideration of the effects of D2-targeting therapeutic drugs.

Neuronal identity control at the resolution of a single transcription factor isoform.

The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. 'Terminal selectors' are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. Here, we establish a novel role for the transcription factor broad as a terminal selector in Drosophila melanogaster. We capitalize on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. We find a single isoform broad-z4 serves as the switch between the identity of two visual projection neurons LPLC1 and LPLC2. Broad-z4 is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. Our comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.

Compensation between FOXP transcription factors maintains proper striatal function.

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN-specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type-specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral-mediated re-expression of Foxp1 into the double knockouts is sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

TNFR1/p38αMAPK signaling in Nex + supraspinal neurons regulates estrogen-dependent chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Role of Posterior Medial Thalamus in the Modulation of Striatal Circuitry and Choice Behavior.

The posterior medial (POm) thalamus is heavily interconnected with sensory and motor circuitry and is likely involved in behavioral modulation and sensorimotor integration. POm provides axonal projections to the dorsal striatum, a hotspot of sensorimotor processing, yet the role of POm-striatal projections has remained undetermined. Using optogenetics with slice electrophysiology, we found that POm provides robust synaptic input to direct and indirect pathway striatal spiny projection neurons (D1- and D2-SPNs, respectively) and parvalbumin-expressing fast spiking interneurons (PVs). During the performance of a whisker-based tactile discrimination task, POm-striatal projections displayed learning-related activation correlating with anticipatory, but not reward-related, pupil dilation. Inhibition of POm-striatal axons across learning caused slower reaction times and an increase in the number of training sessions for expert performance. Our data indicate that POm-striatal inputs provide a behaviorally relevant arousal-related signal, which may prime striatal circuitry for efficient integration of subsequent choice-related inputs.

Postmortem neuropathology in early Huntington disease.

Two aspects of the neuropathology of early Huntington disease (HD) are examined. Neurons of the neostriatum are counted to determine relative loss in striosomes versus matrix at early stages, including for the first time in preclinical cases. An immunohistochemical procedure is described that tentatively distinguishes early HD from HD mimic disorders in postmortem brains. Counts of striatal projection neurons (SPNs) in striosomes defined by calbindin immunohistochemistry versus counts in the surrounding matrix are reported for 8 Vonsattel grade 0 (including 5 premanifest), 8 grade 1, 2 grade 2 HD, and for 8 control postmortem brains. Mean counts of striosome and matrix SPNs were significantly lower in premanifest grade 0 versus controls, with striosome counts significantly lower than matrix. In 8 grade 1 and 2 grade 2 brains, no striosomes with higher SPN counts than in the surrounding matrix were observed. Comparing dorsal versus ventral neostriatum, SPNs in dorsal striosomes and matrix declined more than ventral, making clear the importance of the dorsoventral site of tissue selection for research studies. A characteristic pattern of expanded polyglutamine-immunopositive inclusions was seen in all HD cases. Inclusions were always present in some SPNs and some pontine nucleus neurons and were absent in Purkinje cells, which showed no obvious cell loss.

Role of M4 -receptor cholinergic signaling in direct pathway striatal projection neurons during dopamine depletion.

Direct pathway striatal projection neurons (dSPNs) are characterized by the expression of dopamine (DA) class 1 receptors (D1 R), as well as cholinergic muscarinic M1 and M4 receptors (M1 R, M4 R). D1 R enhances neuronal firing through phosphorylation of voltage-gate calcium channels (CaV 1 Ca2+ channels) activating Gs proteins and protein kinase A (PKA). Concurrently, PKA suppresses phosphatase PP-1 through DARPP-32, thus extending this facilitatory modulation. M1 R also influences Ca2+ channels in SPNs through Gq proteins and protein kinase C. However, the signaling mechanisms of M4 R in dSPNs are less understood. Two pathways are attributed to M4 R: an inhibitory one through Gi/o proteins, and a facilitatory one via the cyclin Cdk5. Our study reveals that a previously observed facilitatory modulation via CaV 1 Ca2+ channels is linked to the Cdk5 pathway in dSPNs. This result could be significant in treating parkinsonism. Therefore, we questioned whether this effect persists post DA-depletion in experimental parkinsonism. Our findings indicate that in such conditions, M4 R activation leads to a decrease in Ca2+ current and an increased M4 R protein level, contrasting with the control response. Nevertheless, parkinsonian and control actions are inhibited by the Cdk5 inhibitor roscovitine, suggesting Cdk5's role in both conditions. Cdk5 may activate PP-1 via PKA inhibition in DA depletion. Indeed, we found that inhibiting PP-1 restores control M4 R actions, implying that PP-1 is overly active via M4 Rs in DA-depleted condition. These insights contribute to understanding how DA-depletion alters modulatory signaling in striatal neurons. Additional working hypotheses are discussed.

Effective Regulation of Auditory Processing by Parvalbumin Interneurons in the Tail of the Striatum.

Parvalbumin (PV) interneurons in the auditory cortex (AC) play a crucial role in shaping auditory processing, including receptive field formation, temporal precision enhancement, and gain regulation. PV interneurons are also the primary inhibitory neurons in the tail of the striatum (TS), which is one of the major descending brain regions in the auditory nervous system. However, the specific roles of TS-PV interneurons in auditory processing remain elusive. In this study, morphological and slice recording experiments in both male and female mice revealed that TS-PV interneurons, compared with AC-PV interneurons, were present in fewer numbers but exhibited longer projection distances, which enabled them to provide sufficient inhibitory inputs to spiny projection neurons (SPNs). Furthermore, TS-PV interneurons received dense auditory input from both the AC and medial geniculate body (MGB), particularly from the MGB, which rendered their auditory responses comparable to those of AC-PV interneurons. Optogenetic manipulation experiments demonstrated that TS-PV interneurons were capable of bidirectionally regulating the auditory responses of SPNs. Our findings suggest that PV interneurons can effectively modulate auditory processing in the TS and may play a critical role in auditory-related behaviors.

Patterning ganglionic eminences in developing human brain organoids using a morphogen-gradient-inducing device.

In early neurodevelopment, the central nervous system is established through the coordination of various neural organizers directing tissue patterning and cell differentiation. Better recapitulation of morphogen gradient production and signaling will be crucial for establishing improved developmental models of the brain in vitro. Here, we developed a method by assembling polydimethylsiloxane devices capable of generating a sustained chemical gradient to produce patterned brain organoids, which we termed morphogen-gradient-induced brain organoids (MIBOs). At 3.5 weeks, MIBOs replicated dorsal-ventral patterning observed in the ganglionic eminences (GE). Analysis of mature MIBOs through single-cell RNA sequencing revealed distinct dorsal GE-derived CALB2+ interneurons, medium spiny neurons, and medial GE-derived cell types. Finally, we demonstrate long-term culturing capabilities with MIBOs maintaining stable neural activity in cultures grown up to 5.5 months. MIBOs demonstrate a versatile approach for generating spatially patterned brain organoids for embryonic development and disease modeling.

Betz cells of the primary motor cortex.

Betz cells, named in honor of Volodymyr Betz (1834-1894), who described them as "giant pyramids" in the primary motor cortex of primates and other mammalian species, are layer V extratelencephalic projection (ETP) neurons that directly innervate α-motoneurons of the brainstem and spinal cord. Despite their large volume and circumferential dendritic architecture, to date, no single molecular criterion has been established that unequivocally distinguishes adult Betz cells from other layer V ETP neurons. In primates, transcriptional signatures suggest the presence of at least two ETP neuron clusters that contain mature Betz cells; these are characterized by an abundance of axon guidance and oxidative phosphorylation transcripts. How neurodevelopmental programs drive the distinct positional and morphological features of Betz cells in humans remains unknown. Betz cells display a distinct biphasic firing pattern involving early cessation of firing followed by delayed sustained acceleration in spike frequency and magnitude. Few cell type-specific transcripts and electrophysiological characteristics are conserved between rodent layer V ETP neurons of the motor cortex and primate Betz cells. This has implications for the modeling of disorders that affect the motor cortex in humans, such as amyotrophic lateral sclerosis (ALS). Perhaps vulnerability to ALS is linked to the evolution of neural networks for fine motor control reflected in the distinct morphomolecular architecture of the human motor cortex, including Betz cells. Here, we discuss histological, molecular, and functional data concerning the position of Betz cells in the emerging taxonomy of neurons across diverse species and their role in neurological disorders.

Striatal Neurons Are Recruited Dynamically into Collective Representations of Self-Initiated and Learned Actions in Freely Moving Mice.

Striatal spiny projection neurons are hyperpolarized-at-rest (HaR) and driven to action potential threshold by a small number of powerful inputs-an input-output configuration that is detrimental to response reliability. Because the striatum is important for habitual behaviors and goal-directed learning, we conducted a microendoscopic imaging in freely moving mice that express a genetically encoded Ca2+ indicator sparsely in striatal HaR neurons to evaluate their response reliability during self-initiated movements and operant conditioning. The sparse expression was critical for longitudinal studies of response reliability, and for studying correlations among HaR neurons while minimizing spurious correlations arising from contamination by the background signal. We found that HaR neurons are recruited dynamically into action representation, with distinct neuronal subsets being engaged in a moment-by-moment fashion. While individual neurons respond with little reliability, the population response remained stable across days. Moreover, we found evidence for the temporal coupling between neuronal subsets during conditioned (but not innate) behaviors.

Balance Between Projecting Neuronal Populations of the Nucleus Accumbens Controls Social Behavior in Mice.

BACKGROUND: Deficient social interactions are a hallmark of major neuropsychiatric disorders, and accumulating evidence points to altered social reward and motivation as key underlying mechanisms of these pathologies. In the present study, we further explored the role of the balance of activity between D1 and D2 receptor-expressing striatal projection neurons (D1R- and D2R-SPNs) in the control of social behavior, challenging the hypothesis that excessive D2R-SPN activity, rather than deficient D1R-SPN activity, compromises social behavior. METHODS: We selectively ablated D1R- and D2R-SPNs using an inducible diphtheria toxin receptor-mediated cell targeting strategy and assessed social behavior as well as repetitive/perseverative behavior, motor function, and anxiety levels. We tested the effects of optogenetic stimulation of D2R-SPNs in the nucleus accumbens (NAc) and pharmacological compounds repressing D2R-SPN. RESULTS: Targeted deletion of D1R-SPNs in the NAc blunted social behavior in mice, facilitated motor skill learning, and increased anxiety levels. These behaviors were normalized by pharmacological inhibition of D2R-SPN, which also repressed transcription in the efferent nucleus, the ventral pallidum. Ablation of D1R-SPNs in the dorsal striatum had no impact on social behavior but impaired motor skill learning and decreased anxiety levels. Deletion of D2R-SPNs in the NAc produced motor stereotypies but facilitated social behavior and impaired motor skill learning. We mimicked excessive D2R-SPN activity by optically stimulating D2R-SPNs in the NAc and observed a severe deficit in social interaction that was prevented by D2R-SPN pharmacological inhibition. CONCLUSIONS: Repressing D2R-SPN activity may represent a promising therapeutic strategy to relieve social deficits in neuropsychiatric disorders.

The roles of surround inhibition for the intrinsic function of the striatum, analyzed in silico.

The basal ganglia are important for action initiation, selection, and motor learning. The input level, the striatum, receives input preferentially from the cortex and thalamus and is to 95% composed of striatal projection neurons (SPNs) with sparse GABAergic collaterals targeting distal dendrites of neighboring SPNs, in a distance-dependent manner. The remaining 5% are GABAergic and cholinergic interneurons. Our aim here is to investigate the role of surround inhibition for the intrinsic function of the striatum. Large-scale striatal networks of 20 to 40 thousand neurons were simulated with detailed multicompartmental models of different cell types, corresponding to the size of a module of the dorsolateral striatum, like the forelimb area (mouse). The effect of surround inhibition on dendritic computation and network activity was investigated, while groups of SPNs were activated. The SPN-induced surround inhibition in distal dendrites shunted effectively the corticostriatal EPSPs. The size of dendritic plateau-like potentials within the specific dendritic segment was both reduced and enhanced by inhibition, due to the hyperpolarized membrane potential of SPNs and the reversal-potential of GABA. On a population level, the competition between two subpopulations of SPNs was found to depend on the distance between the two units, the size of each unit, the activity level in each subgroup and the dopaminergic modulation of the dSPNs and iSPNs. The SPNs provided the dominating source of inhibition within the striatum, while the fast-spiking interneuron mainly had an initial effect due to short-term synaptic plasticity as shown in with ablation of the synaptic interaction.

TNFR1/p38αMAPK signaling in Nex+ supraspinal neurons regulates sex-specific chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP) [1-4]. To test the hypothesis that supraspinal circuitry is critical to pain chronification, we studied the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that following chronic constriction injury (CCI), pain resolves in males; however, female acute pain transitions to chronic. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38αMAPK and NF-κB activation in male cortical tissue; however, p38αMAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed similar behavioral results following CCI in NexCreERT2::p38αMAPKf/f mice. Previously, we established estrogen's ability to modulate sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP [5-9]. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lend "male-like" therapeutic relief to females following CCI. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Synaptic crossroads: navigating the circuits of movement.

The anterior lateral motor area (ALM) is crucial in preparing and executing voluntary movements through its diverse neuronal subpopulations that target different subcortical areas. A recent study by Xu et al. utilized an elaborate viral tracing strategy in mice to provide comprehensive whole-brain maps of monosynaptic inputs to the major descending pathways of ALM.

Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity.

Identities of distinct neuron subtypes are specified during embryonic development, then maintained during post-natal maturation. In cerebral cortex, mechanisms controlling early acquisition of neuron-subtype identities have become increasingly understood. However, mechanisms controlling neuron-subtype identity stability during post-natal maturation are largely unexplored. We identify that Tle4 is required for both early acquisition and post-natal stability of corticothalamic neuron-subtype identity. Embryonically, Tle4 promotes acquisition of corticothalamic identity and blocks emergence of core characteristics of subcerebral/corticospinal projection neuron identity, including gene expression and connectivity. During the first post-natal week, when corticothalamic innervation is ongoing, Tle4 is required to stabilize corticothalamic neuron identity, limiting interference from differentiation programs of developmentally related neuron classes. We identify a deacetylation-based epigenetic mechanism by which TLE4 controls Fezf2 expression level by corticothalamic neurons. This contributes to distinction of cortical output subtypes and ensures identity stability for appropriate maturation of corticothalamic neurons.