The Anterolateral Barrel Subfield Differs from the Posteromedial Barrel Subfield in the Morphology and Cell Density of Parvalbumin-Positive GABAergic Interneurons.

Layer 4 of the rodent somatosensory cortex has unitary structures called barrels that receive tactile information from individual vibrissae. Barrels in the anterolateral barrel subfield (ALBSF) are much smaller and have gained less attention than larger barrels in the posteromedial barrel subfield (PMBSF), though the former outnumber the latter. We compared the morphological features of barrels between the ALBSF and PMBSF in male mice using deformation-free tangential sections and confocal optical slice-based, precise reconstructions of barrels. The average volume of a single barrel in the ALBSF was 34.7% of that in the PMBSF, but the numerical density of parvalbumin (PV)-positive interneurons in the former was 1.49 times higher than that in the latter. Moreover, PV neuron density in septa was 2.08 times higher in the ALBSF than that in the PMBSF. The proportions of PV neuron number to both all neuron number and all GABAergic neuron number in the ALBSF were also higher than those in the PMBSF. Somata of PV neurons in barrels and septa in the ALBSF received 1.64 and 1.50 times more vesicular glutamate transporter Type 2-labeled boutons than those in the PMBSF, suggesting more potent feedforward inhibitory circuits in the ALBSF. The mode of connectivity through dendritic gap junctions among PV neurons also differed between the ALBSF and PMBSF. Clusters of smaller unitary structures containing a higher density of representative GABAergic interneurons with differential morphological features in the ALBSF suggest a division of functional roles in the two vibrissa-barrel systems, as has been demonstrated by behavioral studies.

Total Synthesis of Caldorazole, a Potent Mitochondrial Respiratory Chain Inhibitor without Chiral Centers.

Caldorazole (1) is a novel polyketide that was isolated from a marine cyanobacterium in 2022. It is a unique natural product that exhibits potent inhibitory activity against mitochondrial respiratory chain complex I despite having no chiral centers. To establish a method for obtaining caldorazole without relying on biological resources and for constructing a useful synthetic route for studies of its structure-activity relationship, we achieved the first total synthesis of caldorazole using a convergent synthetic route.

The Tail of the Mouse Striatum Contains a Novel Large Type of GABAergic Neuron Incorporated in a Unique Disinhibitory Pathway That Relays Auditory Signals to Subcortical Nuclei.

The most caudal part of the striatum in rodents, the tail of the striatum (TS), has many features that distinguish it from the rostral striatum, such as its biased distributions of dopamine receptor subtypes, lack of striosomes and matrix compartmentalization, and involvement in sound-driven behaviors. However, information regarding the TS is still limited. We demonstrate in this article that the TS of the male mouse contains GABAergic neurons of a novel type that were detected immunohistochemically with the neurofilament marker SMI-32. Their somata were larger than cholinergic giant aspiny neurons, were located in a narrow space adjacent to the globus pallidus (GP), and extended long dendrites laterally toward the intermediate division (ID) of the trilaminar part of the TS, the region targeted by axons from the primary auditory cortex (A1). Although vesicular glutamate transporter 1-positive cortical axon terminals rarely contacted these TS large (TSL) neurons, glutamic acid decarboxylase-immunoreactive and enkephalin-immunoreactive boutons densely covered somata and dendrites of TSL neurons, forming symmetrical synapses. Analyses of GAD67-CrePR knock-in mice revealed that these axonal boutons originated from nearby medium spiny neurons (MSNs) in the ID. All MSNs examined in the ID in turn received inputs from the A1. Retrograde tracers injected into the rostral zona incerta and ventral medial nucleus of the thalamus labeled somata of TSL neurons. TSL neurons share many morphological features with GP neurons, but their strategically located dendrites receive inputs from closely located MSNs in the ID, suggesting faster responses than distant GP neurons to facilitate auditory-evoked, prompt disinhibition in their targets.SIGNIFICANCE STATEMENT This study describes a newly found population of neurons in the mouse striatum, the brain region responsible for appropriate behaviors. They are large GABAergic neurons located in the most caudal part of the striatum [tail of the striatum (TS)]. These TS large (TSL) neurons extended dendrites toward a particular region of the TS where axons from the primary auditory cortex (A1) terminated. These dendrites received direct synaptic inputs heavily from nearby GABAergic neurons of the striatum that in turn received inputs from the A1. TSL neurons sent axons to two subcortical regions outside basal ganglia, one of which is related to arousal. Specialized connectivity of TSL neurons suggests prompt disinhibitory actions on their targets to facilitate sound-evoked characteristic behaviors.

New Subregions of the Mouse Entopeduncular Nucleus Defined by the Complementary Immunoreactivities for Substance P and Cannabinoid Type-1 Receptor Combined with Distributions of Different Neuronal Types.

The entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr) constitute the output nuclei of the basal ganglia, but studies on the EPN are limited compared with those on the SNr. Both nuclei receive projections from the striatum with axons containing substance P (SP) and cannabinoid type-1 receptor (CB1R), and immunoreactivities for these substances show complementary patterns in the striatum and SNr. In this study, we revealed a similar complementarity in the mouse EPN, combined it with region-specific neuronal distributions, and defined subregions of the EPN. First, the EPN was divided into two areas, one showing low SP and high CB1R (lSP/hCB1R) immunoreactivities, and the other showing high SP and low CB1R (hSP/lCB1R). The former received inputs from the dorsolateral striatum that are innervated by sensorimotor cortices, whereas the latter received inputs from the medial striatum that are innervated by limbic/association cortices. Then, the lSP/hCB1R area was further divided into the dorsolateral subregion in the rostral EPN and the core subregion in the caudal EPN, the latter characterized by the concentration of parvalbumin-positive neurons targeting the ventral anterior-ventral lateral thalamic nucleus. The hSP/lCB1R area was divided into the ventromedial subregion in the rostral EPN and the shell subregion in the caudal EPN, the former characterized by the concentration of nitric oxide synthase-positive neurons targeting the lateral habenula (LHb). Somatostatin-positive neurons targeting the LHb were located diffusely in three subregions other than the core. These findings illuminate structural organization inside the basal ganglia, suggesting mechanisms for sorting diverse information through parallel loops with differing synaptic modulation by CB1R.

The endothelial Dll4-muscular Notch2 axis regulates skeletal muscle mass.

Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell-cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell-cell contact. Inhibition of the Dll4-Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4-muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.

Immunohistochemical Analysis of Neurotransmitters in Neurosecretory Protein GL-Producing Neurons of the Mouse Hypothalamus.

We recently discovered a novel neuropeptide of 80 amino acid residues: neurosecretory protein GL (NPGL), in the hypothalamus of birds and rodents. NPGL is localized in the lateral posterior part of the arcuate nucleus (ArcLP), and it enhances feeding behavior and fat accumulation in mice. Various neurotransmitters, such as catecholamine, glutamate, and γ-aminobutyric acid (GABA), produced in the hypothalamus are also involved in energy metabolism. The colocalization of neurotransmitters and NPGL in neurons of the ArcLP leads to the elucidation of the regulatory mechanism of NPGL neurons. In this study, we performed double immunofluorescence staining to elucidate the relationship between NPGL and neurotransmitters in mice. The present study revealed that NPGL neurons did not co-express tyrosine hydroxylase as a marker of catecholaminergic neurons and vesicular glutamate transporter-2 as a marker of glutamatergic neurons. In contrast, NPGL neurons co-produced glutamate decarboxylase 67, a marker for GABAergic neurons. In addition, approximately 50% of NPGL neurons were identical to GABAergic neurons. These results suggest that some functions of NPGL neurons may be related to those of GABA. This study provides insights into the neural network of NPGL neurons that regulate energy homeostasis, including feeding behavior and fat accumulation.

Subregion-Specific Regulation of Dopamine D1 Receptor Signaling in the Striatum: Implication for L-DOPA-Induced Dyskinesia.

The striatum is the main structure of the basal ganglia. The striatum receives inputs from various cortical areas, and its subregions play distinct roles in motor and emotional functions. Recently, striatal maps based on corticostriatal connectivity and striosome-matrix compartmentalization were developed, and we were able to subdivide the striatum into seven subregions. Dopaminergic modulation of the excitability of medium spiny neurons (MSNs) is critical for striatal function. In this study, we investigated the functional properties of dopamine signaling in seven subregions of the striatum from male mice. By monitoring the phosphorylation of PKA substrates including DARPP-32 in mouse striatal slices, we identified two subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Low D1 receptor signaling in the two subregions was maintained by phosphodiesterase (PDE)10A and muscarinic M4 receptors. In an animal model of 6-hydroxydopamine (6-OHDA)-induced hemi-parkinsonism, D1 receptor signaling was upregulated in almost all subregions including the DL-IR, but not in the IC. When L-DOPA-induced dyskinesia (LID) was developed, D1 receptor signaling in the IC was upregulated and correlated with the severity of LID. Our results suggest that the function of the striatum is maintained through the subregion-specific regulation of dopamine D1 receptor signaling and that the aberrant activation of D1 receptor signaling in the IC is involved in LID. Future studies focusing on D1 receptor signaling in the IC of the striatum will facilitate the development of novel therapeutics for LID.SIGNIFICANCE STATEMENT Recent progress in striatal mapping based on corticostriatal connectivity and striosome-matrix compartmentalization allowed us to subdivide the striatum into seven subregions. Analyses of D1 receptor signaling in the seven subregions identified two unique subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Aberrant activation of D1 receptor signaling in the IC is involved in L-DOPA-induced dyskinesia (LID). Previous studies of LID have mainly focused on the DL-IR, but not on the IC of the striatum. Future studies to clarify aberrant D1 receptor signaling in the IC are required to develop novel therapeutics for LID.

The habenula-targeting neurons in the mouse entopeduncular nucleus contain not only somatostatin-positive neurons but also nitric oxide synthase-positive neurons.

The entopeduncular nucleus (EPN) in rodents is one of the two major output nuclei of the basal ganglia and corresponds to the internal segment of the globus pallidus in primates. Previous studies have shown that the EPN contains three types of neurons that project to different targets, namely, parvalbumin (PV)-, somatostatin (SOM)-, and choline acetyltransferase-positive neurons. However, we have recently reported that neurons lacking immunoreactivities for these substances are present in the EPN. Here, we demonstrate that 27.7% of all EPN neurons showed immunoreactivity for nitric oxide synthase (NOS). Among them, NOS-only positive and NOS/SOM double-positive neurons accounted for 20.1% and 6.8%, respectively, whereas NOS/PV double-positive neurons were rarely observed. NOS-containing neurons were distributed in a shell region surrounding the thalamus-targeting, PV-rich core region of the EPN, especially in the ventromedial part of the shell. The retrograde tracer fluoro-gold (FG) was injected into several target regions of EPN neurons. Among FG-labeled EPN neurons after injection into the lateral habenula (LHb), NOS-only positive, NOS/SOM double-positive, and SOM-only positive neurons accounted for 25.7%, 15.2%, and 59.1%, respectively. We conclude that NOS-positive neurons are the second major population of LHb-targeting EPN neurons, suggesting their possible involvement in behaviors in response to aversive stimuli.

Three divisions of the mouse caudal striatum differ in the proportions of dopamine D1 and D2 receptor-expressing cells, distribution of dopaminergic axons, and composition of cholinergic and GABAergic interneurons.

The greater part of the striatum is composed of striosomes and matrix compartments, but we recently demonstrated the presence of a region that has a distinct structural organization in the ventral half of the mouse caudal striatum (Miyamoto et al. in Brain Struct Funct 223:4275-4291, 2018). This region, termed the tri-laminar part based upon its differential immunoreactivities for substance P and enkephalin, consists of medial, intermediate, and lateral divisions. In this study, we quantitatively analyzed the distributions of both projection neurons and interneurons in each division using immunohistochemistry. Two types of projection neurons expressing either the dopamine D1 receptor (D1R) or D2 receptor (D2R) showed complementary distributions throughout the tri-laminar part, but the proportions significantly differed among the three divisions. The proportion of D1R-expressing neurons in the medial, intermediate, and lateral divisions was 88.6 ± 8.2% (651 cells from 3 mice), 14.7 ± 3.8% (1025 cells), and 49.3 ± 4.5% (873 cells), respectively. The intermediate division was further characterized by poor innervation of tyrosine hydroxylase immunoreactive axons. The numerical density of choline acetyltransferase immunoreactive neurons differed among the three divisions following the order from the medial to lateral divisions. In contrast, PV-positive somata were distributed throughout all three divisions at a constant density. Two types of GABAergic interneurons labeled for nitric oxide synthase and calretinin showed the highest cell density in the medial division. The present results characterize the three divisions of the mouse caudal striatum as distinct structures, which will facilitate studies of novel functional loops in the basal ganglia.

Striosome-based map of the mouse striatum that is conformable to both cortical afferent topography and uneven distributions of dopamine D1 and D2 receptor-expressing cells.

The striatum is critically involved in execution of appropriate behaviors, but its internal structures remain unmapped due to its unique structural organization, leading to ambiguity when interpreting heterogeneous properties of striatal neurons that differ by location. We focused on site-specific diversity of striosomes/matrix compartmentalization to draw the striatum map. Five types of striosomes were discriminated according to diverse immunoreactivities for the µ-opioid receptor, substance P (SP) and enkephalin, and each type occupied a particular domain inside the striatum. Furthermore, there was an additional domain lacking striosomes. This striosome-free space was located at the dorsolateral region and received afferents preferentially from the primary motor and sensory cortices, whereas the striosome-rich part received afferents from associational/limbic cortices, with topography inside both innervations. The proportion of dopamine D1 receptor-expressing, presumptive striatonigral neurons was approximately 70% in SP-positive striosomes, 40% in SP-deficient striosomes, 30% in the striosome-free space, and 50% in the matrix. In contrast, the proportion of D2 receptor-expressing, presumptive striatopallidal neurons was complementary to that of D1 receptor-expressing cells, indicating a close relationship between the map and the direct and indirect parallel circuitry. Finally, the most caudal part of the striatum lacked compartmentalization and consisted of three lamina characterized by intense and mutually exclusive immunoreactivities for SP and enkephalin. This tri-laminar part also received specific afferents from the cortex. The newly obtained map will facilitate broad fields of research in the basal ganglia with higher resolution of the three-dimensional anatomy of the striatum.

Immunohistochemical study on the neuronal diversity and three-dimensional organization of the mouse entopeduncular nucleus.

The entopeduncular nucleus (EPN) is one of the major output nuclei of the basal ganglia in rodents. Previous studies have divided it into rostral and caudal halves, with the former containing somatostatin (SOM)-immunoreactive neurons and the latter dominated by parvalbumin (PV)-containing neurons, respectively. However, it is unclear whether this simple rostrocaudal segmentation is appropriate, and the possibility of the existence of other neuronal populations remains to be investigated. In this study the cytoarchitecture of the mouse EPN was analyzed immunohistochemically. Substance P (SP)-immunoreactivity determined the extent of the EPN, which was 800 µm-long along the rostrocaudal axis. PV-positive neurons were concentrated in the caudal two-thirds of this range. PV-negative neurons were abundant in the rostral half but were further located caudally around the PV neuron-rich core. PV(+)/SOM(-) and PV(-)/SOM(+) neurons constituted 28.6% and 45.7% of EPN neurons, respectively, whereas the remaining population (25.7%) exhibited neither immunoreactivity. Eleven percent of EPN neurons lacked immunoreactivity for glutamic acid decarboxylase, indicating their non-GABAergic nature. Three-dimensional reconstruction revealed that PV-rich/SP-poor core was surrounded by PV-poor/SP-rich shell region. Therefore, presumptive thalamus-targeting PV neurons are outnumbered by other populations, and the regional heterogeneity shown here might be related to functionally distinct pathways through the basal ganglia.