Phenotypic variation of transcriptomic cell types in mouse motor cortex.

Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties1,2. Most existing neural taxonomies are based on either transcriptomic3,4 or morpho-electric5,6 criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells7. Here we used Patch-seq8 to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of more than 1,300 neurons in adult mouse primary motor cortex, providing a morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (those expressing Vip, Pvalb, Sst and so on) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neuronal types in the neocortex do not always form discrete entities. Instead, neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.

Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

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.

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons.

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ±â€¯0.7% of serotonergic and 48.9 ±â€¯5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.

The GABAergic Gudden's dorsal tegmental nucleus: A new relay for serotonergic regulation of sleep-wake behavior in the mouse.

Serotonin (5-HT) neurons are involved in wake promotion and exert a strong inhibitory influence on rapid eye movement (REM) sleep. Such effects have been ascribed, at least in part to the action of 5-HT at post-synaptic 5-HT1A receptors (5-HT1AR) in the brainstem, a major wake/REM sleep regulatory center. However, the neuroanatomical substrate through which 5-HT1AR influence sleep remains elusive. We therefore investigated whether a brainstem structure containing a high density of 5-HT1AR mRNA, the GABAergic Gudden's dorsal tegmental nucleus (DTg), may contribute to 5-HT-mediated regulatory mechanisms of sleep-wake stages. We first found that bilateral lesions of the DTg promote wake at the expense of sleep. In addition, using local microinjections into the DTg in freely moving mice, we showed that local activation of 5-HT1AR by the prototypical agonist 8-OH-DPAT enhances wake and reduces deeply REM sleep duration. The specific involvement of 5-HT1AR in the latter effects was further demonstrated by ex vivo extracellular recordings showing that the selective 5-HT1AR antagonist WAY 100635 prevented DTg neuron inhibition by 8-OH-DPAT. We next found that GABAergic neurons of the ventral DTg exclusively targets glutamatergic neurons of the lateral mammillary nucleus (LM) in the posterior hypothalamus by means of anterograde and retrograde tracing techniques using cre driver mouse lines and a modified rabies virus. Altogether, our findings strongly support the idea that 5-HT-driven enhancement of wake results from 5-HT1AR-mediated inhibition of DTg GABAergic neurons that would in turn disinhibit glutamatergic neurons in the mammillary bodies. We therefore propose a Raphe→DTg→LM pathway as a novel regulatory circuit underlying 5-HT modulation of arousal.

Activation of Parvalbumin Neurons in the Rostro-Dorsal Sector of the Thalamic Reticular Nucleus Promotes Sensitivity to Pain in Mice.

The calcium-binding protein, parvalbumin (PV), is highly expressed in thalamic reticular nucleus (TRN) GABAergic neurons, which receive input from the cerebral cortex and thalamus and send inhibitory output to the thalamic relay nucleus. Previous studies suggest that the TRN is involved in pain regulation as an important relay nucleus of the ascending pain pathway. However, little is known about its functional role in pain regulation and interconnectivity. In our study, the role of rostro-dorsal sector of TRN (TRNrd) PV-positive neurons in pain regulation was studied using chemogenetics based on designer receptors exclusively activated by designer drugs (DREADD). Then, projections from the TRNrd PV-positive neurons were explored using PV-Cre transgenic mice, conditional anterograde axonal tract tracing, and optogenetics, combined with immunohistochemistry and electrophysiology. The results showed that activation of PV-positive neurons in the TRNrd decreased the mechanical threshold and thermal latency of behaving mice during the light period when neuronal activity was low. Furthermore, the anterodorsal and paratenial thalamic nucleus received innervation from PV-positive neurons in the TRNrd. They were specifically inhibited by GABA, which is released from local axonal endings of PV neurons. These findings indicate that activation of PV neurons in the TRNrd increases pain sensitivity in PV-Cre transgenic mice.

Global and local excitation and inhibition shape the dynamics of the cortico-striatal-thalamo-cortical pathway.

The cortico-striatal-thalamo-cortical (CSTC) pathway is a brain circuit that controls movement execution, habit formation and reward. Hyperactivity in the CSTC pathway is involved in obsessive compulsive disorder (OCD), a neuropsychiatric disorder characterized by the execution of repetitive involuntary movements. The striatum shapes the activity of the CSTC pathway through the coordinated activation of two classes of medium spiny neurons (MSNs) expressing D1 or D2 dopamine receptors. The exact mechanisms by which balanced excitation/inhibition (E/I) of these cells controls the network dynamics of the CSTC pathway remain unclear. Here we use non-linear modeling of neuronal activity and bifurcation theory to investigate how global and local changes in E/I of MSNs regulate the activity of the CSTC pathway. Our findings indicate that a global and proportionate increase in E/I pushes the system to states of generalized hyper-activity throughout the entire CSTC pathway. Certain disproportionate changes in global E/I trigger network oscillations. Local changes in the E/I of MSNs generate specific oscillatory behaviors in MSNs and in the CSTC pathway. These findings indicate that subtle changes in the relative strength of E/I of MSNs can powerfully control the network dynamics of the CSTC pathway in ways that are not easily predicted by its synaptic connections.

Genetically Distinct Parallel Pathways in the Entopeduncular Nucleus for Limbic and Sensorimotor Output of the Basal Ganglia.

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.

Hypothalamic Vasopressinergic Projections Innervate Central Amygdala GABAergic Neurons: Implications for Anxiety and Stress Coping.

The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

Intrathecal Transplantation of Embryonic Stem Cell-Derived Spinal GABAergic Neural Precursor Cells Attenuates Neuropathic Pain in a Spinal Cord Injury Rat Model.

Neuropathic pain following spinal cord injury (SCI) is a devastating disease characterized by spontaneous pain such as hyperalgesia and allodynia. In this study, we investigated the therapeutic potential of ESC-derived spinal GABAergic neurons to treat neuropathic pain in a SCI rat model. Mouse embryonic stem cell-derived neural precursor cells (mESC-NPCs) were cultured in media supplemented with sonic hedgehog (SHH) and retinoic acid (RA) and efficiently differentiated into GABAergic neurons. Interestingly, low doses of SHH and RA induced MGE-like progenitors, which expressed low levels of DARPP32 and Nkx2.1 and high levels of Irx3 and Pax6. These cells subsequently generated the majority of the DARPP32(-) GABAergic neurons after in vitro differentiation. The spinal mESC-NPCs were intrathecally transplanted into the lesion area of the spinal cord around T10-T11 at 21 days after SCI. The engrafted spinal GABAergic neurons remarkably increased both the paw withdrawal threshold (PWT) below the level of the lesion and the vocalization threshold (VT) to the level of the lesion (T12, T11, and T10 vertebrae), which indicates attenuation of chronic neuropathic pain by the spinal GABAergic neurons. The transplanted cells were positive for GABA antibody staining in the injured region, and cells migrated to the injured spinal site and survived for more than 7 weeks in L4-L5. The mESC-NPC-derived spinal GABAergic neurons dramatically attenuated the chronic neuropathic pain following SCI, suggesting that the spinal GABAergic mESC-NPCs cultured with low doses of SHH and RA could be alternative cell sources for treatment of SCI neuropathic pain by stem cell-based therapies.

Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons.

Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2-containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi-like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2-positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections.

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex.

BACKGROUND AND OBJECTIVE: Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice. STUDY DESIGN/MATERIALS AND METHODS: We applied 532 nm LLI (Nd:YVO4, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques. RESULTS: In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons. CONCLUSION: It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.

Effective induction of cells expressing GABAergic neuronal markers from mouse embryonic stem cell.

Successful derivations of specific neuronal and glial cells from embryonic stem cells have enormous potential for cell therapies and regenerative medicine. However, the low efficiency, the complexity of induction method, and the need for purification represent obstacles that make their application impractical. In this study, we found that PDGFRα(+) cells derived from mouse embryonic stem cells (mESC) can serve as a useful source from which to induce cells that express γ-aminobutyric-acid (GABA)-releasing (GABAergic) neuronal markers. PDGFRα(+) cells were induced from mESC on collagen IV-coated plates in mesenchymal stem cell (MSC) culture medium with limited exposure to retinoic acid, sorted by fluorescence-activated cell sorter and maintained in MSC culture medium containing Y-27632, a Rho-associated kinase inhibitor. We found that supplementation of vascular endothelial growth factor, fibroblast growth factor-basic, and sodium azide (NaN3) to MSC culture medium effectively differentiated PDGFRα(+) cells into cells that express GABAergic neuronal markers, such as Pax2, Dlx2, GAD67 NCAM, and tubulin-ßIII, while markers for oligodendrocyte (Sox2) and astrocyte (Glast) were suppressed. Immunostaining for GABA showed the majority (86 ± 5%) of the induced cells were GABA-positive. We also found that the PDGFRα(+) cells retained such differentiation potential even after more than ten passages and cryopreservation. In summary, this study presents a simple and highly efficient method of inducing cells that express GABAergic neuronal markers from mESC. Together with its ease of maintenance in vitro, PDGFRα(+) cells derived from mESC may serve as a useful source for such purpose.

A new multistep induction protocol for the transdifferentiation of bone marrow stromal stem cells into GABAergic neuron-like cells.

BACKGROUND: Bone marrow stromal stem cells (BMSC) are appropriate source of multipotent stem cells that are ideally suited for use in various cell-based therapies. It can be differentiated into neuronal-like cells under appropriate conditions. This study examined the effectiveness of co-stimulation of creatine and retinoic acid in increasing the differentiation of BMSC into GABAergic neuron-like cells (GNLC). METHODS: BMSC isolated from the femurs and tibias of adult rats were cultured in DMEM/F12 medium supplemented with 10% FBS, pre-induced using ß-mercaptoethanol ß-ME) and induced using retinoic acid (RA) and creatine. Immunostaining of neurofilament 200 kDa, neurofilament 160 kDa, nestin, fibronectin, Gamma-amino butyric acid (GABA) and glutamic acid decarboxylase (GAD) 65/67 were used to evaluate the transdifferentiation of BMSC into GNLC and to evaluate the effectiveness of pre-induction and induction assays. The expression of genes that encode fibronectin, octamer-binding transcription factor 4 (Oct-4), GAD 65/67 and the vesicular GABA transporter was examined in BMSC and GNLC by using RT-PCR assays during transdifferentiation of BMSC into GLNC. RESULTS: Co-stimulation with RA and creatine during the induction stage doubled the rates of GABAergic differentiation compared with induction using creatine alone, resulting in a 71.6% yield for GLNC. RT-PCR showed no expression of Oct-4 and fibronectin after the induction stage. CONCLUSION: The results of this study showed that the application of ß-ME, RA, and creatine induced the transdifferentiation of BMSC into GLNC.

Presynaptic inhibition of GABAergic synaptic transmission by adenosine in mouse hypothalamic hypocretin neurons.

Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine-regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin neurons by activation of presynaptic adenosine A1 receptors, which may finely regulate the excitability of these neurons as well as eventually modulate the sleep-wakefulness.

Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons.

Whether neocortical γ-aminobutyric acid (GABA) cells are composed of a limited number of distinct classes of neuron, or whether they are continuously differentiated with much higher diversity, remains a contentious issue for the field. Most GABA cells of rat frontal cortex have at least 1 of 6 chemical markers (parvalbumin, calretinin, alpha-actinin-2, somatostatin, vasoactive intestinal polypeptide, and cholecystokinin), with each chemical class comprising several distinct neuronal subtypes having specific physiological and morphological characteristics. To better clarify GABAergic neuron diversity, we assessed the colocalization of these 6 chemical markers with corticotropin-releasing factor (CRF), neuropeptide Y (NPY), the substance P receptor (SPR), and nitric oxide synthase (NOS); these 4 additional chemical markers suggested to be expressed diversely or specifically among cortical GABA cells. We further correlated morphological and physiological characteristics of identified some chemical subclasses of inhibitory neurons. Our results reveal expression specificity of CRF, NPY, SPR, and NOS in morphologically and physiologically distinct interneuron classes. These observations support the existence of a limited number of functionally distinct subtypes of GABA cells in the neocortex.