Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D-28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67-kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types.

Centre-surround inhibition--the suppression of activity of neighbouring cells by a central group of neurons--is a fundamental mechanism that increases contrast in patterned sensory processing. The initial stage of neural processing in olfaction occurs in olfactory bulb glomeruli, but evidence for functional interactions between glomeruli is fragmentary. Here we show that the so-called 'short axon' cells, contrary to their name, send interglomerular axons over long distances to form excitatory synapses with inhibitory periglomerular neurons up to 20-30 glomeruli away. Interglomerular excitation of these periglomerular cells potently inhibits mitral cells and forms an on-centre, off-surround circuit. This interglomerular centre-surround inhibitory network, along with the well-established mitral-granule-mitral inhibitory circuit, forms a serial, two-stage inhibitory circuit that could enhance spatiotemporal responses to odours.

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Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic–GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65 + GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic–GABAergic cells have extensive interglomerular projections. DAergic–GABAergic SA cells comprise two subgroups. One subpopulation contacts 5–12 glomeruli and is referred to as “oligoglomerular.” Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON→ET→SA circuit. The second population of DAergic–GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive “polyglomerular” network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

The molecular mechanisms that regulate the production and diversity of olfactory bulb interneurons remain poorly understood. With the exception of the GABAergic/dopaminergic subtype in the glomerular layer, no information exists concerning the generation of the other subtypes. Here we show that the recently identified zinc finger transcription factor Sp8 is expressed in neurogenic regions, which give rise to olfactory bulb interneurons at embryonic and postnatal time points and remains expressed in the calretinin-expressing and GABAergic/nondopaminergic interneurons of the glomerular layer. Conditional inactivation of Sp8 in the embryonic ventral telencephalon reveals a requirement for the normal generation of these interneuron subtypes. Sp8 conditional mutants exhibit an increase in cell death within the lateral ganglionic eminence and rostral migratory stream. Moreover, mutant neuroblasts/interneurons are misspecified and display abnormal migration patterns in the olfactory bulb, indicating that Sp8 contributes to olfactory bulb interneuron diversity by regulating the survival, migration, and molecular specification of neuroblasts/interneurons.

The adult mouse subependymal zone (SEZ) harbours neural stem cells that are thought to generate exclusively GABAergic interneurons of the olfactory bulb. Here we describe the adult generation of glutamatergic juxtaglomerular neurons, with dendritic arborizations that project into adjacent glomeruli identifying them as short-axon cells. Fate mapping revealed that these originate from Neurogenin2- and Tbr2-expressing progenitors located in the dorsal region of the SEZ. Progenitors of these glutamatergic interneurons recapitulate the sequential expression of transcription factors that hallmark glutamatergic neurogenesis in the developing cerebral cortex and adult hippocampus. Indeed, the molecular specification of these SEZ progenitors allows for their recruitment into the cerebral cortex upon lesion. Taken together, our data show that SEZ progenitors not only produce a novel population of adult-born glutamatergic juxtaglomerular neurons, but may also provide a new source of progenitors for endogenous repair.

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia–neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia–neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury–induced changes at somatic junctions triggered P2Y12 receptor–dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.