Probes for VGLUT2

For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.

In the brain, purines such as ATP and adenosine can function as neurotransmitters and co-transmitters, or serve as signals in neuron-glial interactions. In thalamocortical (TC) projections to sensory cortex, adenosine functions as a negative regulator of glutamate release via activation of the presynaptic adenosine A1 receptor (A1 R). In the auditory forebrain, restriction of A1 R-adenosine signaling in medial geniculate (MG) neurons is sufficient to extend LTP, LTD, and tonotopic map plasticity in adult mice for months beyond the critical period. Interfering with adenosine signaling in primary auditory cortex (A1) does not contribute to these forms of plasticity, suggesting regional differences in the roles of A1 R-mediated adenosine signaling in the forebrain. To advance understanding of the circuitry, in situ hybridization was used to localize neuronal and glial cell types in the auditory forebrain that express A1 R transcripts (Adora1), based on co-expression with cell-specific markers for neuronal and glial subtypes. In A1, Adora1 transcripts were concentrated in L3/4 and L6 of glutamatergic neurons. Subpopulations of GABAergic neurons, astrocytes, oligodendrocytes, and microglia expressed lower levels of Adora1. In MG, Adora1 was expressed by glutamatergic neurons in all divisions, and subpopulations of all glial classes. The collective findings imply that A1 R-mediated signaling broadly extends to all subdivisions of auditory cortex and MG. Selective expression by neuronal and glial subpopulations suggests that experimental manipulations of A1 R-adenosine signaling could impact several cell types, depending on their location. Strategies to target Adora1 in specific cell types can be developed from the data generated here.

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, non-cholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Nmb mRNA. Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, Kcnk5 and Gpr4 These Nmb-low RTN neurons also express varying levels of transcripts for Gal, Penk and Adcyap1,and receptors for substance P, orexin, serotonin and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb-high RTN neurons do not express Fos after hypercapnia, have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1, but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb-low neurons, the vast majority, are central respiratory chemoreceptors whereas Nmb-high neurons likely have other functions.SIGNIFICANCE STATEMENTCentral respiratory chemoreceptors regulate arterial PCO2 by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed in situ hybridization, we show here that a single marker, Neuromedin B mRNA (Nmb), identifies RTN neurons in rodents. We also suggest that >75% of these Nmb neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (Kcnk5 and Gpr4). The other RTN neurons express very high levels of Nmb, but low levels of Kcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin, and do not respond to hypercapnia. Their function is unknown.

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG) /dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

Significance Statement Clinicians have long had the goal of separating analgesia from anxiety when using deep brain electrical stimulation of the periaqueductal gray (PAG) for difficult to treat pain. Here we show that selective activation of dopamine neurons within the PAG produces analgesia without other behavioral effects, while stimulating glutamate neurons mediates stress-induced anxiety and analgesia. Our results suggest that dopamine agonists may represent a novel class of analgesic drugs and elucidate target neurons that could mediate their effect.