Glycemic Challenge Is Associated with the Rapid Cellular Activation of the Locus Ceruleus and Nucleus of Solitary Tract: Circumscribed Spatial Analysis of Phosphorylated MAP Kinase Immunoreactivity.

Rodent studies indicate that impaired glucose utilization or hypoglycemia is associated with the cellular activation of neurons in the medulla (Winslow, 1733) (MY), believed to control feeding behavior and glucose counterregulation. However, such activation has been tracked primarily within hours of the challenge, rather than sooner, and has been poorly mapped within standardized brain atlases. Here, we report that, within 15 min of receiving 2-deoxy-d-glucose (2-DG; 250 mg/kg, i.v.), which can trigger glucoprivic feeding behavior, marked elevations were observed in the numbers of rhombic brain (His, 1893) (RB) neuronal cell profiles immunoreactive for the cellular activation marker(s), phosphorylated p44/42 MAP kinases (phospho-ERK1/2), and that some of these profiles were also catecholaminergic. We mapped their distributions within an open-access rat brain atlas and found that 2-DG-treated rats (compared to their saline-treated controls) displayed greater numbers of phospho-ERK1/2+ neurons in the locus ceruleus (Wenzel and Wenzel, 1812) (LC) and the nucleus of solitary tract (>1840) (NTS). Thus, the 2-DG-activation of certain RB neurons is more rapid than perhaps previously realized, engaging neurons that serve multiple functional systems and which are of varying cellular phenotypes. Mapping these populations within standardized brain atlas maps streamlines their targeting and/or comparable mapping in preclinical rodent models of disease.

Human Brainstem and Cerebellum Atlas: Chemoarchitecture and Cytoarchitecture Paired to MRI.

Lesion localization is the basis for understanding neurologic disease, which is predicated on neuroanatomical knowledge carefully cataloged from histology and imaging atlases. However, it is often difficult to correlate clinical images of brainstem injury obtained by MRI scans with the details of human brainstem neuroanatomy represented in atlases, which are mostly based on cytoarchitecture using Nissl stain or a single histochemical stain, and usually do not include the cerebellum. Here, we report a high-resolution (200 µm) 7T MRI of a cadaveric male human brainstem and cerebellum paired with detailed, coregistered histology (at 2 µm single-cell resolution) of the immunohistochemically stained cholinergic, serotonergic, and catecholaminergic (dopaminergic, noradrenergic, and adrenergic) neurons, in relationship to each other and to the cerebellum. These immunohistochemical findings provide novel insights into the spatial relationships of brainstem cell types and nuclei, including subpopulations of melanin and TH+ neurons, and allows for more informed structural annotation of cell groups. Moreover, the coregistered MRI-paired histology helps validate imaging findings. This is useful for interpreting both scans and histology, and to understand the cell types affected by lesions. Our detailed chemoarchitecture and cytoarchitecture with corresponding high-resolution MRI builds on previous atlases of the human brainstem and cerebellum, and makes precise identification of brainstem and cerebellar cell groups involved in clinical lesions accessible for both laboratory scientists and clinicians alike.SIGNIFICANCE STATEMENT Clinicians and neuroscientists frequently use cross-sectional anatomy of the human brainstem from MRI scans for both clinical and laboratory investigations, but they must rely on brain atlases to neuroanatomical structures. Such atlases generally lack both detail of brainstem chemical cell types, and the cerebellum, which provides an important spatial reference. Our current atlas maps the distribution of key brainstem cell types (cholinergic, serotonergic, and catecholaminergic neurons) in relationship to each other and the cerebellum, and pairs this histology with 7T MR images from the identical brain. This atlas allows correlation of the chemoarchitecture with corresponding MRI, and makes the identification of cell groups that are often discussed, but rarely identifiable on MRI scan, accessible to clinicians and clinical researchers.

Input-output connections of LJA5 prodynorphin neurons.

Sensory information is transmitted from peripheral nerves, through the spinal cord, and up to the brain. Sensory information may be modulated by projections from the brain to the spinal cord, but the neural substrates for top-down sensory control are incompletely understood. We identified a novel population of inhibitory neurons in the mouse brainstem, distinguished by their expression of prodynorphin, which we named LJA5. Here, we identify a similar group of Pdyn+ neurons in the human brainstem, and we define the efferent and afferent projection patterns of LJA5 neurons in mouse. Using specific genetic tools, we selectively traced the projections of the Pdyn-expressing LJA5 neurons through the brain and spinal cord. Terminal fields were densest in the lateral and ventrolateral periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), caudal pressor area, and lamina I of the spinal trigeminal nucleus and all levels of the spinal cord. We then labeled cell types in the PAG, LPB, medulla, and spinal cord to better define the specific targets of LJA5 boutons. LJA5 neurons send the only known inhibitory descending projection specifically to lamina I of the spinal cord, which transmits afferent pain, temperature, and itch information up to the brain. Using retrograde tracing, we found LJA5 neurons receive inputs from sensory and stress areas such as somatosensory/insular cortex, preoptic area, paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus, PAG, and LPB. This pattern of inputs and outputs suggest LJA5 neurons are uniquely positioned to be activated by sensation and stress, and in turn, inhibit pain and itch.

Novel inhibitory brainstem neurons with selective projections to spinal lamina I reduce both pain and itch.

Sensory information is transmitted from peripheral nerves, through the spinal cord, and up to the brain ("bottom up" pathway). Some of this information may be modulated by "top-down" projections from the brain to the spinal cord. Discovering endogenous mechanisms for reducing pain and itch holds enormous potential for developing new treatments. However, neurons mediating the top-down inhibition of pain are not well understood, nor has any such pathway been identified for itch sensation. Here, we identify a novel population of GABAergic neurons in the ventral brainstem, distinguished by prodynorphin expression, which we named LJA5. LJA5 neurons provide the only known inhibitory projection specifically to lamina I of the spinal cord, which contains sensory neurons that transmit pain and itch information up to the brain. Chemogenetically activating LJA5 neurons in male mice reduces capsaicin-induced pain and histamine-induced itch. Identifying this new pathway opens new treatment opportunities for chronic, refractory pain, and pruritis.

Basal forebrain subcortical projections.

The basal forebrain (BF) contains at least three distinct populations of neurons (cholinergic, glutamatergic, and GABA-ergic) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Much attention has focused on the BF's ascending projections to cortex, but less is known about descending projections to subcortical regions. Given the neurochemical and anatomical heterogeneity of the BF, we used conditional anterograde tracing to map the patterns of subcortical projections from multiple BF regions and neurochemical cell types using mice that express Cre recombinase only in cholinergic, glutamatergic, or GABAergic neurons. We confirmed that different BF regions innervate distinct subcortical targets, with more subcortical projections arising from neurons in the caudal and lateral BF (substantia innominata and magnocellular preoptic area). Additionally, glutamatergic and GABAergic BF neurons have distinct patterns of descending projections, while cholinergic descending projections are sparse. Considering the intensity of glutamatergic and GABAergic descending projections, the BF likely acts through subcortical targets to promote arousal, motivation, and other behaviors.

GABAergic Neurons of the Central Amygdala Promote Cataplexy.

Narcolepsy is characterized by chronic sleepiness and cataplexy-sudden muscle paralysis triggered by strong, positive emotions. This condition is caused by a lack of orexin (hypocretin) signaling, but little is known about the neural mechanisms that mediate cataplexy. The amygdala regulates responses to rewarding stimuli and contains neurons active during cataplexy. In addition, lesions of the amygdala reduce cataplexy. Because GABAergic neurons of the central nucleus of the amygdala (CeA) target brainstem regions known to regulate muscle tone, we hypothesized that these cells promote emotion-triggered cataplexy. We injected adeno-associated viral vectors coding for Cre-dependent DREADDs or a control vector into the CeA of orexin knock-out mice crossed with vGAT-Cre mice, resulting in selective expression of the excitatory hM3 receptor or the inhibitory hM4 receptor in GABAergic neurons of the CeA. We measured sleep/wake behavior and cataplexy after injection of saline or the hM3/hM4 ligand clozapine-N-oxide (CNO) under baseline conditions and under conditions that should elicit positive emotions. In mice expressing hM3, CNO approximately doubled the amount of cataplexy in the first 3 h after dosing under baseline conditions. Rewarding stimuli (chocolate or running wheels) also increased cataplexy, but CNO produced no further increase. In mice expressing hM4, CNO reduced cataplexy in the presence of chocolate or running wheels. These results demonstrate that GABAergic neurons of the CeA are sufficient and necessary for the production of cataplexy in mice, and they likely are a key part of the mechanism through which positive emotions trigger cataplexy.SIGNIFICANCE STATEMENT Cataplexy is one of the major symptoms of narcolepsy, but little is known about how strong, positive emotions trigger these episodes of muscle paralysis. Prior research shows that amygdala neurons are active during cataplexy and cataplexy is reduced by lesions of the amygdala. We found that cataplexy is substantially increased by selective activation of GABAergic neurons in the central nucleus of the amygdala (CeA). We also demonstrate that inhibition of these neurons reduces reward-promoted cataplexy. These results build upon prior work to establish the CeA as a crucial element in the neural mechanisms of cataplexy. These results demonstrate the importance of the CeA in regulating responses to rewarding stimuli, shedding light on the broader neurobiology of emotions and motor control.

Descending projections from the basal forebrain to the orexin neurons in mice.

The orexin (hypocretin) neurons play an essential role in promoting arousal, and loss of the orexin neurons results in narcolepsy, a condition characterized by chronic sleepiness and cataplexy. The orexin neurons excite wake-promoting neurons in the basal forebrain (BF), and a reciprocal projection from the BF back to the orexin neurons may help promote arousal and motivation. The BF contains at least three different cell types (cholinergic, glutamatergic, and γ-aminobutyric acid (GABA)ergic neurons) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Given the neurochemical and anatomical heterogeneity of the BF, we mapped the pattern of BF projections to the orexin neurons across multiple BF regions and neuronal types. We performed conditional anterograde tracing using mice that express Cre recombinase only in neurons producing acetylcholine, glutamate, or GABA. We found that the orexin neurons are heavily apposed by axon terminals of glutamatergic and GABAergic neurons of the substantia innominata (SI) and magnocellular preoptic area, but there was no innervation by the cholinergic neurons. Channelrhodopsin-assisted circuit mapping (CRACM) demonstrated that glutamatergic SI neurons frequently form functional synapses with the orexin neurons, but, surprisingly, functional synapses from SI GABAergic neurons were rare. Considering their strong reciprocal connections, BF and orexin neurons likely work in concert to promote arousal, motivation, and other behaviors. J. Comp. Neurol. 525:1668-1684, 2017. © 2016 Wiley Periodicals, Inc.

Genetic identity of thermosensory relay neurons in the lateral parabrachial nucleus.

The parabrachial nucleus is important for thermoregulation because it relays skin temperature information from the spinal cord to the hypothalamus. Prior work in rats localized thermosensory relay neurons to its lateral subdivision (LPB), but the genetic and neurochemical identity of these neurons remains unknown. To determine the identity of LPB thermosensory neurons, we exposed mice to a warm (36°C) or cool (4°C) ambient temperature. Each condition activated neurons in distinct LPB subregions that receive input from the spinal cord. Most c-Fos+ neurons in these LPB subregions expressed the transcription factor marker FoxP2. Consistent with prior evidence that LPB thermosensory relay neurons are glutamatergic, all FoxP2+ neurons in these subregions colocalized with green fluorescent protein (GFP) in reporter mice for Vglut2, but not for Vgat. Prodynorphin (Pdyn)-expressing neurons were identified using a GFP reporter mouse and formed a caudal subset of LPB FoxP2+ neurons, primarily in the dorsal lateral subnucleus (PBdL). Warm exposure activated many FoxP2+ neurons within PBdL. Half of the c-Fos+ neurons in PBdL were Pdyn+, and most of these project into the preoptic area. Cool exposure activated a separate FoxP2+ cluster of neurons in the far-rostral LPB, which we named the rostral-to-external lateral subnucleus (PBreL). These findings improve our understanding of LPB organization and reveal that Pdyn-IRES-Cre mice provide genetic access to warm-activated, FoxP2+ glutamatergic neurons in PBdL, many of which project to the hypothalamus.