The influence of castration on intramural neurons of the urinary bladder trigone in male pigs.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the intramural ganglia of the urinary bladder trigone (UBT) in male pigs using double-labeling immunohistochemistry. The ganglia were examined in intact (IP) 7-day-old (castration day) pigs, and at 3 and 6 months after surgery. In IP and control (3- and 6-month-old noncastrated pigs) groups, virtually, all neurons were adrenergic (68%) or cholinergic (32%) in nature. Many of them (32%, 51%, and 81%, respectively; 56%, 75%, and 85% adrenergic; and 32%, 52%, and 65% cholinergic, respectively) stained for the androgen receptor (AR), and only a small number of nerve cells were caspase-3 (CASP-3)-positive. In 3- and 6-month-old castrated pigs, an excessive loss (87.6% and 87.5%, respectively) of neurons and intraganglionic nerve fibers was observed. The majority of the surviving adrenergic (61% and 72%, respectively) and many cholinergic (41% and 31%, respectively) neurons expressed CASP-3 and were also AR-positive (61% and 66%, and 40% and 36%, respectively). This study revealed for the first time the excessive loss of intramural UBT neurons following castration, which could have resulted from apoptosis induced by androgen deprivation.

Lack of Cannabinoid Receptor Type-1 Leads to Enhanced Age-Related Neuronal Loss in the Locus Coeruleus.

Our laboratory and others have previously shown that cannabinoid receptor type-1 (CB1r) activity is neuroprotective and a modulator of brain ageing; a genetic disruption of CB1r signaling accelerates brain ageing, whereas the pharmacological stimulation of CB1r activity had the opposite effect. In this study, we have investigated if the lack of CB1r affects noradrenergic neurons in the locus coeruleus (LC), which are vulnerable to age-related changes; their numbers are reduced in patients with neurodegenerative diseases and probably also in healthy aged individuals. Thus, we compared LC neuronal numbers between cannabinoid 1 receptor knockout (Cnr1-/-) mice and their wild-type littermates. Our results reveal that old Cnr1-/- mice have less noradrenergic neurons compared to their age-matched wild-type controls. This result was also confirmed by the analysis of the density of noradrenergic terminals which proved that Cnr1-/- mice had less compared to the wild-type controls. Additionally, we assessed pro-inflammatory glial activity in the LC. Although the density of microglia in Cnr1-/- mice was enhanced, they did not show enhanced inflammatory profile. We hypothesize that CB1r activity is necessary for the protection of noradrenergic neurons, but its anti-inflammatory effect probably only plays a minor role in it.

Sex-Related Differential Whole-Brain Input Atlas of Locus Coeruleus Noradrenaline Neurons.

As the most important organ in our bodies, the brain plays a critical role in deciding sex-related differential features; however, the underlying neural circuitry basis remains unclear. Here, we used a cell-type-specific rabies virus-mediated monosynaptic tracing system to generate a sex differences-related whole-brain input atlas of locus coeruleus noradrenaline (LC-NE) neurons. We developed custom pipelines for brain-wide comparisons of input sources in both sexes with the registration of the whole-brain data set to the Allen Mouse Brain Reference Atlas. Among 257 distinct anatomical regions, we demonstrated the differential proportions of inputs to LC-NE neurons in male and female mice at different levels. Locus coeruleus noradrenaline neurons of two sexes showed general similarity in the input patterns, but with differentiated input proportions quantitatively from major brain regions and diverse sub-regions. For instance, inputs to male LC-NE neurons were found mainly in the cerebrum, interbrain, and cerebellum, whereas inputs to female LC-NE neurons were found in the midbrain and hindbrain. We further found that specific subsets of nuclei nested within sub-regions contributed to overall sex-related differences in the input circuitry. Furthermore, among the totaled 123 anatomical regions with proportion of inputs >0.1%, we also identified 11 sub-regions with significant statistical differences of total inputs between male and female mice, and seven of them also showed such differences in ipsilateral hemispheres. Our study not only provides a structural basis to facilitate our understanding of sex differences at a circuitry level but also provides clues for future sexually differentiated functional studies related to LC-NE neurons.

Tyrosine hydroxylase immunolabeling reveals the distribution of catecholaminergic neurons in the central nervous systems of the spiders Hogna lenta (Araneae: Lycosidae) and Phidippus regius (Araneae: Salticidae).

With over 48,000 species currently described, spiders (Arthropoda: Chelicerata: Araneae) comprise one of the most diverse groups of animals on our planet, and exhibit an equally wide array of fascinating behaviors. Studies of central nervous systems (CNSs) in spiders, however, are relatively sparse, and no reports have yet characterized catecholaminergic (dopamine [DA]- or norepinephrine-synthesizing) neurons in any spider species. Because these neuromodulators are especially important for sensory and motor processing across animal taxa, we embarked on a study to identify catecholaminergic neurons in the CNS of the wolf spider Hogna lenta (Lycosidae) and the jumping spider Phidippus regius (Salticidae). These neurons were most effectively labeled with an antiserum raised against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. We found extensive catecholamine-rich neuronal fibers in the first- and second-order optic neuropils of the supraesophageal mass (brain), as well as in the arcuate body, a region of the brain thought to receive visual input and which may be involved in higher order sensorimotor integration. This structure likely shares evolutionary origins with the DA-enriched central complex of the Mandibulata. In the subesophageal mass, we detected an extensive filigree of TH-immunoreactive (TH-ir) arborizations in the appendage neuromeres, as well as three prominent plurisegmental fiber tracts. A vast abundance of TH-ir somata were located in the opisthosomal neuromeres, the largest of which appeared to project to the brain and decorate the appendage neuromeres. Our study underscores the important roles that the catecholamines likely play in modulating spider vision, higher order sensorimotor processing, and motor patterning.

Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal Lung.

Young children are more susceptible to developing allergic asthma than adults. As neural innervation of the peripheral tissue continues to develop after birth, neurons may modulate tissue inflammation in an age-related manner. Here we showed that sympathetic nerves underwent a dopaminergic-to-adrenergic transition during post-natal development of the lung in mice and humans. Dopamine signaled through a specific dopamine receptor (DRD4) to promote T helper 2 (Th2) cell differentiation. The dopamine-DRD4 pathway acted synergistically with the cytokine IL-4 by upregulating IL-2-STAT5 signaling and reducing inhibitory histone trimethylation at Th2 gene loci. In murine models of allergen exposure, the dopamine-DRD4 pathway augmented Th2 inflammation in the lungs of young mice. However, this pathway operated marginally after sympathetic nerves became adrenergic in the adult lung. Taken together, the communication between dopaminergic nerves and CD4+ T cells provides an age-related mechanism underlying the susceptibility to allergic inflammation in the early lung.

Conversion of Astrocytes and Fibroblasts into Functional Noradrenergic Neurons.

Dysfunction of noradrenergic (NA) neurons is associated with a number of neuronal disorders. Diverse neuronal subtypes can be generated by direct reprogramming. However, it is still unknown how to convert non-neuronal cells into NA neurons. Here, we show that seven transcription factors (TFs) (Ascl1, Phox2b, AP-2α, Gata3, Hand2, Nurr1, and Phox2a) are able to convert astrocytes and fibroblasts into induced NA (iNA) neurons. These iNA neurons express the genes required for the biosynthesis, release, and re-uptake of noradrenaline. Moreover, iNA neurons fire action potentials, receive synaptic inputs, and control the beating rate of co-cultured ventricular myocytes. Furthermore, iNA neurons survive and integrate into neural circuits after transplantation. Last, human fibroblasts can be converted into functional iNA neurons as well. Together, iNA neurons are generated by direct reprogramming, and they could be potentially useful for disease modeling and cell-based therapies.

Noradrenergic terminal density varies among different groups of hypoglossal premotor neurons.

In obstructive sleep apnea (OSA) patients, contraction of the muscles of the tongue is needed to protect the upper airway from collapse. During wakefulness, norepinephrine directly excites motoneurons that innervate the tongue and other upper airway muscles but its excitatory effects decline during sleep, thus contributing to OSA. In addition to motoneurons, NE may regulate activity in premotor pathways but little is known about these upstream effects. To start filling this void, we injected a retrograde tracer (beta-subunit of cholera toxin-CTb; 5-10 nl, 1%) into the hypoglossal (XII) motor nucleus in 7 rats. We then used dual immunohistochemistry and brightfield microscopy to count dopamine beta-hydroxylase (DBH)-positive axon terminals closely apposed to CTb cells located in five anatomically distinct XII premotor regions. In different premotor groups, we found on the average 2.2-4.3 closely apposed DBH terminals per cell, with ˜60% more terminals on XII premotor neurons located in the ventrolateral pontine parabrachial region and ventral medullary gigantocellular region than on XII premotor cells of the rostral or caudal intermediate medullary reticular regions. This difference suggests stronger control by norepinephrine of the interneurons that mediate complex behavioral effects than of those mediating reflexes or respiratory drive to XII motoneurons.

Distinct regional patterns in noradrenergic innervation of the rat prefrontal cortex.

The anatomy and functions of the rodent prefrontal cortex (PFC) have been extensively studied. It is now clear that the PFC is at the core of various executive functions and that these functions depend on monoaminergic neuromodulation. The PFC receives extensive projections from monoaminergic nuclei and, in particular, from the locus cœruleus (LC) which is the major source of noradrenaline (NA) in the cortex. Projections of this nucleus have long been considered to act diffusely and uniformly throughout the entire brain. However, recent studies have revealed a separate innervation of prefrontal sub-regions by non-collateralizing LC neurons, suggesting a specific modulation of their functions. Following this idea, we aimed at describing more precisely the pattern of noradrenergic innervation into different orbital (OFC) and medial (mPFC) sub-regions of the PFC. We focused on the lateral (LO), ventral (VO) and medial (MO) portions of the OFC, and on areas 32d (A32d), 32v (A32v) and 25 (A25) in the mPFC. Using Dopamine-ß-Hydroxylase as a specific noradrenergic marker, we performed an automatic quantification of noradrenergic fibers and varicosities in each of these sub-regions. The results indicate that noradrenergic innervation is heterogeneous in some prefrontal sub-regions along the rostro-caudal axis. Functional dissociations have been recently reported in prefrontal sub-regions along the rostro-caudal direction. Our findings add neuroanatomical support to this emergent idea.

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells.

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of ß-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that ß-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum.

Temporal requirements for ISL1 in sympathetic neuron proliferation, differentiation, and diversification.

Malformations of the sympathetic nervous system have been associated with cardiovascular instability, gastrointestinal dysfunction, and neuroblastoma. A better understanding of the factors regulating sympathetic nervous system development is critical to the development of potential therapies. Here, we have uncovered a temporal requirement for the LIM homeodomain transcription factor ISL1 during sympathetic nervous system development by the analysis of two mutant mouse lines: an Isl1 hypomorphic line and mice with Isl1 ablated in neural crest lineages. During early development, ISL1 is required for sympathetic neuronal fate determination, differentiation, and repression of glial differentiation, although it is dispensable for initial noradrenergic differentiation. ISL1 also plays an essential role in sympathetic neuron proliferation by controlling cell cycle gene expression. During later development, ISL1 is required for axon growth and sympathetic neuron diversification by maintaining noradrenergic differentiation, but repressing cholinergic differentiation. RNA-seq analyses of sympathetic ganglia from Isl1 mutant and control embryos, together with ISL1 ChIP-seq analysis on sympathetic ganglia, demonstrated that ISL1 regulates directly or indirectly several distinct signaling pathways that orchestrate sympathetic neurogenesis. A number of genes implicated in neuroblastoma pathogenesis are direct downstream targets of ISL1. Our study revealed a temporal requirement for ISL1 in multiple aspects of sympathetic neuron development, and suggested Isl1 as a candidate gene for neuroblastoma.

Effect of Noradrenergic Neurotoxin DSP-4 and Maprotiline on Heart Rate Spectral Components in Stressed and Resting Rats.

The effects of intraperitoneal DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a noradrenergic neurotoxin) and maprotiline (an inhibitor of norepinephrine reuptake in synapses) on spectral components of heart rhythm variability were examined in outbred male and female rats treated with these agents in daily doses of 10 mg/kg for 3 days. At rest, DSP-4 elevated LF and VLF spectral components in male and female rats. Maprotiline elevated LF and VLF components in males at rest, increased HR and reduced all spectral components in resting females. Stress against the background of DSP-4 treatment sharply increased heart rate and reduced the powers of all spectral components (especially LF and VLF components). In maprotiline-treated rats, stress increased the powers of LF and VLF components. Thus, the central noradrenergic system participates in the formation of LF and VLF spectral components of heart rate variability at rest and especially during stressful stimulation, which can determine the phasic character of changes in the heart rate variability observed in stressed organism.

Neuroblastoma is composed of two super-enhancer-associated differentiation states.

Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.

Effects of endogenous nitric oxide on adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide-containing nerve-mediated vasodilation in pithed rats.

Vascular adrenergic nerves mainly regulate the tone of blood vessels. Calcitonin gene-related peptide-containing (CGRPergic) vasodilator nerves also participate in the regulation of vascular tone. Furthermore, there are nitric oxide (NO)-containing (nitrergic) nerves, which include NO in blood vessels as vasodilator nerves, but it remains unclear whether nitrergic nerves participate in vascular regulation. The present study investigated the role of nitrergic nerves in vascular responses to spinal cord stimulation (SCS) and vasoactive agents in pithed rats. Wistar rats were anesthetized and pithed, and vasopressor responses to SCS and injections of norepinephrine were observed. To evaluate vasorelaxant responses, the BP was increased by a continuous infusion of methoxamine with hexamethonium to block autonomic outflow. After the elevated BP stabilized, SCS and injections of acetylcholine (ACh), sodium nitroprusside (SNP), and CGRP were intravenously administered. We then evaluated the effects of the NO synthase (NOS) inhibitor, N-ω-nitro-L-arginine methylester hydrochloride (L-NAME), on these vascular responses. Pressor responses to SCS and norepinephrine in pithed rats were enhanced by L-NAME, while the combined infusion of L-NAME and L-arginine had no effect on these responses. L-NAME infusion significantly increased the release of norepinephrine evoked by SCS. In pithed rats with artificially increased BP and L-NAME infusion, depressor response to ACh (except for 0.05nmol/kg) was suppressed and SNP (only 2nmol/kg) was enhanced. However, depressor responses to SCS and CGRP were similar to control responses. The present results suggest endogenous NO regulates vascular tone through endothelium function and inhibition of adrenergic neurotransmission, but not through CGRPergic nerves.

Enhanced noradrenergic axon regeneration into schwann cell-filled PVDF-TrFE conduits after complete spinal cord transection.

Schwann cell (SC) transplantation has been utilized for spinal cord repair and demonstrated to be a promising therapeutic strategy. In this study, we investigated the feasibility of combining SC transplantation with novel conduits to bridge the completely transected adult rat spinal cord. This is the first and initial study to evaluate the potential of using a fibrous piezoelectric polyvinylidene fluoride trifluoroethylene (PVDF-TrFE) conduit with SCs for spinal cord repair. PVDF-TrFE has been shown to enhance neurite growth in vitro and peripheral nerve repair in vivo. In this study, SCs adhered and proliferated when seeded onto PVDF-TrFE scaffolds in vitro. SCs and PVDF-TrFE conduits, consisting of random or aligned fibrous inner walls, were transplanted into transected rat spinal cords for 3 weeks to examine early repair. Glial fibrillary acidic protein (GFAP)+ astrocyte processes and GFP (green fluorescent protein)-SCs were interdigitated at both rostral and caudal spinal cord/SC transplant interfaces in both types of conduits, indicative of permissivity to axon growth. More noradrenergic/DßH+ (dopamine-beta-hydroxylase) brainstem axons regenerated across the transplant when greater numbers of GFAP+ astrocyte processes were present. Aligned conduits promoted extension of DßH+ axons and GFAP+ processes farther into the transplant than random conduits. Sensory CGRP+ (calcitonin gene-related peptide) axons were present at the caudal interface. Blood vessels formed throughout the transplant in both conduits. This study demonstrates that PVDF-TrFE conduits harboring SCs are promising for spinal cord repair and deserve further investigation. Biotechnol. Bioeng. 2017;114: 444-456. © 2016 Wiley Periodicals, Inc.

Localization of the delta opioid receptor and corticotropin-releasing factor in the amygdalar complex: role in anxiety.

It is well established that central nervous system norepinephrine (NE) and corticotropin-releasing factor (CRF) systems are important mediators of behavioral responses to stressors. More recent studies have defined a role for delta opioid receptors (DOPR) in maintaining emotional valence including anxiety. The amygdala plays an important role in processing emotional stimuli, and has been implicated in the development of anxiety disorders. Activation of DOPR or inhibition of CRF in the amygdala reduces baseline and stress-induced anxiety-like responses. It is not known whether CRF- and DOPR-containing amygdalar neurons interact or whether they are regulated by NE afferents. Therefore, this study sought to better define interactions between the CRF, DOPR and NE systems in the basolateral (BLA) and central nucleus of the amygdala (CeA) of the male rat using anatomical and functional approaches. Irrespective of the amygdalar subregion, dual immunofluorescence microscopy showed that DOPR was present in CRF-containing neurons. Immunoelectron microscopy confirmed that DOPR was localized to both dendritic processes and axon terminals in the BLA and CeA. Semi-quantitative dual immunoelectron microscopy analysis of gold-silver labeling for DOPR and immunoperoxidase labeling for CRF revealed that 55 % of the CRF neurons analyzed contained DOPR in the BLA while 67 % of the CRF neurons analyzed contained DOPR in the CeA. Furthermore, approximately 41 % of DOPR-labeled axon terminals targeted BLA neurons that expressed CRF while 29 % of DOPR-labeled axon terminals targeted CeA neurons that expressed CRF. Triple label immunofluorescence microscopy revealed that DOPR and CRF were co-localized in common cellular profiles that were in close proximity to NE-containing fibers in both subregions. These anatomical results indicate significant interactions between DOPR and CRF in this critical limbic region and reveal that NE is poised to regulate these peptidergic systems in the amygdala. Functional studies were performed to determine if activation of DOPR could inhibit the anxiety produced by elevation of NE in the amygdala using the pharmacological stressor yohimbine. Administration of the DOPR agonist, SNC80, significantly attenuated elevated anxiogenic behaviors produced by yohimbine as measured in the rat on the elevated zero maze. Taken together, results from this study demonstrate the convergence of three important systems, NE, CRF, and DOPR, in the amygdala and provide insight into their functional role in modulating stress and anxiety responses.

Cerebellar sub-divisions differ in exercise-induced plasticity of noradrenergic axons and in their association with resilience to activity-based anorexia.

The vermis or "spinocerebellum" receives input from the spinal cord and motor cortex for controlling balance and locomotion, while the longitudinal hemisphere region or "cerebro-cerebellum" is interconnected with non-motor cortical regions, including the prefrontal cortex that underlies decision-making. Noradrenaline release in the cerebellum is known to be important for motor plasticity but less is known about plasticity of the cerebellar noradrenergic (NA) system, itself. We characterized plasticity of dopamine ß-hydroxylase-immunoreactive NA fibers in the cerebellum of adolescent female rats that are evoked by voluntary wheel running, food restriction (FR) or by both, in combination. When 8 days of wheel access was combined with FR during the last 4 days, some responded with excessive exercise, choosing to run even during the hours of food access: this exacerbated weight loss beyond that due to FR alone. In the vermis, exercise, with or without FR, shortened the inter-varicosity intervals and increased varicosity density along NA fibers, while excessive exercise, due to FR, also shortened NA fibers. In contrast, the hemisphere required the FR-evoked excessive exercise to evoke shortened inter-varicosity intervals along NA fibers and this change was exhibited more strongly by rats that suppressed the FR-evoked excessive exercise, a behavior that minimized weight loss. Presuming that shortened inter-varicosity intervals translate to enhanced NA release and synthesis of norepinephrine, this enhancement in the cerebellar hemisphere may contribute towards protection of individuals from the life-threatening activity-based anorexia via relays with higher-order cortical areas that mediate the animal's decision to suppress the innate FR-evoked hyperactivity.

Cholinergic and glutamatergic transmission at synapses between pedunculopotine tegmental nucleus axonal terminals and A7 catecholamine cell group noradrenergic neurons in the rat.

We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.

Selectivity of Neuromodulatory Projections from the Basal Forebrain and Locus Ceruleus to Primary Sensory Cortices.

UNLABELLED: Acetylcholine and noradrenaline are major neuromodulators that affect sensory processing in the cortex. Modality-specific sensory information is processed in defined areas of the cortex, but it is unclear whether cholinergic neurons in the basal forebrain (BF) and noradrenergic neurons in the locus ceruleus (LC) project to and modulate these areas in a sensory modality-selective manner. Here, we mapped BF and LC projections to different sensory cortices of the mouse using dual retrograde tracing. We found that while the innervation of cholinergic neurons into sensory cortices is predominantly modality specific, the projections of noradrenergic neurons diverge onto multiple sensory cortices. Consistent with this anatomy, optogenetic activation of cholinergic neurons in BF subnuclei induces modality-selective desynchronization in specific sensory cortices, whereas activation of noradrenergic LC neurons induces broad desynchronization throughout multiple sensory cortices. Thus, we demonstrate a clear distinction in the organization and function of cholinergic BF and noradrenergic LC projections into primary sensory cortices: cholinergic BF neurons are highly selective in their projections and modulation of specific sensory cortices, whereas noradrenergic LC neurons broadly innervate and modulate multiple sensory cortices. SIGNIFICANCE STATEMENT: Neuromodulatory inputs from the basal forebrain (BF) and locus ceruleus (LC) are widespread in the mammalian cerebral cortex and are known to play important roles in attention and arousal, but little is known about the selectivity of their cortical projections. Using a dual retrobead tracing technique along with optogenetic stimulation, we have identified anatomic and functional differences in the way cholinergic BF neurons and noradrenergic LC neurons project into primary sensory cortices. While BF projections are highly selective to individual sensory cortices, LC projections diverge into multiple sensory cortices. To our knowledge, this is the first definitive proof that BF and LC projections to primary sensory cortices show both anatomic and functional differences in selectivity for modulating cortical activity.

Afferent and efferent connections of C1 cells with spinal cord or hypothalamic projections in mice.

The axonal projections and synaptic input of the C1 adrenergic neurons of the rostral ventrolateral medulla (VLM) were examined using transgenic dopamine-beta hydroxylase Cre mice and modified rabies virus. Cre-dependent viral vectors expressing TVA (receptor for envelopeA) and rabies glycoprotein were injected into the left VLM. EnvelopeA-pseudotyped rabies-EGFP glycoprotein-deficient virus (rabies-EGFP) was injected 4-6 weeks later in either thoracic spinal cord (SC) or hypothalamus. TVA immunoreactivity was detected almost exclusively (95 %) in VLM C1 neurons. In mice with SC injections of rabies-EGFP, starter cells (expressing TVA + EGFP) were found at the rostral end of the VLM; in mice with hypothalamic injections starter C1 cells were located more caudally. C1 neurons innervating SC or hypothalamus had other terminal fields in common (e.g., dorsal vagal complex, locus coeruleus, raphe pallidus and periaqueductal gray matter). Putative inputs to C1 cells with SC or hypothalamic projections originated from the same brain regions, especially the lower brainstem reticular core from spinomedullary border to rostral pons. Putative input neurons to C1 cells were also observed in the nucleus of the solitary tract, caudal VLM, caudal spinal trigeminal nucleus, cerebellum, periaqueductal gray matter and inferior and superior colliculi. In sum, regardless of whether they innervate SC or hypothalamus, VLM C1 neurons receive input from the same general brain regions. One interpretation is that many types of somatic or internal stimuli recruit these neurons en bloc to produce a stereotyped acute stress response with sympathetic, parasympathetic, vigilance and neuroendocrine components.

Uncovering diversity in the development of central noradrenergic neurons and their efferents.

Uncovering the mechanisms that underlie central noradrenergic neuron heterogeneity is essential to understanding selective subtype vulnerability to disease and environmental insult. Using recombinase-based intersectional genetic fate mapping we have previously demonstrated that molecularly distinct progenitor populations give rise to mature noradrenergic neurons differing in their anatomical location, axon morphology and efferent projection pattern. Here we review the findings from our previous study and extend our analysis of the noradrenergic subpopulation defined by transient developmental expression of Hoxb1. Using a combination of intersectional genetic fate mapping and analysis of a targeted loss of function mutation in Hoxb1, we have now uncovered additional heterogeneity based on the requirement of some noradrenergic neurons for Hoxb1 expression. By comparing the distribution of noradrenergic neurons derived from the Hoxb1 expression domain in wild-type and mutant mice, we demonstrate that Hoxb1 expression is required by a subset of neurons in the pons. Additional fate mapping, using a Hoxb1 enhancer element that drives Cre recombinase expression exclusively in rhombomere 4 of the hindbrain, reveals the existence of a subpopulation of noradrenergic neurons in the pons with more restricted axonal targets than the full Hoxb1-derived subpopulation. The unique projection profile of this newly defined subpopulation suggests that it may be functionally distinct. These analyses shed new light on the molecular determinants of noradrenergic identity in the pons and the overall complexity of the central noradrenergic system. This article is part of a Special Issue entitled SI: Noradrenergic System.