Feeding signals inhibit fluid-satiation signals in the mouse lateral parabrachial nucleus to increase intake of highly palatable, caloric solutions.

Chemogenetic activation of oxytocin receptor-expressing neurons in the parabrachial nucleus (OxtrPBN neurons) acts as a satiation signal for water. In this research, we investigated the effect of activating OxtrPBN neurons on satiation for different types of fluids. Chemogenetic activation of OxtrPBN neurons in male and female transgenic OxtrCre mice robustly suppressed the rapid, initial (15-min) intake of several solutions after dehydration: water, sucrose, ethanol and saccharin, but only slightly decreased intake of Ensure®, a highly caloric solution (1 kcal/mL; containing 3.72 g protein, 3.27 g fat, 13.42 g carbohydrates, and 1.01 g dietary fibre per 100 mL). OxtrPBN neuron activation also suppressed cumulative, longer-term (2-h) intake of lower caloric, less palatable solutions, but not highly caloric, palatable solutions. These results suggest that OxtrPBN neurons predominantly control initial fluid-satiation responses after rehydration, but not longer-term intake of highly caloric, palatable solutions. The suppression of fluid intake was not because of anxiogenesis, but because OxtrPBN neuron activation decreased anxiety-like behaviour. To investigate the role of different PBN subdivisions on the intake of different solutions, we examined FOS as a proxy marker of PBN neuron activation. Different PBN subdivisions were activated by different solutions: the dorsolateral PBN similarly by all fluids; the external lateral PBN by caloric but not non-caloric solutions; and the central lateral PBN primarily by highly palatable solutions, suggesting PBN subdivisions regulate different aspects of fluid intake. To explore the possible mechanisms underlying the minimal suppression of Ensure® after OxtrPBN neuron activation, we demonstrated in in vitro slice recordings that the feeding-associated agouti-related peptide (AgRP) inhibited OxtrPBN neuron firing in a concentration-related manner, suggesting possible inhibition by feeding-related neurocircuitry of fluid satiation neurocircuitry. Overall, this research suggests that although palatable beverages like sucrose- and ethanol-containing beverages activate fluid satiation signals encoded by OxtrPBN neurons, these neurons can be inhibited by hunger-related signals (agouti-related peptide, AgRP), which may explain why these fluids are often consumed in excess of what is required for fluid satiation.

Sites and mechanisms of action of colokinetics at dopamine, ghrelin and serotonin receptors in the rodent lumbosacral defecation centre.

Agonists of dopamine D2 receptors (D2R), 5-hydroxytryptamine (5-HT, serotonin) receptors (5-HTR) and ghrelin receptors (GHSR) activate neurons in the lumbosacral defecation centre, and act as 'colokinetics', leading to increased propulsive colonic motility, in vivo. In the present study, we investigated which neurons in the lumbosacral defecation centre express the receptors and whether dopamine, serotonin and ghrelin receptor agonists act on the same lumbosacral preganglionic neurons (PGNs). We used whole cell electrophysiology to record responses from neurons in the lumbosacral defecation centre, following colokinetic application, and investigated their expression profiles and the chemistries of their neural inputs. Fluorescence in situ hybridisation revealed Drd2, Ghsr and Htr2C transcripts were colocalised in lumbosacral PGNs of mice, and immunohistochemistry showed that these neurons have closely associated tyrosine hydroxylase and 5-HT boutons. Previous studies showed that they do not receive ghrelin inputs. Whole cell electrophysiology in adult mice spinal cord revealed that dopamine, serotonin, α-methylserotonin and capromorelin each caused inward, excitatory currents in overlapping populations of lumbosacral PGNs. Furthermore, dopamine caused increased frequency of both IPSCs and EPSCs in a cohort of D2R neurons. Tetrodotoxin blocked the IPSCs and EPSCs, revealing a post-synaptic excitatory action of dopamine. In lumbosacral PGNs of postnatal day 7-14 rats, only dopamine's postsynaptic effects were observed. Furthermore, inward, excitatory currents evoked by dopamine were reduced by the GHSR antagonist, YIL781. We conclude that lumbosacral PGNs are the site where the action of endogenous ligands of D2R and 5-HT2R converge, and that GHSR act as a cis-modulator of D2R expressed by the same neurons. KEY POINTS: Dopamine, 5-hydroxytryptamine (5-HT, serotonin) and ghrelin (GHSR) receptor agonists increase colorectal motility and have been postulated to act at receptors on parasympathetic preganglionic neurons (PGNs) in the lumbosacral spinal cord. We aimed to determine which neurons in the lumbosacral spinal cord express dopamine, serotonin and GHSR receptors, their neural inputs, and whether agonists at these receptors excite them. We show that dopamine, serotonin and ghrelin receptor transcripts are contained in the same PGNs and that these neurons have closely associated tyrosine hydroxylase and serotonin boutons. Whole cell electrophysiology revealed that dopamine, serotonin and GHSR receptor agonists induce an inward excitatory current in overlapping populations of lumbosacral PGNs. Dopamine-induced excitation was reversed by GHSR antagonism. The present study demonstrates that lumbosacral PGNs are the site at which actions of endogenous ligands of dopamine D2 receptors and 5-HT type 2 receptors converge. Ghrelin receptors are functional, but their role appears to be as modulators of dopamine effects at D2 receptors.

Selective transduction and photoinhibition of pre-Bötzinger complex neurons that project to the facial nucleus in rats affects nasofacial activity.

The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases.

While breathing seems to come easy, it is a complex process in which many muscles coordinate to allow air to flow into the lungs. These muscles also control the flow of air we breathe out to allow us to talk, sing, eat, or drink. The brain circuits that control these muscles, can also influence other parts of the brain. The preBötzinger Complex, which is a key region of brainstem circuits that generate and control breathing, contains neurons that also project widely, connecting to other regions of the brain. This helps to modulate the sense of smell, emotional state, heart rate, and even blood pressure. Understanding how the preBötzinger Complex is organized can untangle how breathing can influence these other processes. Melo et al. wanted to learn whether they could manipulate the activity of a subgroup of preBötzinger Complex neurons that project into the facial nucleus ­ a region of the brain that controls the muscles of the face when we breathe ­ without affecting breathing. If this can be done, it might also be possible to affect blood pressure by manipulating selective preBötzinger neurons, and thus the development of hypertension, without having any impact on breathing. To test this hypothesis, Melo et al. used rats in which the activation of preBötzinger Complex neurons that project into the facial nucleus was blocked. This decreased the activity of the muscles around the nose with hardly any effect on breathing. Melo et al. also found that the state of consciousness of the rat (anesthetized or conscious) could affect how preBötzinger Complex neurons control these muscles. Melo et al. also observed that preBötzinger Complex neurons projecting into the facial nucleus had projections into many other regions in the brainstem. This might help to the coordinate respiratory, cardiovascular, orofacial, and potentially other physiological functions. The findings of Melo et al. set a technical foundation for exploring the influence of specific subgroups of preBötzinger Complex neurons on respiratory modulation of other physiological activities, including blood pressure and heart rate and in conditions, such as hypertension and heart failure. More broadly, most brain regions contain complex and heterogeneous groups of neurons and the strategy validated by Melo et. al. could be applied to unravel other brain-function relationships.

Analysis of the distribution of vagal afferent projections from different peripheral organs to the nucleus of the solitary tract in rats.

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.

FGF-MAPK signaling regulates human deep-layer corticogenesis.

Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

Use of a physiological reflex to standardize vagal nerve stimulation intensity improves data reproducibility in a memory extinction assay.

BACKGROUND: Modulating brainstem activity, via electrical vagus nerve stimulation (VNS), influences cognitive functions, including memory. However, controlling for changes in stimulus efficacy during chronic studies, and response variability between subjects, is problematic. OBJECTIVE/HYPOTHESIS: We hypothesized that recruitment of an autonomic reflex, the Hering-Breuer reflex, would provide robust confirmation of VNS efficacy. We compared this to measurement of electrode resistance over time. We also examined whether VNS modulates contextual memory extinction. METHODS: Electrodes for VNS and diaphragm electromyography recording were implanted into anesthetized Sprague Dawley rats. When conscious, we measured the electrode resistance as well as the minimum VNS current required to evoke the Hering-Breuer reflex, before, and after, an inhibitory avoidance assay - a two chamber, dark/light model, where the dark compartment was paired with an aversive foot shock. The extinction of this contextual memory was assessed in sham and VNS treated rats, with VNS administered for 30 s at 1.5 times the Hering-Breuer reflex threshold during extinction memory formation. RESULTS: Assessment of VNS-evoked Hering-Breuer reflex successfully identified defective electrodes. VNS accelerated extinction memory and decreased multiple physiological metrics of fear expression. We observed an inverse relationship between memory extinction and respiratory rate during the behavioural assay. Additionally, no current - response relationship between VNS and extinction memory formation was established. CONCLUSION: These data demonstrate that reliable, experimental VNS studies can be produced by verifying reflex initiation as a consequence of stimulation. Further, studies could be standardised by indexing stimulator efficacy to initiation of autonomic reflexes.

Selective optogenetic stimulation of efferent fibers in the vagus nerve of a large mammal.

BACKGROUND: Electrical stimulation applied to individual organs, peripheral nerves, or specific brain regions has been used to treat a range of medical conditions. In cardiovascular disease, autonomic dysfunction contributes to the disease progression and electrical stimulation of the vagus nerve has been pursued as a treatment for the purpose of restoring the autonomic balance. However, this approach lacks selectivity in activating function- and organ-specific vagal fibers and, despite promising results of many preclinical studies, has so far failed to translate into a clinical treatment of cardiovascular disease. OBJECTIVE: Here we report a successful application of optogenetics for selective stimulation of vagal efferent activity in a large animal model (sheep). METHODS AND RESULTS: Twelve weeks after viral transduction of a subset of vagal motoneurons, strong axonal membrane expression of the excitatory light-sensitive ion channel ChIEF was achieved in the efferent projections innervating thoracic organs and reaching beyond the level of the diaphragm. Blue laser or LED light (>10 mW mm-2; 1 ms pulses) applied to the cervical vagus triggered precisely timed, strong bursts of efferent activity with evoked action potentials propagating at speeds of ∼6 m s-1. CONCLUSIONS: These findings demonstrate that in species with a large, multi-fascicled vagus nerve, it is possible to stimulate a specific sub-population of efferent fibers using light at a site remote from the vector delivery, marking an important step towards eventual clinical use of optogenetic technology for autonomic neuromodulation.

A Chemogenetic Tool that Enables Functional Neural Circuit Analysis.

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight.

PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate.

Heart rate and blood pressure oscillate in phase with respiratory activity. A component of these oscillations is generated centrally, with respiratory neurons entraining the activity of pre-sympathetic and parasympathetic cardiovascular neurons. Using a combination of optogenetic inhibition and excitation in vivo and in situ in rats, as well as neuronal tracing, we demonstrate that preBötzinger Complex (preBötC) neurons, which form the kernel for inspiratory rhythm generation, directly modulate cardiovascular activity. Specifically, inhibitory preBötC neurons modulate cardiac parasympathetic neuron activity whilst excitatory preBötC neurons modulate sympathetic vasomotor neuron activity, generating heart rate and blood pressure oscillations in phase with respiration. Our data reveal yet more functions entrained to the activity of the preBötC, with a role in generating cardiorespiratory oscillations. The findings have implications for cardiovascular pathologies, such as hypertension and heart failure, where respiratory entrainment of heart rate is diminished and respiratory entrainment of blood pressure exaggerated.

Extensive Inhibitory Gating of Viscerosensory Signals by a Sparse Network of Somatostatin Neurons.

Integration and modulation of primary afferent sensory information begins at the first terminating sites within the CNS, where central inhibitory circuits play an integral role. Viscerosensory information is conveyed to the nucleus of the solitary tract (NTS) where it initiates neuroendocrine, behavioral, and autonomic reflex responses that ensure optimal internal organ function. This excitatory input is modulated by diverse, local inhibitory interneurons, whose functions are not clearly understood. Here we show that, in male rats, 65% of somatostatin-expressing (SST) NTS neurons also express GAD67, supporting their likely role as inhibitory interneurons. Using whole-cell recordings of NTS neurons, from horizontal brainstem slices of male and female SST-yellow fluorescent protein (YFP) and SST-channelrhodopsin 2 (ChR2)-YFP mice, we quantified the impact of SST-NTS neurons on viscerosensory processing. Light-evoked excitatory photocurrents were reliably obtained from SST-ChR2-YFP neurons (n = 16) and the stimulation-response characteristics determined. Most SST neurons (57%) received direct input from solitary tract (ST) afferents, indicating that they form part of a feedforward circuit. All recorded SST-negative NTS neurons (n = 72) received SST-ChR2 input. ChR2-evoked PSCs were largely inhibitory and, in contrast to previous reports, were mediated by both GABA and glycine. When timed to coincide, the ChR2-activated SST input suppressed ST-evoked action potentials at second-order NTS neurons, demonstrating strong modulation of primary viscerosensory input. These data indicate that the SST inhibitory network innervates broadly within the NTS, with the potential to gate viscerosensory input to powerfully alter autonomic reflex function and other behaviors.SIGNIFICANCE STATEMENT Sensory afferent input is modulated according to state. For example the baroreflex is altered during a stress response or exercise, but the basic mechanisms underpinning this sensory modulation are not fully understood in any sensory system. Here we demonstrate that the neuronal processing of viscerosensory information begins with synaptic gating at the first central synapse with second-order neurons in the NTS. These data reveal that the somatostatin subclass of inhibitory interneurons are driven by visceral sensory input to play a major role in gating viscerosensory signals, placing them within a feedforward circuit within the NTS.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

The intersection of stress and reward: BNST modulation of aversive and appetitive states.

The bed nucleus of the stria terminalis (BNST) is widely acknowledged as a brain structure that regulates stress and anxiety states, as well as aversive and appetitive behaviours. The diverse roles of the BNST are afforded by its highly modular organisation, neurochemical heterogeneity, and complex intrinsic and extrinsic circuitry. There has been growing interest in the BNST in relation to psychopathologies such as anxiety and addiction. Although research on the human BNST is still in its infancy, there have been extensive preclinical studies examining the molecular signature and hodology of the BNST and their involvement in stress and reward seeking behaviour. This review examines the neurochemical phenotype and connectivity of the BNST, as well as electrophysiological correlates of plasticity in the BNST mediated by stress and/or drugs of abuse.

Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner.

Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract.

Expression of the large extracellular glycan, polysialic acid (polySia), is restricted in the adult, to brain regions exhibiting high levels of plasticity or remodeling, including the hippocampus, prefrontal cortex, and the nucleus of the solitary tract (NTS). The NTS, located in the dorsal brainstem, receives constant viscerosensory afferent traffic as well as input from central regions controlling sympathetic nerve activity, respiration, gastrointestinal functions, hormonal release, and behavior. Our aims were to determine the ultrastructural location of polySia in the NTS and the functional effects of enzymatic removal of polySia, both in vitro and in vivo polySia immunoreactivity was found throughout the adult rat NTS. Electron microscopy demonstrated polySia at sites that influence neurotransmission: the extracellular space, fine astrocytic processes, and neuronal terminals. Removing polySia from the NTS had functional consequences. Whole-cell electrophysiological recordings revealed altered intrinsic membrane properties, enhancing voltage-gated K+ currents and increasing intracellular Ca2+ Viscerosensory afferent processing was also disrupted, dampening low-frequency excitatory input and potentiating high-frequency sustained currents at second-order neurons. Removal of polySia in the NTS of anesthetized rats increased sympathetic nerve activity, whereas functionally related enzymes that do not alter polySia expression had little effect. These data indicate that polySia is required for the normal transmission of information through the NTS and that changes in its expression alter sympathetic outflow. polySia is abundant in multiple but discrete brain regions, including sensory nuclei, in both the adult rat and human, where it may regulate neuronal function by mechanisms identified here.SIGNIFICANCE STATEMENT All cells are coated in glycans (sugars) existing predominantly as glycolipids, proteoglycans, or glycoproteins formed by the most complex form of posttranslational modification, glycosylation. How these glycans influence brain function is only now beginning to be elucidated. The adult nucleus of the solitary tract has abundant polysialic acid (polySia) and is a major site of integration, receiving viscerosensory information which controls critical homeostatic functions. Our data reveal that polySia is a determinant of neuronal behavior and excitatory transmission in the nucleus of the solitary tract, regulating sympathetic nerve activity. polySia is abundantly expressed at distinct brain sites in adult, including major sensory nuclei, suggesting that sensory transmission may also be influenced via mechanisms described here. These findings hint at the importance of elucidating how other glycans influence neural function.

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents.

Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937-948.

Nucleus incertus promotes cortical desynchronization and behavioral arousal.

Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach-clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs-to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.

Dedicated C-fibre viscerosensory pathways to central nucleus of the amygdala.

KEY POINTS: Emotions are accompanied by concordant changes in visceral function, including cardiac output, respiration and digestion. One major forebrain integrator of emotional responses, the amygdala, is considered to rely on embedded visceral afferent information, although few details are known. In the present study, we retrogradely transported dye from the central nucleus of the amygdala (CeA) to identify CeA-projecting nucleus of the solitary tract (NTS) neurons for synaptic characterization and compared them with unlabelled, near-neighboor NTS neurons. Solitary tract (ST) afferents converged onto NTS-CeA second-order sensory neurons in greater numbers, as well as indirectly via polysynaptic pathways. Unexpectedly, all mono- and polysynaptic ST afferent pathways to NTS-CeA neurons were organized exclusively as either transient receptor potential cation channel subfamily V member 1 (TRPV1)-sensitive or TRPV1-resistant, regardless of whether intervening neurons were excitatory or inhibitory. This strict sorting provides viscerosensory signals to CeA about visceral conditions with respect to being either 'normal' via A-fibres or 'alarm' via TRPV1 expressing C-fibres and, accordingly, this pathway organization probably encodes interoceptive status. ABSTRACT: Emotional state is impacted by changes in visceral function, including blood pressure, breathing and digestion. A main line of viscerosensory information processing occurs first in the nucleus of the solitary tract (NTS). In the present study conducted in rats, we examined the synaptic characteristics of visceral afferent pathways to the central nucleus of the amygdala (CeA) in brainstem slices by recording from retrogradely labelled NTS projection neurons. We simultaneously recorded neuron pairs: one dye positive (i.e. NTS-CeA) and a second unlabelled neighbour. Graded shocks to the solitary tract (ST) always (93%) triggered EPSCs at CeA projecting NTS neurons. Half of the NTS-CeA neurons received at least one primary afferent input (classed 'second order') indicating that viscerosensory information arrives at the CeA conveyed via a pathway involving as few as two synapses. The remaining NTS-CeA neurons received viscerosensory input only via polysynaptic pathways. By contrast, ∼3/4 of unlabelled neighbouring neurons were directly connected to ST. NTS-CeA neurons received greater numbers of ST-related inputs compared to unlabelled NTS neurons, indicating that highly convergent viscerosensory signals reach the CeA. Remarkably, despite multifibre convergence, all single NTS-CeA neurons received inputs derived from only unmyelinated afferents [transient receptor potential cation channel subfamily V member 1 (TRPV1) expressing C-fibres] or only non-TRPV1 ST afferent inputs, and never a combination of both. Such segregation means that visceral afferent information followed separate lines to reach the CeA. Their very different physiological activation profiles mean that these parallel visceral afferent pathways encode viscerosensory signals to the amygdala that may provide interoceptive assessments to impact on behaviours.

Independent transmission of convergent visceral primary afferents in the solitary tract nucleus.

Cranial primary afferents from the viscera enter the brain at the solitary tract nucleus (NTS), where their information is integrated for homeostatic reflexes. The organization of sensory inputs is poorly understood, despite its critical impact on overall reflex performance characteristics. Single afferents from the solitary tract (ST) branch within NTS and make multiple contacts onto individual neurons. Many neurons receive more than one ST input. To assess the potential interaction between converging afferents and proximal branching near to second-order neurons, we probed near the recorded soma in horizontal slices from rats with focal electrodes and minimal shocks. Remote ST shocks evoked monosynaptic excitatory postsynaptic currents (EPSCs), and nearby focal shocks also activated monosynaptic EPSCs. We tested the timing and order of stimulation to determine whether focal shocks influenced ST responses and vice versa in single neurons. Focal-evoked EPSC response profiles closely resembled ST-EPSC characteristics. Mean synaptic jitters, failure rates, depression, and phenotypic segregation by capsaicin responsiveness were indistinguishable between focal and ST-evoked EPSCs. ST-EPSCs failed to affect focal-EPSCs within neurons, indicating that release sites and synaptic terminals were functionally independent and isolated from cross talk or neurotransmitter overflow. In only one instance, focal shocks intercepted and depleted the ST axon generating evoked EPSCs. Despite large numbers of functional contacts, multiple afferents do not appear to interact, and ST axon branches may be limited to close to the soma. Thus single or multiple primary afferents and their presynaptic active release sites act independently when they contact single second-order NTS neurons.

Low-fidelity GABA transmission within a dense excitatory network of the solitary tract nucleus.

Visceral primary afferents enter the CNS at the caudal solitary tract nucleus (NTS), and activate central pathways key to autonomic and homeostatic regulation. Excitatory transmission from primary solitary tract (ST)-afferents consists of multiple contacts originating from single axons that offer a remarkably high probability of glutamate release and high safety factor for ST afferent excitation. ST afferent activation sometimes triggers polysynaptic GABAergic circuits, which feedback onto second-order NTS neurons. Although inhibitory transmission is observed at second-order neurons, much less is known about the organization and mechanisms regulating GABA transmission. Here, we used a focal pipette to deliver minimal stimulus shocks near second-order NTS neurons in rat brainstem slices and directly activated single GABAergic axons. Most minimal focal shocks activated low jitter EPSCs from single axons with characteristics resembling ST afferents. Much less commonly (9% of sites), minimal focal shocks activated monosynaptic IPSCs at fixed latency (low jitter) that often failed (30%) and had no frequency-dependent facilitation or depression. These GABA release characteristics contrasted markedly to the unfailing, large amplitudes for glutamate released during ST-EPCSs recorded from the same neurons. Surprisingly, unitary GABAergic IPSCs were only weakly calcium dependent. In some neurons, strong focal shocks evoked compound IPSCs indicating convergent summation of multiple inhibitory axons. Our studies demonstrate that second-order NTS neurons receive GABAergic transmission from a diffuse network of inhibitory axons that rely on an intrinsically less reliable and substantially weaker release apparatus than ST excitation. Effective inhibition depends on co-activation of convergent inputs to blunt excitatory drive.