Sodium appetite and thirst do not require angiotensinogen production in astrocytes or hepatocytes.

In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.

Parabrachial-insular stimulation does not wake mice.

The parabrachial nucleus (PB) in the upper brainstem receives interoceptive information and sends a massive output projection directly to the cerebral cortex. Its glutamatergic axons primarily target the midinsular cortex, and we have proposed that this PB-insular projection promotes arousal. Here, we test whether stimulating this projection causes wakefulness. We combined optogenetics and video-electroencephalography (vEEG) in mice to test this hypothesis by stimulating PB axons in the insular cortex. Stimulating this projection did not alter the cortical EEG or awaken mice. Also, despite a tendency toward aversion, PB-insular stimulation did not significantly alter real-time place preference (RTPP). These results are not consistent with the hypothesis that the direct PB-insular projection is part of the ascending arousal system.NEW & NOTEWORTHY A brainstem region critical for wakefulness overlaps the medial parabrachial nucleus (PB) and has functional and direct axonal connectivity with the insular cortex. In this study, we hypothesized that this direct projection from the PB to the insular cortex promotes arousal. However, photostimulating PB axons in the insular cortex did not alter the cortical EEG or awaken mice. This information constrains the possible circuit connections through which brainstem neurons may sustain arousal.

Pre-locus coeruleus neurons in rat and mouse.

Previously, we identified a population of neurons in the hindbrain tegmentum, bordering the locus coeruleus (LC). We named this population the pre-locus coeruleus (pre-LC) because in rats its neurons lie immediately rostral to the LC. In mice, however, pre-LC and LC neurons intermingle, making them difficult to distinguish. Here, we use molecular markers and anterograde tracing to clarify the location and distribution of pre-LC neurons in mice, relative to rats. First, we colocalized the transcription factor FoxP2 with the activity marker Fos to identify pre-LC neurons in sodium-deprived rats and show their distribution relative to surrounding catecholaminergic and cholinergic neurons. Next, we used sodium depletion and chemogenetic activation of the aldosterone-sensitive HSD2 neurons in the nucleus of the solitary tract (NTS) to identify the homologous population of pre-LC neurons in mice, along with a related population in the central lateral parabrachial nucleus. Using Cre-reporter mice for Pdyn, we confirmed that most of these sodium-depletion-activated neurons are dynorphinergic. Finally, after confirming that these neurons receive excitatory input from the NTS and paraventricular hypothalamic nucleus, plus convergent input from the inhibitory AgRP neurons in the arcuate hypothalamic nucleus, we identify a major, direct input projection from the medial prefrontal cortex. This new information on the location, distribution, and input to pre-LC neurons provides a neuroanatomical foundation for cell-type-specific investigation of their properties and functions in mice. Pre-LC neurons likely integrate homeostatic information from the brainstem and hypothalamus with limbic, contextual information from the cerebral cortex to influence ingestive behavior.

Direct Parabrachial-Cortical Connectivity.

The parabrachial nucleus (PB) in the upper brain stem tegmentum includes several neuronal subpopulations with a wide variety of connections and functions. A subpopulation of PB neurons projects axons directly to the cerebral cortex, and limbic areas of the cerebral cortex send a return projection directly to the PB. We used retrograde and Cre-dependent anterograde tracing to identify genetic markers and characterize this PB-cortical interconnectivity in mice. Cortical projections originate from glutamatergic PB neurons that contain Lmx1b (81%), estrogen receptor alpha (26%), and Satb2 (20%), plus mRNA for the neuropeptides cholecystokinin (Cck, 48%) and calcitonin gene-related peptide (Calca, 13%), with minimal contribution from FoxP2+ PB neurons (2%). Axons from the PB produce an extensive terminal field in an unmyelinated region of the insular cortex, extending caudally into the entorhinal cortex, and arcing rostrally through the dorsolateral prefrontal cortex, with a secondary terminal field in the medial prefrontal cortex. In return, layer 5 neurons in the insular cortex and other prefrontal areas, along with a dense cluster of cells dorsal to the claustrum, send a descending projection to subregions of the PB that contain cortically projecting neurons. This information forms the neuroanatomical basis for testing PB-cortical interconnectivity in arousal and interoception.

Thalamic strokes that severely impair arousal extend into the brainstem.

In this study, we evaluate the role of the thalamus in the neural circuitry of arousal. Level of consciousness within the first 12 hours of a thalamic stroke is assessed with lesion symptom mapping. Impaired arousal correlates with lesions in the paramedian posterior thalamus near the centromedian and parafascicular nuclei, posterior hypothalamus, and midbrain tegmentum. All patients with severely impaired arousal (coma, stupor) had lesion extension into the midbrain and/or pontine tegmentum, whereas purely thalamic lesions did not severely impair arousal. These results are consistent with growing evidence that pathways most critical for human arousal lie outside the thalamus. Ann Neurol 2018;84:926-930.

Reciprocal Control of Drinking Behavior by Median Preoptic Neurons in Mice.

UNLABELLED: Stimulation of glutamatergic neurons in the subfornical organ drives drinking behavior, but the brain targets that mediate this response are not known. The densest target of subfornical axons is the anterior tip of the third ventricle, containing the median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT), a region that has also been implicated in fluid and electrolyte management. The neurochemical composition of this region is complex, containing both GABAergic and glutamatergic neurons, but the possible roles of these neurons in drinking responses have not been addressed. In mice, we show that optogenetic stimulation of glutamatergic neurons in MnPO/OVLT drives voracious water consumption, and that optogenetic stimulation of GABAergic neurons in the same region selectively reduces water consumption. Both populations of neurons have extensive projections to overlapping regions of the thalamus, hypothalamus, and hindbrain that are much more extensive than those from the subfornical organ, suggesting that the MnPO/OVLT serves as a key link in regulating drinking responses. SIGNIFICANCE STATEMENT: Neurons in the median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT) are known to regulate fluid/electrolyte homeostasis, but few studies have examined this issue with an appreciation for the neurochemical heterogeneity of these nuclei. Using Cre-Lox genetic targeting of Channelrhodospin-2 in transgenic mice, we demonstrate that glutamate and GABA neurons in the MnPO/OVLT reciprocally regulate water consumption. Stimulating glutamatergic MnPO/OVLT neurons induced water consumption, whereas stimulating GABAergic MnPO neurons caused a sustained and specific reduction in water consumption in dehydrated mice, the latter highlighting a heretofore unappreciated role of GABAergic MnPO neurons in thirst regulation. These observations represent an important advance in our understanding of the neural circuits involved in the regulation of fluid/electrolyte homeostasis.

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.

Alpha-Synuclein Pre-Formed Fibrils Injected into Prefrontal Cortex Primarily Spread to Cortical and Subcortical Structures.

BACKGROUND: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are characterized by diffuse spread of alpha-synuclein (α-syn) throughout the brain. Patients with PDD and DLB have a neuropsychological pattern of deficits that include executive dysfunction, such as abnormalities in planning, timing, working memory, and behavioral flexibility. The prefrontal cortex (PFC) plays a major role in normal executive function and often develops α-syn aggregates in DLB and PDD. OBJECTIVE: To investigate the long-term behavioral and cognitive consequences of α-syn pathology in the cortex and characterize pathological spread of α-syn. METHODS: We injected human α-syn pre-formed fibrils into the PFC of wild-type male mice. We then assessed the behavioral and cognitive effects between 12- and 21-months post-injection and characterized the spread of pathological α-syn in cortical, subcortical, and brainstem regions. RESULTS: We report that PFC PFFs: 1) induced α-syn aggregation in multiple cortical and subcortical regions with sparse aggregation in midbrain and brainstem nuclei; 2) did not affect interval timing or spatial learning acquisition but did mildly alter behavioral flexibility as measured by intraday reversal learning; and 3) increased open field exploration. CONCLUSIONS: This model of cortical-dominant pathology aids in our understanding of how local α-syn aggregation might impact some symptoms in PDD and DLB.

Efferent projections of Nps-expressing neurons in the parabrachial region.

In the brain, connectivity determines function. Neurons in the parabrachial nucleus (PB) relay diverse information to widespread brain regions, but the connections and functions of PB neurons that express Nps (neuropeptide S) remain mysterious. Here, we use Cre-dependent anterograde tracing and whole-brain analysis to map their output connections. While many other PB neurons project ascending axons through the central tegmental tract, NPS axons reach the forebrain via distinct periventricular and ventral pathways. Along the periventricular pathway, NPS axons target the tectal longitudinal column and periaqueductal gray then continue rostrally to target the paraventricular nucleus of the thalamus. Along the ventral pathway, NPS axons blanket much of the hypothalamus but avoid the ventromedial and mammillary nuclei. They also project prominently to the ventral bed nucleus of the stria terminalis, A13 cell group, and magnocellular subparafasciular nucleus. In the hindbrain, NPS axons have fewer descending projections, targeting primarily the superior salivatory nucleus, nucleus of the lateral lemniscus, and periolivary region. Combined with what is known about NPS and its receptor, the output pattern of Nps-expressing neurons in the PB region predicts a role in threat response and circadian behavior.

Sustained, Effortless Weight Loss after Damage to the Left Frontoinsular Cortex: A Case Report.

This case report highlights a possible consequence of damage to the left frontoinsular region. A 53-year-old woman with chronic obesity and headaches presented with seizure, leading to the discovery and resection of a large sphenoid wing meningioma. Postoperative brain imaging revealed loss of the left frontoinsular cortex and portions of the underlying white matter, claustrum, and striatum. Throughout her adult life, this patient had tried and failed to lose weight, but after surgery, she no longer desired to eat large meals, and without effort, her body mass index decreased from 38.6 (85th percentile) to 24.9 (25th percentile). Combined with previous research implicating the insular cortex in interoception, appetite, and drug-related urges, her reduced hunger and effortless weight loss after resection of the left frontoinsular cortex suggest that this region of the human brain may play a role in hunger-related urges that contribute to overeating.

Alpha-synuclein pre-formed fibrils injected into prefrontal cortex primarily spread to cortical and subcortical structures and lead to isolated behavioral symptoms.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are characterized by diffuse spread of alpha-synuclein (α-syn) throughout the brain. Patients with PDD and DLB have a neuropsychological pattern of deficits that include executive dysfunction, such as abnormalities in planning, timing, working memory, and behavioral flexibility. The prefrontal cortex (PFC) plays a major role in normal executive function and often develops α-syn aggregates in DLB and PDD. To investigate the consequences of α-syn pathology in the cortex, we injected human α-syn pre-formed fibrils into the PFC of wildtype mice. We report that PFC PFFs: 1) induced α-syn aggregation in multiple cortical and subcortical regions with sparse aggregation in midbrain and brainstem nuclei; 2) did not affect interval timing or spatial learning acquisition but did mildly alter behavioral flexibility as measured by intraday reversal learning; 3) increased open field exploration; and 4) did not affect susceptibility to an inflammatory challenge. This model of cortical-dominant pathology aids in our understanding of how local α-syn aggregation might impact some symptoms in PDD and DLB.

Right Tegmental Hemorrhage with Urinary Retention: A Case Report.

The upper brainstem tegmentum is dense and complex, making it difficult to localize functions to specific subregions. In particular, the precise location and possible laterality of subregions supporting basic functions like consciousness and urinary continence remain unclear. Here, we describe a patient who presented with a right pontine tegmental syndrome caused by intraparenchymal hemorrhage. Despite hemorrhage extension into the fourth ventricle and expansion of both hemorrhage and edema into a large region of the caudal midbrain and right-sided pontine tegmentum, this patient did not lose consciousness. Instead, he developed new and total urinary retention, with residual bladder volumes of more than 1,000 mL. We conclude that injury to the right pontine tegmentum is sufficient to disrupt the micturition reflex pathway.

A Century Searching for the Neurons Necessary for Wakefulness.

Wakefulness is necessary for consciousness, and impaired wakefulness is a symptom of many diseases. The neural circuits that maintain wakefulness remain incompletely understood, as do the mechanisms of impaired consciousness in many patients. In contrast to the influential concept of a diffuse "reticular activating system," the past century of neuroscience research has identified a focal region of the upper brainstem that, when damaged, causes coma. This region contains diverse neuronal populations with different axonal projections, neurotransmitters, and genetic identities. Activating some of these populations promotes wakefulness, but it remains unclear which specific neurons are necessary for sustaining consciousness. In parallel, pharmacological evidence has indicated a role for special neurotransmitters, including hypocretin/orexin, histamine, norepinephrine, serotonin, dopamine, adenosine and acetylcholine. However, genetically targeted experiments have indicated that none of these neurotransmitters or the neurons producing them are individually necessary for maintaining wakefulness. In this review, we emphasize the need to determine the specific subset of brainstem neurons necessary for maintaining arousal. Accomplishing this will enable more precise mapping of wakefulness circuitry, which will be useful in developing therapies for patients with coma and other disorders of arousal.

BoutonNet: an automatic method to detect anterogradely labeled presynaptic boutons in brain tissue sections.

Neurons emit axons, which form synapses, the fundamental unit of the nervous system. Neuroscientists use genetic anterograde tracing methods to label the synaptic output of specific neuronal subpopulations, but the resulting data sets are too large for manual analysis, and current automated methods have significant limitations in cost and quality. In this paper, we describe a pipeline optimized to identify anterogradely labeled presynaptic boutons in brain tissue sections. Our histologic pipeline labels boutons with high sensitivity and low background. To automatically detect labeled boutons in slide-scanned tissue sections, we developed BoutonNet. This detector uses a two-step approach: an intensity-based method proposes possible boutons, which are checked by a neural network-based confirmation step. BoutonNet was compared to expert annotation on a separate validation data set and achieved a result within human inter-rater variance. This open-source technique will allow quantitative analysis of the fundamental unit of the brain on a whole-brain scale.

Neuropeptide S (NPS) neurons: Parabrachial identity and novel distributions.

Neuropeptide S (NPS) increases wakefulness. A small number of neurons in the brainstem express Nps. These neurons are located in or near the parabrachial nucleus (PB), but we know very little about their ontogeny, connectivity, and function. To identify Nps-expressing neurons within the molecular framework of the PB region, we used in situ hybridization, immunofluorescence, and Cre-reporter labeling in mice. The primary concentration of Nps-expressing neurons borders the lateral lemniscus at far-rostral levels of the lateral PB. Caudal to this main cluster, Nps-expressing neurons scatter through the PB and form a secondary concentration medial to the locus coeruleus (LC). Most Nps-expressing neurons in the PB region are Atoh1-derived, Foxp2-expressing, and mutually exclusive with neurons expressing Calca or Lmx1b. Among Foxp2-expressing PB neurons, those expressing Nps are distinct from intermingled subsets expressing Cck or Pdyn. Examining Nps Cre-reporter expression throughout the brain identified novel populations of neurons in the nucleus incertus, anterior hypothalamus, and lateral habenula. This information will help focus experimental questions about the connectivity and function of NPS neurons.

Molecular ontology of the parabrachial nucleus.

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.

Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations.

OBJECTIVE: The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. METHODS: Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. RESULTS: We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. CONCLUSIONS: Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.

Despite increasing aldosterone, elevated potassium is not necessary for activating aldosterone-sensitive HSD2 neurons or sodium appetite.

Restricting dietary sodium promotes sodium appetite in rats. Prolonged sodium restriction increases plasma potassium (pK), and elevated pK is largely responsible for a concurrent increase in aldosterone, which helps promote sodium appetite. In addition to increasing aldosterone, we hypothesized that elevated potassium directly influences the brain to promote sodium appetite. To test this, we restricted dietary potassium in sodium-deprived rats. Potassium restriction reduced pK and blunted the increase in aldosterone caused by sodium deprivation, but did not prevent sodium appetite or the activation of aldosterone-sensitive HSD2 neurons. Conversely, supplementing potassium in sodium-deprived rats increased pK and aldosterone, but did not increase sodium appetite or the activation of HSD2 neurons relative to potassium restriction. Supplementing potassium without sodium deprivation did not significantly increase aldosterone and HSD2 neuronal activation and only modestly increased saline intake. Overall, restricting dietary sodium activated the HSD2 neurons and promoted sodium appetite across a wide range of pK and aldosterone, and saline consumption inactivated the HSD2 neurons despite persistent hyperaldosteronism. In conclusion, elevated potassium is important for increasing aldosterone, but it is neither necessary nor sufficient for activating HSD2 neurons and increasing sodium appetite.