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The canonical view of motor control is that distal musculature is controlled primarily by the contralateral cerebral hemisphere; unilateral brain lesions typically affect contralateral but not ipsilateral musculature. Contralateral-only limb deficits following a unilateral lesion suggest but do not prove that control is strictly contralateral: the loss of a contribution of the lesioned hemisphere to the control of the ipsilesional limb could be masked by the intact contralateral drive from the nonlesioned hemisphere. To distinguish between these possibilities, we serially inactivated the parietal reach region, comprising the posterior portion of medial intraparietal area, the anterior portion of V6a, and portions of the lateral occipital parietal area, in each hemisphere of 2 monkeys (23 experimental sessions, 46 injections total) to evaluate parietal reach region's contribution to the contralateral reaching deficits observed following lateralized brain lesions. Following unilateral inactivation, reach reaction times with the contralesional limb were slowed compared with matched blocks of control behavioral data; there was no effect of unilateral inactivation on the reaction time of either ipsilesional limb reaches or saccadic eye movements. Following bilateral inactivation, reaching was slowed in both limbs, with an effect size in each no different from that produced by unilateral inactivation. These findings indicate contralateral organization of reach preparation in posterior parietal cortex. SIGNIFICANCE STATEMENT Unilateral brain lesions typically affect contralateral but not ipsilateral musculature. Contralateral-only limb deficits following a unilateral lesion suggest but do not prove that control is strictly contralateral: the loss of a contribution of the lesioned hemisphere to the control of the ipsilesional limb could be masked by the intact contralateral drive from the nonlesioned hemisphere. Unilateral lesions cannot distinguish between contralateral and bilateral control, but bilateral lesions can. Here we show similar movement initiation deficits after combined unilateral and bilateral inactivation of the parietal reach region, indicating contralateral organization of reach preparation., (Copyright © 2022 the authors.)

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., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Newborns are at risk for vitamin K deficiency bleeding (VKDB) caused by inadequate prenatal storage and deficiency of vitamin K in breast milk. Systematic review of evidence to date suggests that a single intramuscular (IM) injection of vitamin K at birth effectively prevents VKDB. Current scientific data suggest that single or repeated doses of oral (PO) vitamin K are less effective than IM vitamin K in preventing VKDB. The Canadian Paediatric Society and the College of Family Physicians of Canada recommend routine IM administration of a single dose of vitamin K at 0.5 mg to 1.0 mg to all newborns. Administering PO vitamin K (2.0 mg at birth, repeated at 2 to 4 and 6 to 8 weeks of age) should be confined to newborns whose parents decline IM vitamin K. Health care providers should clarify with parents that newborns are at increased risk of VKDB if such a regimen is chosen. Current evidence is insufficient to recommend routine intravenous vitamin K administration to preterm infants undergoing intensive care. Keywords HDNB; Newborn; Prophylaxis; Vitamin K; VKDB ., (Copyright© the College of Family Physicians of Canada.)

Newborns are at risk for vitamin K deficiency bleeding (VKDB) caused by inadequate prenatal storage and deficiency of vitamin K in breast milk. Systematic review of evidence to date suggests that a single intramuscular (IM) injection of vitamin K at birth effectively prevents VKDB. Current scientific data suggest that single or repeated doses of oral (PO) vitamin K are less effective than IM vitamin K in preventing VKDB. The Canadian Paediatric Society and the College of Family Physicians of Canada recommend routine IM administration of a single dose vitamin K at 0.5 mg to 1.0 mg to all newborns. Administering PO vitamin K (2.0 mg at birth, repeated at 2 to 4 and 6 to 8 weeks of age), should be confined to newborns whose parents decline IM vitamin K. Health care providers should clarify with parents that newborns are at increased risk of VKDB if such a regimen is chosen. Current evidence is insufficient to recommend routine intravenous vitamin K administration to preterm infants undergoing intensive care.

In some patients, renal nerve denervation has been reported to be an effective treatment for essential hypertension. Considerable evidence suggests that afferent renal nerves (ARN) and sodium balance play important roles in the development and maintenance of high blood pressure. ARN are sensitive to sodium concentrations in the renal pelvis. To better understand the role of ARN, we infused isotonic or hypertonic NaCl (308 or 500mOsm) into the left renal pelvis of conscious rats for two 2hours while recording arterial pressure and heart rate. Subsequently, brain tissue was analyzed for immunohistochemical detection of the protein Fos, a marker for neuronal activation. Fos-immunoreactive neurons were identified in numerous sites in the forebrain and brainstem. These areas included the nucleus tractus solitarius (NTS), the lateral parabrachial nucleus, the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). The most effective stimulus was 500mOsm NaCl. Activation of these sites was attenuated or prevented by administration of benzamil (1μM) or amiloride (10μM) into the renal pelvis concomitantly with hypertonic saline. In anesthetized rats, infusion of hypertonic saline but not isotonic saline into the renal pelvis elevated ARN activity and this increase was attenuated by simultaneous infusion of benzamil or amiloride. We propose that renal pelvic epithelial sodium channels (ENaCs) play a role in activation of ARN and, via central visceral afferent circuits, this system modulates fluid volume and peripheral blood pressure. These pathways may contribute to the development of hypertension., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The 1970s saw the introduction of new technologies for tracing axons both anterogradely and retrogradely. These methods allowed us to visualize fine, unmyelinated pathways for the first time, such as the hypothalamic pathways that control the autonomic nervous system. As a result, we were able to identify the paraventricular nucleus and lateral hypothalamus as the key sites that provide direct inputs to the autonomic preganglionic neurons in the medulla and spinal cord. These findings revolutionized our understanding of hypothalamic control of the autonomic nervous system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

By the late 1970׳s, the pathways had been identified from neurons in the nucleus of the solitary tract that control visceral sensory inflow and from the paraventricular nucleus and lateral hypothalamus that directly innervate the autonomic preganglionic neurons, thereby controlling autonomic outflow. However, the connections between the two were not yet clear. This paper identified the parabrachial nucleus as a key intermediary, receiving the bulk of outflow from the nucleus of the solitary tract and distributing it to a set of brainstem and forebrain sites that constituted a central autonomic control network. This work also identified the insular cortex as a key visceral sensory cortical area. This article is part of a Special Issue entitled SI:50th Anniversary Issue., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Epithelial sodium channels (ENaCs) are strongly expressed in the circumventricular organs (CVOs), and these structures may play an important role in sensing plasma sodium levels. Here, the potent ENaC blocker amiloride was injected intraperitoneally in rats and 2h later, the c-Fos activation pattern in the CVOs was studied. Amiloride elicited dose-related activation in the area postrema (AP) but only ~10% of the rats showed c-Fos activity in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). Tyrosine hydroxylase-immunoreactive (catecholamine) AP neurons were activated, but tryptophan hydroxylase-immunoreactive (serotonin) neurons were unaffected. The AP projects to FoxP2-expressing neurons in the dorsolateral pons which include the pre-locus coeruleus nucleus and external lateral part of the parabrachial nucleus; both cell groups were c-Fos activated following systemic injections of amiloride. In contrast, another AP projection target--the aldosterone-sensitive neurons of the nucleus tractus solitarius which express the enzyme 11-β-hydroxysteriod dehydrogenase type 2 (HSD2) were not activated. As shown here, plasma concentrations of amiloride used in these experiments were near or below the IC50 level for ENaCs. Amiloride did not induce changes in blood pressure, heart rate, or regional vascular resistance, so sensory feedback from the cardiovascular system was probably not a causal factor for the c-Fos activity seen in the CVOs. In summary, amiloride may have a dual effect on sodium homeostasis causing a loss of sodium via the kidney and inhibiting sodium appetite by activating the central satiety pathway arising from the AP., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

The sensory circumventricular organs (CVOs) are specialized collections of neurons and glia that lie in the midline of the third and fourth ventricles of the brain, lack a blood-brain barrier, and function as chemosensors, sampling both the cerebrospinal fluid and plasma. These structures, which include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), are sensitive to changes in sodium concentration but the cellular mechanisms involved remain unknown. Epithelial sodium channel (ENaC)-expressing neurons of the CVOs may be involved in this process. Here we demonstrate with immunohistochemical and in situ hybridization methods that ENaC-expressing neurons are densely concentrated in the sensory CVOs. These neurons become c-Fos activated, a marker for neuronal activity, after various manipulations of peripheral levels of sodium including systemic injections with hypertonic saline, dietary sodium deprivation, and sodium repletion after prolonged sodium deprivation. The increases seen c-Fos activity in the CVOs were correlated with parallel increases in plasma sodium levels. Since ENaCs play a central role in sodium reabsorption in kidney and other epithelia, we present a hypothesis here suggesting that these channels may also serve a related function in the CVOs. ENaCs could be a significant factor in modulating CVO neuronal activity by controlling the magnitude of sodium permeability in neurons. Hence, some of the same circulating hormones controlling ENaC expression in kidney, such as angiotensin II and atrial natriuretic peptide, may coordinate ENaC expression in sensory CVO neurons and could potentially orchestrate sodium appetite, osmoregulation, and vasomotor sympathetic drive.

Using a double immunofluorescence procedure, we report the discovery of a novel group of fibrous astrocytes that co-express epithelial sodium channel (ENaC) γ-subunit protein along with glial acidic fibrillary protein (GFAP). These cells are concentrated along the borders of the sensory circumventricular organs (CVOs), embedded in the white matter (e.g., optic nerve/chiasm, anterior commissure, corpus callosum, pyramidal tract) and are components of the pia mater. In the CVOs, a compact collection of ENaC γ-immunoreactive glial fibers form the lamina terminalis immediately rostral to the organum vasculosum of the lamina terminalis (OVLT). Astrocyte processes can be traced into the median preoptic nucleus - a region implicated in regulation of sodium homeostasis. In the subfornical organ (SFO), ENaC γ-GFAP astrocytes lie in its lateral border, but not in the ventromedial core. In the area postrema (AP), a dense ENaC γ-GFAP glial fibers form the interface between the AP and nucleus tractus solitarius; this area is termed the subpostremal region. Antibodies against the ENaC α- or β-subunit proteins do not immunostain these regions. In contrast, the antibodies against the ENaC γ-subunit protein react weakly with neuronal cell bodies in the CVOs. Besides affecting glial-neural functions in the CVOs, the astrocytes found in the white matter may affect saltatory nerve conduction, serving as a sodium buffer. The ENaC γ-expressing astrocytes of the ventral medulla send processes into the raphe pallidus which intermingle with the serotoninergic (5-HT) neurons found in this region as well as with the other nearby 5-HT neurons distributed along ventral medullary surface., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The parabrachial nucleus (PB) is a brainstem cell group that receives a strong input from the nucleus tractus solitarius regarding the physiological status of the internal organs and sends efferent projections throughout the forebrain. Since the neuroanatomical organization of the PB remains unclear, our first step was to use specific antibodies against two neural lineage transcription factors: Forkhead box protein2 (FoxP2) and LIM homeodomain transcription factor 1 beta (Lmx1b) to define the PB in adult rats. This allowed us to construct a cytoarchitectonic PB map based on the distribution of neurons that constitutively express these two transcription factors. Second, the in situ hybridization method combined with immunohistochemistry demonstrated that mRNA for glutamate vesicular transporter Vglut2 (Slc17a6) was present in most of the Lmx1b+ and FoxP2+ parabrachial neurons, indicating these neurons use glutamate as a transmitter. Third, conscious rats were maintained in a hypotensive or hypertensive state for 2h, and then, their brainstems were prepared by the standard c-Fos method which is a measure of neuronal activity. Both hypotension and hypertension resulted in c-Fos activation of Lmx1b+ neurons in the external lateral-outer subdivision of the PB (PBel-outer). Hypotension, but not hypertension, caused c-Fos activity in the FoxP2+ neurons of the central lateral PB (PBcl) subnucleus. The Kölliker-Fuse nucleus as well as the lateral crescent PB and rostral-most part of the PBcl contain neurons that co-express FoxP2+ and Lmx1b+, but none of these were activated after blood pressure changes. Salt-sensitive FoxP2 neurons in the pre-locus coeruleus and PBel-inner were not c-Fos activated following blood pressure changes. In summary, the present study shows that the PBel-outer and PBcl subnuclei originate from two different neural progenitors, contain glutamatergic neurons, and are affected by blood pressure changes, with the PBel-outer reacting to both hypo- and hypertension, and the PBcl signaling only hypotensive changes., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The present study demonstrates that serotonin (5-hydroxytryptamine, 5-HT)-containing axons project to two sets of neurons in the dorsolateral pons that have been implicated in salt appetite regulation. These two neuronal groups are the pre-locus coeruleus (pre-LC) and a region in the parabrachial nucleus termed the external lateral-inner subdivision (PBel-inner). Neurons in both regions constitutively express the transcription factor Forkhead protein2 (FoxP2), and become c-Fos activated after prolonged sodium depletion. They send extensive projections to the midbrain and forebrain, including a strong projection to the ventral tegmental area (VTA)-a reward processing site. The retrograde neuronal tracer cholera toxin β-subunit (CTb) was injected into the VTA region; this was done to label the cell bodies of the pre-LC and PBel-inner neurons. After 1 week, the rats were killed and their brainstems processed by a triple-color immunofluorescence procedure. The purpose was to determine whether the CTb-labeled pre-LC and PBel-inner neurons, which also had FoxP2 immunoreactive nuclei, received close contacts from 5-HT axons. Neurons with these properties were found in both sites. Since the origin of this 5-HT input was unknown, a second set of experiments was carried out in which CTb was injected into the pre-LC or lateral PB. One week later, the rats were perfused and the brainstems from these animals were analyzed for the presence of neurons that co-contained CTb and tryptophan hydroxylase (synthetic enzyme for 5-HT) immunoreactivity. Co-labeled neurons were found mainly in the area postrema and to a lesser degree, in the dorsal raphe nucleus. We propose that the 5-HT inputs to the pre-LC and PBel-inner may modulate the salt appetite-related functions that influence the reward system., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The transcription factor Forkhead box protein 2 (FoxP2) is expressed in two cell groups of the brainstem that have been implicated in sodium appetite regulation: the pre-locus coeruleus (pre-LC) and parabrachial nucleus--external lateral-inner subdivision (PBel-inner). Because the connections of these two groups are unknown, neuroanatomical tracing methods were used to define their central projections. The pre-LC outputs were first analyzed using an anterograde axonal tracer--Phaseolus vulgaris leucoagglutinin (PHAL) to construct a brain map. Next, we examined whether the FoxP2 immunoreactive (FoxP2+) neurons of the pre-LC contribute to these projections using a retrograde neuronal tracer--cholera toxin β-subunit (CTb). CTb was injected into selected brain regions identified in the anterograde tracing study. One week later the rats were killed, and brainstem sections were processed by a double immunohistochemical procedure to determine whether the FoxP2+ neurons in the pre-LC and/or PBel-inner contained CTb. FoxP2+ pre-LC neurons project to: (1) ventral pallidum; (2) substantia innominata and bed nucleus of the stria terminalis; (3) paraventricular, central medial, parafascicular, and subparafascicular parvicellular thalamic nuclei; (4) paraventricular (PVH), lateral, perifornical, dorsomedial (DMH), and parasubthalamic hypothalamic nuclei; and (5) ventral tegmental area (VTA), periaqueductal gray matter (PAG), dorsal and central linear raphe nuclei. FoxP2+ PBel-inner neurons project to the PVH and DMH, with weaker connections to the LHA, VTA, and PAG. Both the pre-LC and PBel-inner project to central sites implicated in sodium appetite, and related issues, including foraging behavior, hedonic responses to salt intake, sodium balance, and cardiovascular regulation, are discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Two specific groups of neurons in the dorsolateral pons are activated by dietary sodium deprivation. These two groups are the pre-locus coeruleus (pre-LC) and the inner subdivision of the external lateral parabrachial nucleus (PBel-inner). In each site, after rats are fed an extremely low-sodium diet for over a week, neurons increase their expression of an activity-induced transcription factor, c-Fos. Here, we confirm this observation and extend it by demonstrating that these two groups of neurons express a common marker gene, the constitutively-expressed transcription factor Forkhead box protein 2 (FoxP2). That is, virtually all of the c-Fos activated neurons in both regions also express FoxP2. The expression of FoxP2 by both these groups of neurons suggests that they are developmentally-related subsets derived from the same basic population. Given that FoxP2, unlike c-Fos, is expressed independent of sodium deprivation, this marker may be useful in future studies of the pre-LC and PBel-inner. The molecular definition of these neurons, which project to circuits in the forebrain that influence visceral, appetitive, and hedonic functions, may allow direct experimental exploration of the functional role of these circuits using genetic tools., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The area postrema (AP) is a circumventricular organ located in the dorsal midline of the medulla. It functions as a chemosensor for blood-borne peptides and solutes, and converts this information into neural signals that are transmitted to the nucleus tractus solitarius (NTS) and parabrachial nucleus (PB). One of its NTS targets in the rat is the aldosterone-sensitive neurons which contain the enzyme 11 β-hydroxysteroid dehydrogenase type 2 (HSD2). The HSD2 neurons are part of a central network involved in sodium appetite regulation, and they innervate numerous brain sites including the pre-locus coeruleus (pre-LC) and PB external lateral-inner (PBel-inner) cell groups of the dorsolateral pons. Both pontine cell groups express the transcription factor FoxP2 and become c-Fos activated following sodium depletion. Because the AP is a component in this network, we wanted to determine whether it also projects to the same sites as the HSD2 neurons. By using a combination of anterograde axonal and retrograde cell body tract-tracing techniques in individual rats, we show that the AP projects to FoxP2 immunoreactive neurons in the pre-LC and PBel-inner. Thus, the AP sends a direct projection to both the first-order medullary (HSD2 neurons of the NTS) and the second-order dorsolateral pontine neurons (pre-LC and PB-el inner neurons). All three sites transmit information related to systemic sodium depletion to forebrain sites and are part of the central neural circuitry that regulates the complex behavior of sodium appetite., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The paraventricular hypothalamic nucleus (PVH) contains many neurons that innervate the brainstem, but information regarding their target sites remains incomplete. Here we labeled neurons in the rat PVH with an anterograde axonal tracer, Phaseolus vulgaris leucoagglutinin (PHAL), and studied their descending projections in reference to specific neuronal subpopulations throughout the brainstem. While many of their target sites were identified previously, numerous new observations were made. Major findings include: 1) In the midbrain, the PVH projects lightly to the ventral tegmental area, Edinger-Westphal nucleus, ventrolateral periaqueductal gray matter, reticular formation, pedunculopontine tegmental nucleus, and dorsal raphe nucleus. 2) In the dorsal pons, the PVH projects heavily to the pre-locus coeruleus, yet very little to the catecholamine neurons in the locus coeruleus, and selectively targets the viscerosensory subregions of the parabrachial nucleus. 3) In the ventral medulla, the superior salivatory nucleus, retrotrapezoid nucleus, compact and external formations of the nucleus ambiguous, A1 and caudal C1 catecholamine neurons, and caudal pressor area receive dense axonal projections, generally exceeding the PVH projection to the rostral C1 region. 4) The medial nucleus of the solitary tract (including A2 noradrenergic and aldosterone-sensitive neurons) receives the most extensive projections of the PVH, substantially more than the dorsal vagal nucleus or area postrema. Our findings suggest that the PVH may modulate a range of homeostatic functions, including cerebral and ocular blood flow, corneal and nasal hydration, ingestive behavior, sodium intake, and glucose metabolism, as well as cardiovascular, gastrointestinal, and respiratory activities., ((c) 2009 Wiley-Liss, Inc.)

The HSD2 (11-beta-hydroxysteroid dehydrogenase-type 2 enzyme) containing neurons of the nucleus tractus solitarius (NTS) become activated during low-sodium and high-aldosterone states such as hypovolemia. This response may be due to hormonal and/or neural signals. Hormonal signals may activate neurons in the area postrema that innervate the HSD2 neurons. The vagus nerve projects directly to the HSD2 neurons and this could be another route whereby these neurons receive information about systemic sodium/aldosterone status. The peripheral sites of origin that contribute to this vagal projection remain unknown, and in the present study, we injected the transganglionic tracer, cholera toxin beta-subunit-horseradish peroxidase (CTb-HRP), into wall of various gastrointestinal organs (stomach, small and large intestine) or liver of rats. Confocal microscopy of brainstem sections stained by a double immunohistochemical procedure was used to analyze whether the HSD2 neurons received axonal contacts from specific gastrointestinal structures. The major source of afferents arose from the stomach, mainly from its pyloric antrum, but a weaker input originated from the fundus region. A trace amount originated from the duodenum. The terminal part of the small intestine and large intestine did not to contribute to this projection. Similarly, no afferent inputs from the liver or portal vein were found. In conclusion, HSD2 neurons receive an input mainly from the stomach and these results are considered as potential sites affecting sodium intake.

Pharmacological and physiological phenomena suggest that cells somewhere inside the central nervous system are responsive to aldosterone. Here, we present the fundamental physiological limitations for aldosterone action in the brain, including its limited blood-brain barrier penetration and its substantial competition from glucocorticoids. Recently, a small group of neurons with unusual sensitivity to circulating aldosterone were identified in the nucleus of the solitary tract. We review the discovery and characterization of these neurons, which express the enzyme 11beta-hydroxysteroid dehydrogenase type 2, and consider alternative proposals regarding sites and mechanisms for mineralocorticoid action within the brain.

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B(12) (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype-phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late-onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late- or early-onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early-onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late-onset c.394C>T and c.482G>A mutations. The early-onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real-time RT-PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early-onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life., ((c) 2009 Wiley-Liss, Inc.)

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines and some tumors in situ. Most cell lines proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 has a cellular phenotype identical to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonylCoA mutase. Inability of cblC cells to complement the defect in MeWo-LC1 suggested that it was caused by decreased activity of the MMACHC gene. However, no potentially disease causing mutations were detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression was detected in MeWo-LC1 by quantitative or non-quantitative PCR. There was virtually complete methylation of a CpG island at the 5'-end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation. The CpG island was partially methylated (30-45%) in MeWo and only lightly methylated (2-11%) in control fibroblasts. Infection of MeWo-LC1 with wild type MMACHC resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

The nucleus of the solitary tract (NTS) contains a unique subpopulation of aldosterone-sensitive neurons. These neurons express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) and are activated by sodium deprivation. They are located in the caudal NTS, a region which is densely innervated by the vagus nerve, suggesting that they could receive direct viscerosensory input from the periphery. To test this possibility, we injected the highly sensitive axonal tracer biotinylated dextran amine (BDA) into the left nodose ganglion in rats. Using confocal microscopy, we observed a sparse input from the vagus to most HSD2 neurons. Roughly 80% of the ipsilateral HSD2 neurons exhibited at least one close contact with a BDA-labeled vagal bouton, although most of these cells received only a few total contacts. Most of these contacts were axo-dendritic (approximately 80%), while approximately 20% were axo-somatic. In contrast, the synaptic vesicular transporters VGLUT2 or GAD7 labeled much larger populations of boutons contacting HSD2-labeled dendrites and somata, suggesting that direct input from the vagus may only account for a minority of the information integrated by these neurons. In summary, the aldosterone-sensitive HSD2 neurons in the NTS appear to receive a small amount of direct viscerosensory input from the vagus nerve. The peripheral sites of origin and functional significance of this projection remain unknown. Combined with previously-identified central sources of input to these cells, the present finding indicates that the HSD2 neurons integrate humoral information with input from a variety of neural afferents.

The ventrolateral bed nucleus of the stria terminalis (BSTvl) receives direct input from two specific subpopulations of neurons in the nucleus tractus solitarius (NTS). It is heavily innervated by aldosterone-sensitive NTS neurons, which are selectively activated by sodium depletion, and by the A2 noradrenergic neurons, which are activated by visceral and immune- and stress-related stimuli. Here, we used a retrograde neuronal tracer to identify other brain sites that innervate the BSTvl. Five general brain regions contained retrogradely labeled neurons: cerebral cortex (infralimbic and insular regions), rostral forebrain structures (subfornical organ, organum vasculosum of the lamina terminalis, taenia tecta, nucleus accumbens, lateral septum, endopiriform nucleus, dorsal BST, substantia innominata, and, most prominently the amygdala--primarily its basomedial and central subnuclei), thalamus (central medial, intermediodorsal, reuniens, and, most prominently the paraventricular thalamic nucleus), hypothalamus (medial preoptic area, perifornical, arcuate, dorsomedial, parasubthalamic, and posterior hypothalamic nuclei), and brainstem (periaqueductal gray matter, dorsal and central superior raphe nuclei, parabrachial nucleus, pre-locus coeruleus region, NTS, and A1 noradrenergic neurons in the caudal ventrolateral medulla). In the arcuate hypothalamic nucleus, some retrogradely labeled neurons contained either agouti-related peptide or cocaine/amphetamine-regulated transcript. Of the numerous retrogradely labeled neurons in the perifornical hypothalamic area, few contained melanin-concentrating hormone or orexin. In the brainstem, many retrogradely labeled neurons were either serotoninergic or catecholaminergic. In summary, the BSTvl receives inputs from a variety of brain sites implicated in hunger, salt and water intake, stress, arousal, and reward., (Copyright 2008 Wiley-Liss, Inc.)

The transcription factor Phox2b is necessary for the development of the nucleus of the solitary tract (NTS). In this brainstem nucleus, Phox2b is expressed exclusively within a subpopulation of glutamatergic neurons. The present experiments in the adult rat were designed to test whether this subpopulation includes the aldosterone-sensitive NTS neurons, which express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). Nuclear Phox2b was found in virtually all the HSD2 neurons (95-99%, n = 6 cases). Unlike the activity-related transcription factor c-Fos, Phox2b expression in the HSD2 neurons was not influenced by dietary sodium deprivation. The ubiquitous expression of Phox2b by the HSD2 neurons suggests that they are developmentally related to other Phox2b-dependent neurons of the NTS and that they release the excitatory neurotransmitter glutamate. This finding also suggests that human Phox2b mutations, which cause the central congenital hypoventilation syndrome (CCHS, also known as Ondine's curse), may also produce deficits in central aldosterone signaling and appetitive or autonomic responses to sodium deficiency.

Sodium appetite, the behavioural drive to ingest salt, is stimulated by prolonged physiological sodium deficiency in many animal species. The same neural mechanisms that are responsible for sodium appetite in laboratory animals may influence human behaviour as well, with particular relevance to the dietary salt intake of patients with diseases such as heart failure, renal failure, liver failure and salt-sensitive hypertension. Since the original experimental work of Curt Richter in the 1930s, much has been learned about the regulation of salt-ingestive behaviour. Here, we review data from physiology, pharmacology, neuroanatomy and neurobehavioural investigations into the stimulatory and inhibitory signals that regulate sodium appetite. A rudimentary framework is proposed for the brain circuits that integrate peripheral information representing the need for sodium with neural signals for the gustatory detection of salt in order to drive a motivated ingestive response. Based on this model, areas of remaining uncertainty are highlighted where future information would allow a more detailed understanding of the neural circuitry responsible for sodium appetite.

Salt intake is an established response to sodium deficiency, but the brain circuits that regulate this behavior remain poorly understood. We studied the activation of neurons in the nucleus of the solitary tract (NTS) and their efferent target nuclei in the pontine parabrachial complex (PB) in rats during sodium deprivation and after salt intake. After 8-day dietary sodium deprivation, immunoreactivity for c-Fos (a neuronal activity marker) increased markedly within the aldosterone-sensitive neurons of the NTS, which express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). In the PB, c-Fos labeling increased specifically within two sites that relay signals from the HSD2 neurons to the forebrain--the pre-locus coeruleus and the innermost region of the external lateral parabrachial nucleus. Then, 1-2 hours after sodium-deprived rats ingested salt (a hypertonic 3% solution of NaCl), c-Fos immunoreactivity within the HSD2 neurons was virtually eliminated, despite a large increase in c-Fos activation in the surrounding NTS (including the A2 noradrenergic neurons) and area postrema. Also after salt intake, c-Fos activation increased within pontine nuclei that relay gustatory (caudal medial PB) and viscerosensory (rostral lateral PB) information from the NTS to the forebrain. Thus, sodium deficiency and salt intake stimulate separate subpopulations of neurons in the NTS, which then transmit this information to the forebrain via largely separate relay nuclei in the PB complex. These findings offer new perspectives on the roles of sensory information from the brainstem in the regulation of sodium appetite., ((c) 2007 Wiley-Liss, Inc.)

The nucleus accumbens (NAc) is part of a forebrain system implicated in reward, motivation, and learning. NAc neurons become activated during various ingestive activities, including salt intake. A subset of neurons within the nucleus tractus solitarius (NTS) shows c-Fos activation during prolonged sodium deprivation in rats. These neurons express mineralocorticoid receptors and the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them selectively sensitive to aldosterone-an adrenal hormone that modulates sodium appetite. Here we tested whether these neurons project multisynaptically to the core or shell subregions of the NAc. Pseudorabies virus (PRV)-a retrograde transneuronal tracer-was injected into the NAc in rats and after 3-4 days PRV-infected HSD2 neurons were identified. PRV injections into the NAc core yielded greater numbers of PRV-labeled HSD2 neurons than did comparable injections into the NAc shell. Transneuronal labeling was also found in brainstem sites that receive direct projections from HSD2 neurons, namely, lateral parabrachial and prelocus coeruleus nuclei. In other experiments a retrograde neural tracer (cholera toxin beta-subunit) was injected into the NAc. Extensive retrograde labeling was found in the midline thalamus and frontal cortical regions, but no cells were labeled in the NTS or parabrachial region. These findings indicate that the HSD2 neurons project via a multisynaptic pathway to the NAc, which may be relayed sequentially through two sites: the dorsolateral pons and the paraventricular thalamic nucleus. HSD2 neurons may be part of an ascending pathway involved in the salt-seeking behavior of sodium-depleted rats.

Thirst and sodium appetite are both critical for restoring blood volume. Because these two behavioral drives can arise under similar physiological conditions, some of the brain sensory sites that stimulate thirst may also drive sodium appetite. However, the physiological and temporal dynamics of these two appetites exhibit clear differences, suggesting that they involve separate brain circuits. Unlike thirst-associated sensory neurons in the hypothalamus, the 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) neurons in the rat nucleus tractus solitarius (NTS) are activated in close association with sodium appetite (16). Here, we tested whether the HSD2 neurons are also activated in response to either of the two physiological stimuli for thirst: hyperosmolarity and hypovolemia. Hyperosmolarity, produced by intraperitoneal injection of hypertonic saline, stimulated a large increase in water intake and a substantial increase in immunoreactivity for the neuronal activity marker c-Fos within the medial NTS, but not in the HSD2 neurons. Hypovolemia, produced by subcutaneous injection of hyperoncotic polyethylene glycol (PEG), stimulated an increase in water intake within 1-4 h without elevating c-Fos expression in the HSD2 neurons. The HSD2 neurons were, however, activated by prolonged hypovolemia, which also stimulated sodium appetite. Twelve hours after PEG was injected in rats that had been sodium deprived for 4 days, the HSD2 neurons showed a consistent increase in c-Fos immunoreactivity. In summary, the HSD2 neurons are activated specifically in association with sodium appetite and appear not to function in thirst.

The nucleus of the solitary tract (NTS) contains a unique subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). These neurons are mineralocorticoid-sensitive and are activated in association with salt appetite during sodium deficiency. In the absence of sodium deficiency, the HSD2 neurons and sodium appetite are both stimulated by chronic mineralocorticoid administration. After 7 days of treatment with deoxycorticosterone (2 mg/day), an increased number of HSD2 neurons became immunoreactive for the neuronal activity marker c-Fos. When given access to concentrated saline (3% NaCl), deoxycorticosterone-treated rats drank eight times more than vehicle-treated rats. Saline ingestion increased neuronal activation within the medial subdivision of the NTS, but the number of c-Fos-immunoreactive HSD2 neurons was reduced. This finding suggests that the HSD2 neurons are inhibited by signals directly related to saline ingestion, and not simply by the alleviation of sodium deficiency, which does not occur during mineralocorticoid administration.

The nucleus of the solitary tract (NTS) contains a subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them uniquely sensitive to aldosterone. These neurons may drive sodium appetite, which is enhanced by aldosterone. Anterograde and retrograde neural tracing techniques were used to reveal the efferent projections of the HSD2 neurons in the rat. First, the anterograde tracer Phaseolus vulgaris leucoagglutinin was used to label axonal projections from the medial NTS. Then, NTS-innervated brain regions were injected with a retrograde tracer, cholera toxin beta subunit, to determine which sites are innervated by the HSD2 neurons. The HSD2 neurons project mainly to the ventrolateral bed nucleus of the stria terminalis (BSTvl), the pre-locus coeruleus (pre-LC), and the inner division of the external lateral parabrachial nucleus (PBel). They also send minor axonal projections to the midbrain ventral tegmental area, lateral and paraventricular hypothalamic nuclei, central nucleus of the amygdala, and periaqueductal gray matter. The HSD2 neurons do not innervate the ventrolateral medulla, a key brainstem autonomic site. Additionally, our tracing experiments confirmed that the BSTvl receives direct axonal projections from the neighboring A2 noradrenergic neurons in the NTS, and from the same pontine sites that receive major inputs from the HSD2 neurons (PBel and pre-LC). The efferent projections of the HSD2 neurons may provide new insights into the brain circuitry responsible for sodium appetite.

Aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) become activated during sodium depletion and could be key neural elements regulating sodium intake. The afferent inputs to these neurons have not yet been defined, but one source may be neurons in the area postrema, a neighboring circumventricular organ that innervates the NTS and exerts a powerful inhibitory influence on sodium appetite [Contreras RJ, Stetson PW (1981) Changes in salt intake after lesions of the area postrema and the nucleus of the solitary tract in rats. Brain Res 211:355-366]. After an anterograde axonal tracer was injected into the area postrema in rats, sections through the NTS were immunolabeled for the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a marker for aldosterone-sensitive neurons, and examined by confocal microscopy. We found that some of the aldosterone-sensitive neurons received close appositions from processes originating in the area postrema, suggesting that input to the HSD2 neurons could be involved in the inhibition of sodium appetite by this site. Axonal varicosities originating from the area postrema also made close appositions with other neurons in the medial NTS, including the neurotensin-immunoreactive neurons in the dorsomedial NTS. Besides these projections, a dense field of neurotensinergic axon terminals overlapped the distribution of the HSD2 neurons. Neurotensin-immunoreactive axon terminals were identified in close apposition to the dendrites and cell bodies of some HSD2 neurons, as well as unlabeled neurons lying in the same zone within the medial NTS. A local microcircuit involving the area postrema, HSD2 neurons, and neurotensinergic neurons may play a major role in the regulation of sodium appetite.

The HSD2 (11-beta-hydroxysteroid dehydrogenase type 2-expressing) neurons in the nucleus of the solitary tract (NTS) of the rat are aldosterone-sensitive and have been implicated in sodium appetite. The central nucleus of the amygdala (CeA) has been shown to modulate salt intake in response to aldosterone, so we investigated the connections between these two sites. A prior retrograde tracing study revealed only a minor projection from the HSD2 neurons directly to the CeA, but these experiments suggested that a more substantial projection may be relayed through the parabrachial nucleus. Small injections of cholera toxin beta subunit (CTb) into the external lateral parabrachial subnucleus (PBel) produced both retrograde cell body labeling in the HSD2 neurons and anterograde axonal labeling in the lateral subdivision of the CeA. Also, injections of either CTb or Phaseolus vulgaris leucoagglutinin into the medial subdivision of the CeA labeled a descending projection from the amygdala to the medial NTS. Axons from the medial CeA formed numerous varicosities and terminals enveloping the HSD2 neurons. Complementary CTb injections, centered in the HSD2 subregion of the NTS, retrogradely labeled neurons in the medial CeA. These bidirectional projections could form a functional circuit between the HSD2 neurons and the CeA. The HSD2 neurons may represent one of the functional inputs to the lateral CeA, and their activity may be modulated by a return projection from the medial CeA. This circuit could provide a neuroanatomical basis for the modulation of salt intake by the CeA., (Copyright 2006 Wiley-Liss, Inc.)

The nucleus of the solitary tract (NTS) contains a subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them uniquely sensitive to aldosterone. These neurons may drive sodium appetite, which is enhanced by aldosterone. Anterograde and retrograde neural tracing techniques were used to reveal the efferent projections of the HSD2 neurons in the rat. First, the anterograde tracer Phaseolus vulgaris leucoagglutinin was used to label axonal projections from the medial NTS. Then, NTS-innervated brain regions were injected with a retrograde tracer, cholera toxin beta subunit, to determine which sites are innervated by the HSD2 neurons. The HSD2 neurons project mainly to the ventrolateral bed nucleus of the stria terminalis (BSTvl), the pre-locus coeruleus (pre-LC), and the inner division of the external lateral parabrachial nucleus (PBel). They also send minor axonal projections to the midbrain ventral tegmental area, lateral and paraventricular hypothalamic nuclei, central nucleus of the amygdala, and periaqueductal gray matter. The HSD2 neurons do not innervate the ventrolateral medulla, a key brainstem autonomic site. Additionally, our tracing experiments confirmed that the BSTvl receives direct axonal projections from the neighboring A2 noradrenergic neurons in the NTS, and from the same pontine sites that receive major inputs from the HSD2 neurons (PBel and pre-LC). The efferent projections of the HSD2 neurons may provide new insights into the brain circuitry responsible for sodium appetite., (Copyright 2006 Wiley-Liss, Inc.)

The purpose of this study was to identify brain sites that may be sensitive to the adrenal steroid aldosterone. After a survey of the entire brain for mineralocorticoid receptor (MR) immunoreactivity, we discovered unique clusters of dense nuclear and perinuclear MR in a restricted distribution within the nucleus of the solitary tract (NTS). These same cells were found to contain the glucocorticoid-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a signature of aldosterone-sensitive tissues. Immunoreactivity for various other NTS marker molecules failed to colocalize with HSD2 in these putative aldosterone target neurons, so they may represent a unique neuronal phenotype. Finally, the entire rat CNS was examined for evidence of HSD2 protein expression. Outside the NTS, HSD2-immunoreactive neurons were found in only two other sites: the ventrolateral division of the ventromedial hypothalamic nucleus and a few scattered neurons in the medial vestibular nucleus, just rostral to the NTS. HSD2 immunoreactivity was also found in the ependymal cells that form the subcommissural organ. In summary, few brain sites contain neurons that may be aldosterone sensitive, and only one of these sites, the NTS, contains neurons that express HSD2 and contain dense nuclear MR. The HSD2 neurons in the NTS may represent an important target for aldosterone action in the brain.

Sodium appetite can be enhanced by the adrenal steroid aldosterone via an unknown brain mechanism. A novel group of neurons in the nucleus tractus solitarius expresses the enzyme 11-beta-hydroxysteroid dehydrogenase type 2, which makes them selectively responsive to aldosterone. Their activation parallels sodium appetite in different paradigms of salt loss even in the absence of aldosterone. These unique aldosterone target neurons may represent a previously unrecognized central convergence point at which hormonal and neural signals can be integrated to drive sodium appetite.

Dahl salt-sensitive rats develop severe hypertension during a high-sodium diet, but the basis of their salt-sensitive phenotype is not completely understood. A subset of neurons in the nucleus tractus solitarius (NTS) are uniquely sensitive to the adrenal steroid hormone aldosterone, which is critically involved in sodium homeostasis, due to their expression of the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). The number of HSD2 neurons in the NTS was counted in prehypertensive 7-week-old Dahl salt-sensitive rats and compared with two control strains: Dahl salt-resistant and Sprague-Dawley rats. Dahl salt-sensitive rats had more HSD2 neurons than age-matched Dahl salt-resistant and Sprague-Dawley rats (24% and 21%, respectively). Cell counts were also made in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats; the number of HSD2 neurons in both of these strains was similar to the values obtained for Sprague-Dawley rats. The increased number of HSD2-immunoreactive neurons counted in Dahl salt-sensitive rats suggests that they may have a greater number of aldosterone-sensitive NTS neurons. Alternatively, an increase in HSD2 expression in Dahl salt-sensitive rats could increase the overall immunoreactivity, permitting detection of more of these neurons. In either case, the roughly 20% increase in HSD2 neurons in the NTS of prehypertensive Dahl salt-sensitive rats is a novel factor associated with their salt-sensitive phenotype. These neurons may play a role in regulating sodium appetite, which is abnormally suppressed in Dahl salt-sensitive rats.

Cardiovascular changes occur during mental stress and in certain types of mood disorders. The neural basis for this phenomenon is unknown but it may be dependent on CNS neurons that provide branched projections to affective processing regions of the brain, such as the medial prefrontal cortex, and to the sympathetic outflow system. Because these putative neurons may be connected to these two target sites by chains of neurons, we performed double virus transneuronal tracing experiments and show here that a select subset of neurons in the medial preoptic nucleus (MPN), lateral hypothalamic area (LHA), and nucleus tractus solitarius (NTS) are co-linked to these two sites. Neurotensin MPN, orexin-containing LHA, and catecholamine NTS neurons were the major phenotypes involved in these projections. This novel class of neurons may coordinate cardiovascular changes seen in different emotional states.

Vagal nerve stimulation has been reported to enhance memory in both rats and humans, and to be an effective treatment for epilepsy in some patients, but the underlying neuroanatomical substrate(s) responsible for these effects remains unknown. Since there is no direct anatomical projection from the nucleus tractus solitarius, the main vagal relay site of the brain, to the hippocampus, we tested whether a multisynaptic pathway exists. Pseudorabies virus, a pig herpesvirus that can be used as a retrograde transneuronal tracer, was injected into the ventral CA1 hippocampus of rats, and after 4 days, pseudorabies virus infected neurons were identified in the general visceral portion of the nucleus tractus solitarius, with the majority being localized in the A2 noradrenergic cell group. Other autonomic brainstem nuclei, including the parabrachial nucleus, locus coeruleus, A1 and A5 noradrenergic cell groups, and C1 adrenergic cell group, were labeled. In order to identify some of the potential relay sites of the nucleus tractus solitarius-->hippocampal pathway, immunotoxin lesions of the ventral CA1 region were made that selectively destroyed either the noradrenergic or cholinergic fibers. After 2 weeks' recovery, pseudorabies virus was injected in this same CA1 area, and 4 days later, the transneuronal labeling in the nucleus tractus solitarius was reduced by approximately 65%. These findings suggest that the noradrenergic neurons of the locus coeruleus and cholinergic neurons of the medial septum/diagonal band are likely to be relay sites for this pathway. Other potential linkages are discussed. In summary, this is the first anatomical report to show that the general visceral region of nucleus tractus solitarius is linked via multisynaptic relays to the hippocampus.

Two anatomical experiments were performed to test the hypothesis that single CNS neurons link the central areas that regulate the somatomotor and sympathetic systems. First, the retrograde neuronal tracer cholera toxin beta-subunit was injected into the lateral parafascicular thalamic nucleus, a region that projects to both the motor cortex and striatum. Several days later, a second injection of the retrograde transneuronal tracer, pseudorabies virus (PRV), was made in the same rats in the stellate ganglion, which provides the main sympathetic supply to the heart. Using immunohistochemical methods, we demonstrate that the cholinergic neurons of the pedunculopontine tegmental nucleus (PPN) are connected to both systems. The second experiment used two isogenic strains of Bartha PRV as double transneuronal tracers. One virus contained the unique gene for green fluorescent protein (GFP) and the other had the unique gene for beta-galactosidase (beta-gal). GFP-PRV was injected in the stellate ganglion and beta-gal-PRV was injected into the primary motor cortex. Double-labeled neurons were found in the lateral hypothalamic area (50% contained orexin) and PPN (approximately 95% were cholinergic). Other double-labeled neurons were identified in the deep temporal lobe (viz., amygdalohippocampal zone and lateral entorhinal cortex), posterior hypothalamus, ventral tuberomammillary nucleus, locus coeruleus, laterodorsal tegmental nucleus, periaqueductal gray matter, dorsal raphe nucleus, and nucleus tractus solitarius. These results suggest these putative command neurons integrate the somatomotor and cardiosympathetic functions and may affect different behaviors (viz., arousal, sleep, and/or locomotion).

The viral transneuronal labeling method was used to demonstrate that orexin-containing neurons of the lateral hypothalamic area (LHA) are linked via multisynaptic connections to different sympathetic outflow systems. Two different types of transneuronal tracing experiments were performed: single- and double-virus studies. In the first series of experiments, Bartha pseudorabies virus (PRV), a retrograde transneuronal tracer, was injected into single sympathetic targets, viz., stellate ganglion, adrenal gland, celiac ganglion, and kidney. Six to 7 days post-injection, orexin (hypocretin) neurons were transneuronally labeled. In a second set of experiments, the double-virus tracing method was used to determine whether single orexin LHA neurons are linked to two different sympathetic outflow systems. Two isogenic forms of Bartha PRV were used that differed by a single gene. beta-Galactosidase Bartha PRV was injected into the stellate ganglion and green fluorescent protein Bartha PRV into the adrenal gland of the same rat. The reverse placement of viral injections was made in another set of rats. In both paradigms, some orexin LHA neurons were transneuronally labeled with both viruses, indicating that they are capable of modulating multiple sympathetic outflow systems. These findings raise the possibility that orexin LHA neurons regulate general sympathetic functions, such as those that occur during arousal or the fight-or-flight response.

The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin beta -subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nuclei-the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental field. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem-thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions., (Copyright 2002 Wiley-Liss, Inc.)

This paper describes the use of a high-resolution flatbed scanner for neuroanatomical studies. Individual neurons that had been labeled using gold intensified diaminobenzidine (DAB) could be resolved in thionin-stained rat brain sections. One of the strengths of this method is that it permits the simultaneous visualization of labeled cells and their position relative to underlying cytoarchitectonic structures, allowing for highly accurate neuroanatomical analysis. High resolution maps of complete rat brains ( approximately 100 sections spaced at 250 microm intervals) can be obtained in 10 h. Since much of this method can be automated, the scanning of brain sections is approximately 50% faster than conventional X-Y mapping, camera lucida, or photographic procedures. The method may have many other applications, particularly in evaluating the distribution of other types of labeled cells. For example, Fos immunoreactive neurons, cells labeled with reporter genes such as beta-galactosidase in brains from transgenic animals, and unique histological inclusions such as plaques found in Alzheimer's disease could all be directly imaged without a microscope. In addition, the exchange of primary data between labs via the Internet will provide additional opportunities for collaboration.