Graciela Sanchez-Watts

Comparison of melanin-concentrating hormone and hypocretin/orexin mRNA expression patterns in a new parceling scheme of the lateral hypothalamic zone

A high-resolution spatial distribution analysis of hypothalamic neurons expressing melanin-concentrating hormone or hypocretin/orexin was performed in adult male rats with in situ hybridization cytochemistry. For the analysis, a new parcellation of the lateral zone with some two-dozen regions was used, and distributions were plotted on 15 transverse reference levels through the hypothalamus. Qualitatively the results confirm earlier, much lower resolution mapping studies, although some discrepancies are clarified. Previous work indicates that each of these cell populations is far from homogeneous, and the present results should help establish a framework for clarifying more precisely how they are differentiated and organized in terms of axonal input-output relationships and gene expression patterns, and for defining precise relationships with other hypothalamic neuron populations.

Catecholaminergic Control of Mitogen-Activated Protein Kinase Signaling in Paraventricular Neuroendocrine Neurons In Vivo and In Vitro: A Proposed Role during Glycemic Challenges

Paraventricular hypothalamic (PVH) corticotropin-releasing hormone (CRH) neuroendocrine neurons mount neurosecretory and transcriptional responses to glycemic challenges [intravenous 2-deoxyglucose (2-DG) or insulin]. Although these responses require signals from intact afferents originating from hindbrain CA (catecholaminergic) neurons, the identity of these signals and the mechanisms by which they are transduced by PVH neurons during glycemic challenge remain unclear. Here, we tested whether the prototypical catecholamine, norepinephrine (NE), can reproduce PVH neuroendocrine responses to glycemic challenge. Because these responses include phosphorylation of p44/42 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases 1/2 (ERK1/2)], we also determined whether NE activates ERK1/2 in PVH neurons and, if so, by what mechanism. We show that systemic insulin and 2-DG, and PVH-targeted NE microinjections, rapidly elevated PVH phospho-ERK1/2 levels. NE increased Crh and c-fos expression, together with circulating ACTH/corticosterone. However, because injections also increased c-Fos mRNA in other brain regions, we used hypothalamic slices maintained in vitro to clarify whether NE activates PVH neurons without contribution of inputs from distal regions. In slices, bath-applied NE triggered robust phospho-ERK1/2 immunoreactivity in PVH (including CRH) neurons, which attenuated markedly in the presence of the α1 adrenoceptor antagonist, prazosin, or the MAP kinase kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Therefore, at a systems level, local PVH delivery of NE is sufficient to account for hindbrain activation of CRH neuroendocrine neurons during glycemic challenge. At a cellular level, these data provide the first demonstration that MAP kinase signaling cascades (MEK→ERK) are intracellular transducers of noradrenergic signals in CRH neurons, and implicate this transduction mechanism as an important component of central neuroendocrine responses during glycemic challenge.

Neuropeptides and thirst: the temporal response of corticotropin-releasing hormone and neurotensin/neuromedin N gene expression in rat limbic forebrain neurons to drinking hypertonic saline.

The authors have demonstrated in rats that the ingestion of hypertonic saline for 5 days provides an increasingly complex dehydrating stimulus to the rats. Initially, the stimulus leads to cellular dehydration, but extracellular dehydration develops as ingestion continues beyond 3 days. The initial cellular dehydration provokes modifications to corticotropin-releasing hormone and neurotensin/neuromedin N messenger RNAs (mRNAs) in some neurons of the limbic forebrain, changes that are either maintained or are modified as extracellular dehydration develops. These changes in mRNA content occur in neurosecretory neurons as well as in neurons in hypothalamic and telencephalic regions associated with behavior and autonomic regulation. The authors propose that alterations in peptide mRNAs are allied to altered neuronal signaling processes that direct the different components of the homeostatic response to dehydration.

Salt-Inducible Kinase Is Involved in the Regulation of Corticotropin-Releasing Hormone Transcription in Hypothalamic Neurons in Rats

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

Rapid and preferential activation of Fos protein in hypocretin/orexin neurons following the reversal of dehydration-anorexia.

Dehydration (DE)‐anorexia is stimulated by chronic consumption of hypertonic saline. Spontaneous nocturnal food intake is markedly reduced with this treatment but is rapidly reversed upon the return of drinking water. Here we examined the neurons in the lateral hypothalamic area (LHA) of chronically dehydrated rats for their peptidergic phenotype, colocalization, and activation profiles following the rapid reversal of anorexia. To do this, we used double‐labeling combinations of Fos immunocytochemistry and radioisotopic‐ and digoxigenin‐labeled in situ hybridization. We found that lateral hypothalamic corticotropin‐releasing hormone (CRH) neurons show extensive coexpression with neurotensin mRNA, but they are distinct from hypocretin/orexin and melanin‐concentrating hormone (MCH) neurons. Chronic dehydration increases Fos‐ir in large numbers of neurons in dorsal regions of the LHA. Some of these LHA neurons also show increased CRH, but not hypocretin/orexin or MCH gene expression, as dehydration‐anorexia develops. Furthermore, the behavioral sequence of eating and increased activity exhibited by DE animals in the minutes following water drinking is accompanied by a further increase in the number of Fos‐ir nuclei in the LHA. Increased Fos activation occurs in a significant number of LHA hypocretin/orexin neurons, but not CRH or MCH neurons, in the LHA. Together these data implicate CRH but not hypocretin/orexin or MCH neurons in the LHA in the motor events associated with dehydration. However, when water is returned, contributions to the network controlling responses evidently come from hypocretin/orexin, but not CRH or MCH, neurons in the LHA. J. Comp. Neurol. 502:768–782, 2007.

MAP Kinases Couple Hindbrain-Derived Catecholamine Signals To Hypothalamic Adrenocortical Control Mechanisms During Glycemia-Related Challenges

Physiological responses to hypoglycemia, hyperinsulinemia, and hyperglycemia include a critical adrenocortical component that is initiated by hypothalamic control of the anterior pituitary and adrenal cortex. These adrenocortical responses ensure appropriate long-term glucocorticoid-mediated modifications to metabolism. Despite the importance of these mechanisms to disease processes, how hypothalamic afferent pathways engage the intracellular mechanisms that initiate adrenocortical responses to glycemia-related challenges are unknown. This study explores these mechanisms using network- and cellular-level interventions in in vivo and ex vivo rat preparations. Results show that a hindbrain-originating catecholamine afferent system selectively engages a MAP kinase pathway in rat paraventricular hypothalamic CRH (corticotropin-releasing hormone) neuroendocrine neurons shortly after vascular insulin and 2-deoxyglucose challenges. In turn, this MAP kinase pathway can control both neuroendocrine neuronal firing rate and the state of CREB phosphorylation in a reduced ex vivo paraventricular hypothalamic preparation, making this signaling pathway an ideal candidate for coordinating CRH synthesis and release. These results establish the first clear structural and functional relationships linking neurons in known nutrient-sensing regions with intracellular mechanisms in hypothalamic CRH neuroendocrine neurons that initiate the adrenocortical response to various glycemia-related challenges.

Structural organization and chromosomal localization of the human Na,K-ATPase β3 subunit gene and pseudogene

We have cloned and characterized the Na,K-ATPase β3 subunit gene (ATP1B3), and a β3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The β3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the β3 subunit gene is identical to that of the Na,K-ATPase β3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse β3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the β3 subunit gene are likely to be located within this conserved region. Dot blot analysis of β3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of β3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22 → 23 region of Chromosome (Chr) 3, and the β3 pseudogene to the pl3 → 15 region of Chr 2.

The Distribution of Messenger RNAs Encoding the Three Isoforms of the Transducer of Regulated cAMP Responsive Element Binding Protein Activity in the Rat Forebrain

Increasing evidence indicates that the cAMP responsive element binding protein (CREB)‐dependent transcriptional activation of a number of genes requires the CREB co‐activator: transducer of regulated CREB activity (TORC). Because of the central importance of CREB in many brain functions, we examined the topographic distribution of TORC1, 2, and 3 mRNAs in specific regions of the rat forebrain. In situ hybridisation analysis revealed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. All three TORC isoforms were found in a number of brain nuclei, the ventricular ependyma and pia mater. Although high levels of TORC1 were widely distributed in the forebrain, TORC2 was found in discrete nuclei and TORC3 mostly in the ependyma, and pia mater. The relative expression of TORC isoforms was confirmed by quantitative reverse transcriptase‐polymerase chain reaction analysis in the hippocampus and hypothalamus. In the paraventricular nucleus of the hypothalamus, TORC1 and 2 mRNAs were abundant in the parvicellular and magnocellular neuroendocrine compartments, whereas TORC3 expression was low. All three isoform mRNAs were found elsewhere in the hypothalamus, with the most prominent expression of TORC1 in the ventromedial nucleus, TORC2 in the dorsomedial and arcuate nuclei, TORCs 1 and 2 in the supraoptic nucleus, and TORC2 in the suprachiasmatic nucleus. These differential distribution patterns are consistent with complex roles for all three TORC isoforms in diverse brain structures, and provide a foundation for further studies on the mechanisms of CREB/TORC signalling on brain function.

Activation in neural networks controlling ingestive behaviors: what does it mean, and how do we map and measure it?

Over the past thirty years many of different methods have been developed that use markers to track or image the activity of the neurons within the central networks that control ingestive behaviors. The ultimate goal of these experiments is to identify the location of neurons that participate in the response to an identified stimulus, and more widely to define the structure and function of the networks that control specific aspects of ingestive behavior. Some of these markers depend upon the rapid accumulation of proteins, while others reflect altered energy metabolism as neurons change their firing rates. These methods are widely used in behavioral neuroscience, but the way results are interpreted within the context of defining neural networks is constrained by how we answer the following questions. How well can the structure of the behavior be documented? What do we know about the processes that lead to the accumulation of the marker? What is the function of the marker within the neuron? How closely in time does the marker accumulation track the stimulus? How long does the marker persist after the stimulus is removed? We will review how these questions can be addressed with regard to ingestive and related behaviors. We will also discuss the importance of plotting the location of labeled cells using standardized atlases to facilitate the presentation and comparison of data between experiments and laboratories. Finally, we emphasize the importance of comprehensive and accurate mapping for using newly emerging technologies in neuroinfomatics.

A cell-specific role for the adrenal gland in regulating CRH mRNA levels in rat hypothalamic neurosecretory neurons after cellular dehydration

In the rat, the cellular dehydration induced by water deprivation rapidly increases CRH mRNA in magnocellular neurosecretory neurons, but gradually reduces mRNA levels in hypothalamic paraventricular parvicellular neurosecretory neurons. Using in situ hybridization we investigated a possible role for corticosterone as a mediator of the effects of water deprivation on the levels of CRH mRNA in the paraventricular and supraoptic nuclei. Following adrenalectomy and water deprivation, the reduction of CRH mRNA in the medial parvicellular part of the paraventricular nucleus was inhibited. However, replacement of low-doses of corticosterone to dehydrated adrenalectomized animals was not sufficient to reduce parvicellular CRH mRNA levels to those seen in intact dehydrated animals. Neither adrenalectomy nor corticosterone replacement had any effect on the increased CRH mRNA levels in magnocellular neurosecretory neurons. We conclude that an intact adrenal gland is required for the decreased levels of CRH mRNA seen during water deprivation in parvicellular paraventricular neurosecretory neurons, but not magnocellular neurosecretory neurons. These effects may be mediated by the increased corticosterone secretion seen during water deprivation.