Jeffrey D. White

Increased hypothalamic neuropeptide Y expression following food deprivation

Neuropeptide Y potently stimulates food intake by acting within the hypothalamus. In this study we examined the possibility that hypothalamic neuropeptide Y expression is increased following food deprivation, as assessed by measuring the hypothalamic content of the mRNA coding for preproneuropeptide Y. Adult male Sprague-Dawley rats were allowed free access to water but were subjected to food withdrawal either overnight or for 72 h or not. Total RNA was isolated from whole hypothalamic dissections and the content of mRNA coding for preproneuropeptide Y was determined by RNase protection analysis. This study revealed a 2.6-fold increase in hypothalamic preproneuropeptide Y mRNA content in 72-h-food- deprived versus control rats with a small increase (40%), which did not reach statistical significance, in overnight-fasted versus control rats. In situ hybridization analysis was used to determine the cellular localization of this increased mRNA content. This study revealed an increase in hybridization in the arcuate nucleus of the hypothalamus of a magnitude similar to that observed using nuclease protection with no change observed over neocortex or reticular nucleus of the thalamus. These data are consistent with the hypothesis that expression of hypothalamic neuropeptide Y is modulated by peripheral metabolic status and support a role for neuropeptide Y in the control of food intake.

Differential regulation of neuropeptide and proto-oncogene mRNA content in the hippocampus following recurrent seizures

The amounts of the mRNAs for the neuropeptide precursor proteins preproenkephalin, preprocholecystokinin and preproneuropeptide Y were measured in the entorhinal cortex of normal rats and rats that had experienced recurrent limbic seizures induced by a small contralateral lesion of the dentate gyrus hilus. Additionally, the amount of mRNAs for preproenkephalin as well as for the cellular proto-oncogenes c-myc, c-fos and c-H-ras, which are thought to be mediators of intracellular signal transduction, was determined in hippocampus in these same animals. It was determined that the hilus lesion led to a dramatic (18-fold) increase in the content of preproenkephalin mRNA in the entorhinal cortex whereas only a modest increase in preproneuropeptide Y mRNA content and no change in preprocholecystokinin mRNA was detected in this same brain region. In hippocampus a large and very rapid increase in c-fos mRNA was observed to precede the previously reported increase in preproenkephalin mRNA following hilus lesion-induced seizures. Like the increase in opioid peptide mRNA, the increase in c-fos mRNA began early in the period of seizure activity and could be blunted by maintaining the animals under anesthesia with the anticonvulsant sodium pentobarbital. Messenger RNA for c-H-ras was not altered at any time following the lesion and c-myc mRNA was not reliably detected in either control or hilus lesioned rats. These data demonstrate that neuropeptide genes within the entorhinal cortex and proto-oncogenes within the hippocampus are differentially regulated by seizure activity and suggest that the c-fos proto-oncogene may be involved in events which mediate the physiological regulation of enkephalin gene expression.

Neuropeptide Y: a central regulator of energy homeostasis

Neuropeptide Y (NPY) is a 36 amino acid peptide belonging to the pancreatic polypeptide family of neuroendocrine hormones. It is the most abundant peptide yet discovered in the mammalian brain and is widely expressed by neurons in the central and peripheral nervous systems as well as adrenal medullary cells. Recently, a large number of studies have focussed on the potential roles played by NPY within the hypothalamus and pituitary with respect to the control of food intake and energy homeostasis. It is now clear that NPY is a potent stimulator of food intake in models of hyperphagia, that hypothalamic NPY also regulates sympathetic neural activity and it appears that NPY may also influence the glucocorticoid, growth hormone and thyroid hormone axes. Taken together, current data suggest that hypothalamic and pituitary NPY-expressing cells represent an important and critical site of integration of peripheral hormonal signals with regulation of energy homeostasis.

Hypothalamic neuropeptide Y gene expression in rats on scheduled feeding regimen

Recent evidence suggests that neuropeptide Y (NPY) is an important signal in the neural circuitry that controls feeding behavior. Previously we observed that in rats entrained to 4 h daily scheduled feeding regimen (SFR), NPY content and release in the paraventricular nucleus (PVN) was elevated but decreased rapidly in association with food consumption. In the present study, we investigated the pattern of hypothalamic NPY gene expression in SFR rats before and after food consumption by measuring the content of preproNPY mRNA in the medial basal hypothalamus (MBH). Adult male rats were maintained on either ad libitum diet (control) or on SFR. Rats were killed before food presentation at 11.00 h and at the end of 4 h food consumption at 15.00 h. The levels of preproNPY mRNA in the MBH were determined by solution hybridization/RNase protection assay using a cRNA probe complementary to rat NPY precursor mRNA. We observed that, as compared to that in control rats on ad libitum diet, preproNPY mRNA levels in the MBH were increased two-fold in the SFR rat at 11.00 h and remained elevated even after 4 h of food consumption. These results show a simultaneous enhancement in PVN NPY release and hypothalamic gene expression in advance of scheduled feeding time, but food intake rapidly decreases PVN NPY release and content, with little impact on hypothalamic gene expression.

Pentylenetetrazole-induced seizures stimulate transcription of early and late response genes

Cellular immediate early gene and neuropeptide gene expression have each been demonstrated to be modulated in hippocampus in response to a variety of seizure-inducing stimuli. In this study, gene transcription for three immediate early genes, c-fos, c-jun and NGFI-A, and three neuropeptide genes, enkephalin, dynorphin and neuropeptide Y, was investigated using nuclear run-on assays following a single injection of the convulsant pentylenetetrazole (PTZ). At 15 min following PTZ injection, only transcription for c-fos was increased. By 6 h following PTZ treatment, transcription for all immediate early genes and for dynorphin and neuropeptide Y was increased; however, this increase was transient in that transcription of all genes returned to control values by 48 h following PTZ treatment. Thus, regulation of immediate early and neuropeptide gene mRNA levels and immunoreactivity occurs, at least in part, at the level of transcription for the genes encoding neuropeptide Y, dynorphin, c-fos, c-jun, and NGFI-A. Moreover, the difference between increased transcription rates reported here and increased mRNA levels reported here and elsewhere suggests that additional post-transcriptional regulation of gene expression occurs in hippocampal neurons.

Biosynthesis and metabolism of native and oxidized Neuropeptide Y in the hippocampal mossy fiber system

Abstract: Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole‐induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY‐like immunoreactive (NPY‐LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole‐treated animals confirmed the increased total NPY‐LI, and demonstrated that the increased NPY‐LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY‐LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti‐C‐terminal flanking peptide of NPY, further HPLC purification, and matrix‐assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY‐LI is methionine sulfoxide NPY. NPY and NPY‐sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high‐affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

Abstract: The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5‐and for Leu5‐enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5‐ and Leu5‐enkephalin and with substance P. The ontogeny of Met5‐enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5‐enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.

Regulation of hypothalamic neuropeptide expression by peripheral metabolism.

The mechanisms of appetite and body-weight regulation by peripheral signals are highly complex in vertebrates and remain poorly understood. It is intuitively apparent that such regulation must involve interactions between peripheral metabolic status and the brain, but what are the signals recognized by the brain to initiate feeding? The hypothalamus has long been recognized as central in recognition of peripheral nutrient and metabolic signals (and, perhaps, body weight status) and in regulation of hunger and satiety responses and, therefore, is a logical site on which to focus research aimed at understanding interactions between and regulation of the periphery and central nervous system. Recent studies demonstrating modulation of hypothalamic neurotransmitter expression by peripheral metabolic status may yield insights into regulation of appetite and metabolism in obesity and aberrant metabolic homeostasis. This review concentrates on summarizing data regarding regulation of expression of neuropeptide Y and growth hormone-releasing hormone as model peptide systems for addressing questions relating peripheral metabolism and hypothalamic neuropeptide expression.

In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysin from the hypothalamus to the spinal cord

The biosynthesis of oxytocin, vasopressin and their associated neurophysins were studied in the projection from the paraventricular nucleus of the hypothalamus to the spinal cord in individual freely-moving adult male rats. Neuropeptide biosynthesis was studied in vivo by the delivery of [35S]cysteine through stereotaxically implanted indwelling cannulae using an osmotic minipump delivery system. Following the appropriate chase times, the neural lobe and spinal cord segments T1-T4 and T12-L2 were removed from fresh tissue; in addition, the nucleus of the solitary tract was punched from frozen coronal sections. The radiolabeled peptides were purified from the tissue homogenates by sequential linear and exponential gradient elution from reverse-phase high performance liquid chromatography columns. This approach has allowed us to purify radiolabeled oxytocin and vasopressin from both the upper and lower spinal cord. However, the kinetics of oxytocin and vasopressin biosynthesis appeared to be remarkably different, as judged by their differential labeling with different pulse and chase times. Additionally, the use of different chase periods following the pulse of radiolabel has allowed us to determine that oxytocin reaches the spinal cord via the fast component of axonal transport (greater than 8 mm h-1). Using immunoprecipitation and purification by high performance liquid chromatography, we were also able to purify radiolabeled neurophysins from spinal cord tissue homogenates. These results lend further support to a role for oxytocin and vasopressin in the modulation of autonomic nervous system function and to the role of the paraventricular nucleus as an integration center for endocrine and autonomic function.

Limbic seizures increase cyclophilin mRNA levels in rat hippocampus

Limbic seizures lead to dramatic and specific modulation of mRNA levels for many genes in the hippocampus including immediate early, growth factor and neuropeptide genes. In the present study, the influence of hilus lesion (HL)-induced seizures on the abundance of mRNA coding for cyclophilin, a peptide prolyl isomerase, in rat hippocampus was analyzed. By nuclease protection analysis a significant increase in cyclophilin mRNA levels was observed in the hippocampal dentate gyrus/CA1 subfield following HL-induced seizures. The increase began 6 h post-HL, reached a maximum (2.5-fold) at 12 h post-HL and returned to control values by 48 h post-HL. Cyclophilin mRNA levels remained stable in the cerebral cortex throughout the same seizure and post-seizure activity time span.