Impact of aging on heat shock protein expression in the substantia nigra and striatum of the female rat.

Many heat shock proteins are chaperones that help refold or degrade misfolded proteins and battle apoptosis. Because of their capacity to protect against protein misfolding, they may help keep diseases of aging at bay. A few reports have examined heat shock proteins (eg. Hsp25, Hsp60, Hsp70, and heat shock cognate 70 or Hsc70) as a function of age in the striatum and nigra. In the present study, we examined the impact of aging on Hsp25, heme oxygenase 1 (HO1 or Hsp32), Hsp40, Hsp60, Hsc70, Hsc/Hsp70 interacting protein (Hip), 78 kDa glucose-regulated protein (GRP78), Hsp90, and ubiquitinated proteins in the nigra and striatum of the female rat by infrared immunoblotting. Female animals are not typically examined in aging studies, adding further to the novelty of our study. Striatal HO1 and Hsp40 were both higher in middle-aged females than in the oldest group. Hsp60 levels were also highest in middle age in the nigra, but were highest in the oldest animals in the striatum. Striatal levels of Hsc70 and the co-chaperone Hip were lower in the oldest group relative to the youngest animals. In contrast, Hsp25 rose with advancing age in both regions. Hsp25 was also colocalized with tyrosine hydroxylase in nigral neurons. Ubiquitinated proteins exhibited a trend to rise in the oldest animals in both regions, and K48 linkage-specific ubiquitin rose significantly from 4-6 to 16-19 months in the striatum. Our study reveals a complex array of age-related changes in heat shock proteins. Furthermore, the age-related rises in some proteins, such as Hsp25, may reflect endogenous adaptations to cellular stress.

Topographic organization of suprachiasmatic nucleus projection neurons.

The mammalian circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), has two subdivisions. The core is located above the optic chiasm, receives primary and secondary visual afferents, and contains neurons producing vasoactive intestinal polypeptide and gastrin-releasing peptide. The shell largely surrounds the core, receives input from nonvisual sources and contains neurons producing arginine vasopressin and calretinin. In this study, we tested the hypothesis that SCN efferent projections are topographically organized with respect to the subdivision of origin. Injections of retrograde tracers were placed in major sites of efferent termination, described from prior studies that used anterograde tracers (Watts and Swanson, [1987] J. Comp. Neurol. 258:230-252; Watts et al. [1987] J. Comp. Neurol. 258:204-229). After retrograde tracer injections in the medial preoptic area, dorsomedial and paraventricular hypothalamic nuclei, bed nucleus of stria terminalis, paraventricular thalamic nucleus, zona incerta, and medial subparaventricular zone, retrogradely labeled SCN cells are clustered in the shell with few labeled neurons in the core. After injections centered in the lateral subparaventricular zone, peri-suprachiasmatic region, lateral septum, or ventral tuberal area, the majority of neuronal label is in the core with moderate to sparse neuronal label in the shell. Both subdivisions are labeled after injections in the paratenial thalamic nucleus. The same pattern of retrograde labeling is found with four tracers, cholera toxin-beta subunit, Fluoro-Gold, the Bartha strain of pseudorabies virus, and biotinylated dextran amine. These data extend our understanding of the significance of the division of the SCN into shell and core by demonstrating that the subdivisions differ in the pattern of projections. Together with prior observations that the subdivisions differ with respect to afferents, local connections, and neuroactive substances, the present study provides an anatomic basis for discrete control of circadian function by the SCN core and shell. In this novel view, the nature of the signal conveyed to areas receiving core or shell projections varies as a function of the subdivision from which innervation is derived.

Detection and transduction of daylength in birds.

Daylength is an important environmental cue used by temperate zone avian species to time the onset of seasonal reproductive activity. Photic cues are detected by extra-retinal, extra-pineal central nervous system elements, and are rapidly transduced to an efferent signal. In this paper, we describe the brain locus of putative encephalic photoreceptors in birds, and explore the pathway of information transfer from photic input to the reproductive axis. To this end, we examine how photoreceptors might communicate with the hypothalamic-pituitary axis, and how brain peptides vary seasonally. Recent studies indicate that brain photoreceptors lie in the lateral septum and in the tuberal hypothalamus, and co-express proteins characteristic of retinal photoreceptors, as well as vasoactive-intestinal polypeptide (VIP). At the light microscopic level, photoreceptor cells appear to communicate with gonadotropin-releasing hormone (GnRH) neurons, and vice versa. Expression of VIP-like immunoreactivity is highest in photorefractory animals while GnRH-like immunoreactivity is highest in photosensitive birds. Expression of these CNS peptides is correlated with changes in plasma prolactin and luteinizing hormone (LH), suggesting a mechanism mediating seasonal cyclicity.

The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems.

The suprachiasmatic nucleus (SCN) temporally organizes behavior in part by sustaining arousal during the wake period of the sleep/wake cycle to consolidate adaptive waking behavior. In this study, we demonstrate direct projections from the SCN, in both the rat and the human brains, to perikarya and proximal dendrites of two groups of posterior hypothalamic neurons with axonal projections that suggest they are important in the regulation of arousal, one producing hypocretins (HCT) and the other melanin-concentrating hormone (MCH). In addition, we demonstrate that both HCT and MCH-producing neurons are immunoreactive for glutamate (GLU). These observations support the hypothesis that direct projections from the SCN to the posterior hypothalamus mediate the arousal function of the circadian timing system.

Calbindin-D28K cells in the hamster SCN express light-induced Fos.

Although the suprachiasmatic nuclei (SCN) have been intensively analyzed, they contain a population of cells that has not yet been characterized. In this study, we examined the distribution of cells immunoreactive (ir) for calbindin-D28K (CaBP), calretinin (CR), parvalbumin, vasopressin-associated neurophysin (NP), substance P (SP), vasoactive intestinal peptide (VIP), and light-induced Fos-like protein. Previously unidentified cells in the core of the hamster SCN contained CaBP. Photic stimulation during the night induced Fos expression in about 75% of the CaBP-positive SCN cells, and about 50% of the Fos-positive cells in the core region expressed CaBP. These findings provide new information in the search for the cellular localization of pacemaker cells in the SCN, as photic input entrains the circadian system, and cells that receive photic input must be either part of the clock itself, or an upstream component of the clock.

Identification of retinal ganglion cells projecting to the lateral hypothalamic area of the rat.

The objective of the present study was to identify the retinal ganglion cells projecting to the lateral hypothalamic area of the rat. The retinohypothalamic tract has been divided into a medial and a lateral component on anatomical and developmental grounds. The medial component projects to the suprachiasmatic nucleus and adjacent structures such as the anterior hypothalamic and retrochiasmatic areas. The lateral component terminates in the lateral hypothalamic are dorsal to the supraoptic nucleus. Injections of the retrograde tracer FluoroGold were made into the retinorecipient region of the lateral hypothalamic area and retinal whole mounts were immunohistochemically processed for retrogradely labeled retinal ganglion cells. With FluoroGold injections confined to the lateral hypothalamic area, retrogradely labeled retinal ganglion cells are located almost exclusively in the superior temporal quadrant of the retina. Their size and morphology indicates that they are a homogeneous subset of type III cells, but a definitive classification would require a more complete fill of dendritic arbors than is available in our retrograde material. In contrast, injections involving fibers of passage in the optic tract, or centered in the medial terminal nucleus of the accessory optic system, label cells distributed across the entire retinal surface. Unlike the retinal ganglion cells projecting to the suprachiasmatic nucleus [Moore et al., J. Comp. Neurol., 352 (1995) 351-366], the cells labeled after restricted lateral hypothalamic injections are not distributed evenly across the retinal surface. The difference in location of the retinal ganglion cells projecting to the lateral hypothalamic area supports the view that this retinohypothalamic projection is anatomically and functionally distinct from the projection to the suprachiasmatic nucleus and adjacent medial hypothalamus.

Suprachiasmatic pacemaker organization analyzed by viral transynaptic transport.

The suprachiasmatic nucleus (SCN) of the hypothalamus, the principal circadian pacemaker, is a paired structure with two subdivisions, a ventral core receiving photic input and a dorsal shell receiving non-photic input. Rhythmicity is thought to be generated by individual SCN neurons which are coupled to achieve synchrony [D.K. Welsh, D.E. Logothetis, M. Meister, S.M. Reppert, Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing patterns, Neuron, 14 (1995) 697-706]. Normally, the core and shell, and the nuclei on each side, act in unison to transmit rhythmicity to effector systems. It is not known how coupling between neurons in the two subdivisions, and between the two SCNs, takes place. In the present study, we analyze the intrinsic, commissural, and efferent projections of the SCN using the swine herpesvirus (pseudorabies virus, PRV) as a tool for transynaptic analysis of circuits and small iontophoretic injections of the conventional tracer horseradish peroxidase (HRP) conjugated to fluorescein. We find that the core and shell each project through commissural efferents to homologous contralateral areas. The core projects densely to shell but we find little reciprocal innervation. The two subdivisions project to different hypothalamic areas, with the core projecting to the lateral subparaventricular zone and shell to the dorsomedial hypothalamic nucleus and medial subparaventricular zone. These data are the first demonstration that connections within the SCN, and from the SCN to effector regions, are topographically organized and lend insight into the flow of information through and out of the pacemaker.

Transgenic overproduction of omega-3 polyunsaturated fatty acids provides neuroprotection and enhances endogenous neurogenesis after stroke.

Strokes are devastating as there are no current therapies to prevent the long term neurological deficits that they cause. Soon after ischemic stroke, there is proliferation and differentiation of neural stem/progenitor cells as an important mechanism for neuronal restoration. However, endogenous neurogenesis by itself is insufficient for effective brain repair after stroke as most newborn neurons do not survive. One fascinating strategy for stroke treatment would thus be maintaining the survival and/or promoting the differentiation of endogenous neural stem/progenitor cells. Using transgenic (Tg) mice over-expressing the C. elegans fat-1 gene encoding an enzyme that converts endogenous omega-6 to omega-3 polyunsaturated fatty acids (n-3 PUFAs), we showed that fat-1 Tg mice with chronically elevated brain levels of n-3 PUFAs exhibited less brain damage and significantly improved long-term neurological performance compared to wild type littermates. Importantly, post-stroke neurogenesis occurred more robustly in fat-1 Tg mice after focal ischemia. This was manifested by enhanced neural stem cell proliferation/differentiation and increased migration of neuroblasts to the ischemic sites where neuroblasts matured into resident neurons. Moreover, these neurogenic effects were accompanied by significantly increased oligodendrogenesis. Our results suggest that n-3 PUFA supplementation is a potential neurogenic and oligodendrogenic treatment to naturally improve post-stroke brain repair and long-term functional recovery.

N-Acetyl cysteine blunts proteotoxicity in a heat shock protein-dependent manner.

N-Acetyl cysteine, a glutathione precursor, has been shown to benefit patients with Alzheimer's disease and reduce the symptoms of traumatic brain injury in soldiers. Parkinson's and Alzheimer's disease are both characterized by stress from protein misfolding, or proteotoxicity. We have developed a high-throughput model of proteotoxicity by treating neuroblastoma N2a cells with the proteasome inhibitor MG132 and performing three independent assays for viability. Our previous study showed that N-acetyl cysteine protects N2a cells against two sequential treatments of MG132 and raises glutathione levels in a two-hit model of synergistic neurodegeneration. In the present study, however, N-acetyl cysteine was found to reduce the toxicity of a single hit of MG132 independent of its effect on glutathione. All three viability assays confirmed this protection. We measured heat shock protein 70 (Hsp70) levels because Hsp70 is a protective chaperone that helps refold proteins or guides ubiquitinated proteins toward degradation by the proteasome. Hsp70 levels were higher in MG132-treated cells when N-acetyl cysteine was applied. No parallel change in heat shock cognate 70 (Hsc70) was elicited. Inhibition of Hsp70/Hsc70 activity with VER 155008 attenuated the protection afforded by N-acetyl cysteine in a dose-responsive manner. MG132 induced a large rise in ubiquitinated proteins and N-acetyl cysteine reduced this effect. Consistent with the chaperone functions of Hsp70, VER 155008 also prevented the reduction in ubiquitin-conjugated proteins by N-acetyl cysteine. These data reveal a new role for N-acetyl cysteine: this compound may reduce misfolded protein levels and ameliorate proteotoxicity through heat shock proteins. These findings broaden the potential mechanisms of action for this dietary supplement in neurodegenerative proteinopathies.

Pharmaceutical development of ondansetron tablets.

Ondansetron tablets contain ondansetron base as the hydrochloride dihydrate, lactose, microcrystalline cellulose, starch and magnesium stearate. Tablets sampled at the beginning and end of the compression process have good content uniformity and drug content, showing that there is no segregation or loss of the drug substance during tabletting. The release of drug substance is related to the tablet disintegration time. Tablets with disintegration times of 3 and 10 min release 85% of the drug substance in approximately 6 and 20 min respectively. Satisfactory bioavailability has been demonstrated. The tablets have good stability, and have a shelf life of 2 years when stored below 30 degrees C.

Pharmaceutical development of ondansetron injection.

Ondansetron injection is an aqueous solution containing ondansetron base as the hydrochloride dihydrate. The pH of the injection was selected to achieve good physical and chemical stability. The shelf life is 3 years when stored below 30 degrees C, protected from light. Ondansetron injection may be diluted for administration by slow intravenous injection or infusion and is compatible with several intravenous infusion fluids. In addition, specific concentrations of cisplatin, 5-fluorouracil, carboplatin, etoposide, ceftazidime, cyclophosphamide and doxorubicin are compatible when administered via a giving set delivering ondansetron by infusion.