Keith A. Gary

Altered neuronal nitric oxide synthase expression contributes to disease progression in Huntington's disease transgenic mice

Reduced neuronal NOS (nNOS) expression and biochemical activity was found in the striatum (P<0.05) and cerebellum P<0.05) of late-stage R6/1 Huntingtons disease (HD) mice. The changes in NOS biochemical activity correlated with body weight (P<0.001), abnormal clasping (P<0.05) and motor functioning (P<0.05) scores. HD transgenic mice missing both copies of the nNOS gene showed accelerated disease progression relative to HD transgenic mice wildtype or heterozygous for the nNOS gene. On the other hand, mice with one copy of the nNOS gene had delayed onset of their HD-related symptoms relative to HD transgenic mice wildtype for nNOS. Administration of an iNOS inhibitor had no effect on behavioral progression. The effects of nNOS genotype on behavior may be related to abnormal expression of nNOS during development, which was increased relative to controls in R6/2 mice 3 weeks of age (presymptomatic), but decreased in R6/2 mice relative to controls at 6 (around the time of symptom onset) and 11 (late-stage disease) weeks of age. Finally, protein expression of calmodulin kinase II and IV, both of which are regulators of nNOS transcription and activation, had a pattern of increased expression early in development, and decreased expression late in development, similar to that seen for nNOS. These findings indicate that nNOS activity is altered in a complex manner in HD transgenic mice and suggest that these abnormalities occur in the setting of a more global disturbance of calcium-regulated proteins.

The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary

The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate‐limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post‐natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By post‐natal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post‐natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post‐natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 ± 0.425 ng at PN 4, 86.31 ± 20.42 ng at PN 7, 168.72 ± 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 ± 0.01% to 6.5 ± 0.002% at PN 4, and continued to decrease until a level of 3 ± 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6‐hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 ± 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 ± 0.104 ng; PN 6 [DA] = 11.60 ± 2.24 ng; PN 7 [DA] = 20.93 + 3.80 ng; and PN 10 [DA] = 27.95 ± 3.46 ng. Melanotrope proliferation also increased in 6‐hydroxydopamine‐treated animals. At PN 4, the onset of innervation reduced the pre‐innervation proliferation index to 8.75 ± 0.002%, only a 30% reduction in contrast to the > 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6‐OHDA administration.

Dietary arginine alters time of symptom onset in Huntington's disease transgenic mice.

Recent neuroimaging studies reported complex changes in cerebral blood flow (CBF) in early-staged Huntingtons disease (HD) patients. Deckel and co-workers [Deckel and Duffy, Brain Res. (in press); Deckel and Cohen, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 24 (2000) 193; Deckel et al., Neurology 51 (1998) 1576; Deckel et al., J. Nucl. Med. 41 (2000) 773] suggested that these findings might be accounted for, in part, by alterations in cerebral nitric oxide (NO) and its byproduct, peroxynitrite. The current experiment tested this hypothesis by altering NO levels via manipulations of dietary L-arginine (ARG), the dietary precursor of NO, in mice transgenic for HD. Seventy-one mice were assigned at 12 weeks of age to one of three isocaloric diets that varied in their content of ARG. These diets included: (a) 0% ARG, (b) 1.2% ARG (i.e. typical mouse chow), or (c) 5% ARG. The 5% ARG diets in HD mice accelerated the time of onset of body weight loss (P<0.05) and motor impairments (P<0.05), and increased resting CBF in HD relative to control (P<0.05). Conversely, the 0% ARG diet demonstrated no loss of body weight and had no changes in CBF relative to controls. However, the 0% ARG HD group continued to show significant deficits on motor testing (P<0. 05). The 1.2% ARG HD group showed reduced body weight loss, better motor functioning, and fewer changes in CBF compared to the 5% ARG HD group. Immunocytochemistry analysis found greater deposition of nitrotyrosine in the cortex, and vasculature, of HD+ mice, 5% and 1. 2%>0% arginine diets. When collapsed across all conditions, CBF inversely correlated (P<0.05) both with the body weight and motor changes suggesting that changes in CBF are associated with behavioral decline in HD mice. Collectively, these findings indicate that dietary consumption of the NO precursor ARG has a measurable, but complex, effect on symptom progression in HD transgenic mice, and implicates NO in the pathophysiology of HD.

Glial-like cells of the rat pituitary intermediate lobe change morphology and shift from vimentin to GFAP expression during development

This study demonstrated morphological changes in glial‐like cells of the rat pituitary intermediate lobe during early postnatal development, and a subsequent shift in protein expression from vimentin to GFAP. Vimentin immunoreactivity was detected in the lobe at embryo day 14 and was localized in radially‐oriented, bipolar cells whose processes spanned the thickness of the intermediate lobe. At electron microscopical resolution, processes contained intermediate filaments, cell nuclei were indented while secretory vesicles characteristic of the endocrine cells were not found. Vimentin immunoreactive intensity began to decrease at postnatal day 5. By postnatal day 7, vimentin‐positive, setellate cells were observed, with few radial processes found by day 10. The intensity of vimentin immunoreactivity decrease through day 25. Within the lobe parenchyma, vimentin was localized in glial‐like cells since double‐label immunohistochemistry revealed no colocalization of β‐endorphin and vimentin, or fibronectin and vimentin. Dopamine‐containing axons were in close apposition to vimentin‐positive processes. GFAP immunoreactivity first appeared on postnatal day 20 and, by day 25, stellate cell bodies with three to six extended processes were evident. Cells were primarily distributed in the caudal third of the lobe. The characteristic adult pattern of cell clusters in latero‐dorsal and ventral portions of the lobe was fully established by postnatal day 55. The transition from vimentin to GFAP expression and concurrent morphological changes resemble those described for radial glia during cerebral cortical development.

Melanotrope dopamine D2 receptor isoform expression in the developing rat pituitary

This study measured melanotrope mRNA and protein expression for the dopamine D2 receptor, and its long isoform, in relation to the appearance of dopamine in axons of the postnatal rat pituitary intermediate lobe. At postnatal day 2, prior to the onset of dopaminergic innervation, D2 receptor (D2T) mRNA was expressed heterogeneously in a subpopulation of melanotropes which also expressed the long isoform (DL). The D2L mRNA appeared to be predominant during early postnatal development, since the D2T probe, which did not discriminate between the isoforms, and the D2L probe hybridized generally to the same cells, as demonstrated in serial sections. Immunohistochemical methods, using two different antisera for the D2T receptor, however, indicated a low level of protein in most melanotropes. Localization of D2L protein corresponded well to D2L receptor mRNA distribution. At day 10, representing a time when dopamine is present in axons throughout the lobe, both D2T receptor mRNA and protein were detected in a significantly larger population of melanotropes than those expressing D2L mRNA and protein. This suggests the appearance of detectable short isoform (D2S) mRNA in virtually all melanotropes and implicates dopamine as a possible signal for increasing D2S isoform mRNA expression.

Transient expression of S-100 by melanotropes of the rat pituitary intermediate lobe during development.

S‐100, an acidic calcium‐binding protein, is co‐localized with vimentin in glial‐like cells in the adult rat pituitary intermediate lobe. S‐100 and melanotrope markers were not co‐localized in the adult. During development, S‐100 and vimentin were not co‐localized but appeared in cells with different morphological characteristics. S‐100 was co‐localized with POMC mRNA and β‐endorphin during prenatal time and the first three postnatal weeks. This was demonstrated by double‐label immunohistochemistry, using combinations of antisera against S‐100, vimentin and β‐endorphin, and in situ hybridization histochemistry for POMC mRNA combined with immunohistochemistry for S‐100. In the second and third weeks of postnatal development, S‐100 was observed in fewer melanotropes and more frequently in stellate cells, which also expressed vimentin. Thus, S‐100 appeared to be transiently expressed in melanotropes during prenatal and early postnatal development. S‐100 serves as a neurotrophic and glial maturation factor in the CNS. Since S‐100 expression in melanotropes coincides with the onset of dopaminergic innervation and morphological changes in glial‐like cells of the lobe, it could have similar functions in the rat pituitary intermediate lobe.

Differential innervation of individual melanotropes suggests a role for nonsynaptic inhibitory regulation of the developing and adult rat pituitary intermediate lobe

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense‐cored vesicles; membrane specializations were not well‐developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to “parasynaptic” [Schmitt (1984) Neuroscience, 13:991–1001] or “volume” [Agnati et al. (1995) Neuroscience, 69:711–726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles.

Thyrotropin-releasing hormone phase shifts circadian rhythms in hamsters

The role of thyrotropin-releasing hormone (TRH) in regulating circadian rhythms was investigated by assessing the ability of TRH microinjections into the suprachiasmatic nucleus (SCN) to induce phase shifts in hamster wheel-running behavior. TRH injected into the SCN at 10 and 100 nM doses produced phase advances in wheel-running activity of 18.3 +/- 1.9 and 34.8 +/- 2.9 minutes, respectively, when administered at circadian time (CT) 6. Injections at CT 18 produced no effects. The temporal sensitivity of the SCN to TRH administration was examined by administering TRH at specific circadian times. TRH produced significant phase advances at CT 4, 6, and 8, while no significant changes in wheel-running onset were observed at other CT times. These studies represent the first evidence of TRHs ability to affect circadian function.