Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: Regulation by NGF

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.     

Nitric oxide production in the basal forebrain is required for recovery sleep

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 +/- 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), completely abolished non-rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L-NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.     

NADPH-Diaphorase reactivity and neurovascular coupling in the basal forebrain and motor cortex

Using NADPH-diaphorase histochemistry, distribution of reactive neurons in the forebrain structures and motor cortex of rats was studied. Some reactive (NO-generating) neurons are associated with microvessels and, thus, can be involved in the regulation of regional blood flow. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 405–407, July–October, 2007.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Transforming growth factor-α expands progenitor cells of the basal forebrain,but does not promote cholinergic differentiation

Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, binds to the EGF-receptor (EGF-R). The early expression and widespread distribution of TGF-α and EGF-R in the developing central nervous system (CNS) suggest that TGF-α may play a role in the developing CNS. To study possible effects of TGF-α on cholinergic differentiation in the basal forebrain, we cultured septal nuclei with adjacent basal forebrain from embryonic rat brain in the presence and absence of TGF-α. At the highest dose of TGF-α used (100 ng/mL), activity of choline acetyltransferase (ChAT; EC 2.3.1.6) and the number of cholinergic neurons doubled. However, because protein levels tripled, specific ChAT activity actually declined. To determine the mechanism accounting for the increase in ChAT, we labeled dividing precursors present in the cultures with a replication-deficient retrovirus expressing β-galactosidase in the presence and absence of TGF-α. By staining the cultures for both LacZ and ChAT, we determined that the precursor population expanded in size (individually labeled clones contained more cells), but the percentage of cholinergic neurons present in the clones was unchanged. Therefore, while TGF-α expands the precursor pool, it does not promote cholinergic differentiation. Interleukin-9, included to prompt neuronal differentiation, did not by itself increase ChAT activity, nor did it enhance the action of TGF-α. This was true even when basic fibroblast growth factor was included. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 405–412, 1998     

Effects of Ginkgolide on the development of NOS and AChE positive neurons in the embryonic basal forebrain

Extract of Ginkgo biloba (EGb) has been therapeutically used for several decades to increase peripheral and cerebral blood flow so as to prevent cardiovascular and neurovascular diseases. However, the role of EGb in neuroprotective effects has received much attention recently. In this study, we investigated the effect of EGb on the development of NOS and AChE positive neurons in the rat embryonic basal forebrain. The results showed that treated with EGb, the OD of MTT staining analysis, and the numbers, the cell sizes and circumferences of NOS and AChE positive neurons were greatly promoted. These data suggest that EGb had similar effects of the neurotrophins such as NGF and BDNF in promoting the development of NOS and AChE positive neurons in the rat embryonic basal forebrain.     

The time course of adenosine, nitric oxide (NO) and inducible NO synthase changes in the brain with sleep loss and their role in the non-rapid eye movement sleep homeostatic cascade

Both adenosine and nitric oxide (NO) are known for their role in sleep homeostasis, with the basal forebrain (BF) wakefulness center as an important site of action. Previously, we reported a cascade of homeostatic events, wherein sleep deprivation (SD) induces the production of inducible nitric oxide synthase (iNOS)-dependent NO in BF, leading to enhanced release of extracellular adenosine. In turn, increased BF adenosine leads to enhanced sleep intensity, as measured by increased non-rapid eye movement sleep EEG delta activity. However, the presence and time course of similar events in cortex has not been studied, although a frontal cortical role for the increase in non-rapid eye movement recovery sleep EEG delta power is known. Accordingly, we performed simultaneous hourly microdialysis sample collection from BF and frontal cortex (FC) during 11 h SD. We observed that both areas showed sequential increases in iNOS and NO, followed by increases in adenosine. BF increases began at 1 h SD, whereas FC increases began at 5 h SD. iNOS and Fos-double labeling indicated that iNOS induction occurred in BF and FC wake-active neurons. These data support the role of BF adenosine and NO in sleep homeostasis and indicate the temporal and spatial sequence of sleep homeostatic cascade for NO and adenosine.     

Gene expression in lung and basal forebrain during influenza infection in mice

Inbred mice develop strain-dependent changes in sleep during the first few days after inoculation with influenza virus. To identify genes with the potential to differentially modulate sleep under this condition, we performed complementary DNA microarray analysis of both lung and basal forebrain (BF) of infected (I) and uninfected BALB/cByJ (C) and C57BL/6J (B6) mice. This analysis showed significant variation in the expression pattern of 667 and 1217 of the surveyed genes in BF and lung, respectively ( P  < 0.01). Applying the additional criterion of an effect size ≥2, 495 genes differed in expression in lung compared with 204 in BF. In BF, more genes were differentially expressed as a function of mouse strain, whereas in lung, more genes were differentially expressed as a function of health status. Significant alterations in expression after infection were more numerous and robust in BALB/cByJ vs. C57BL/6J mice. Some genes showed significant variation in both tissues as a function of strain or condition, but the changes in general were not parallel. Genes that showed significant and robust variation as a function of strain, health status or tissue included those related to immune function, metabolism, signal transduction, cell cycle regulation, apoptosis and other miscellaneous categories. Different patterns of gene expression in BF of uninfected mice suggest the possibility of fundamental mechanistic differences in pathways that modulate vigilance in these strains, whereas differences in expression of lung of infected mice suggests different peripherally generated sleep-modulatory stimuli in the two strains.     

Basal forebrain cholinergic system in the dementias: Vulnerability,resilience, and resistance

The basal forebrain cholinergic neurons (BFCN) provide the primary source of cholinergic innervation of the human cerebral cortex. They are involved in the cognitive processes of learning, memory, and attention. These neurons are differentially vulnerable in various neuropathologic entities that cause dementia. This review summarizes the relevance to BFCN of neuropathologic markers associated with dementias, including the plaques and tangles of Alzheimer's disease (AD), the Lewy bodies of diffuse Lewy body disease, the tauopathy of frontotemporal lobar degeneration (FTLD-TAU) and the TDP-43 proteinopathy of FTLD-TDP. Each of these proteinopathies has a different relationship to BFCN and their corticofugal axons. Available evidence points to early and substantial degeneration of the BFCN in AD and diffuse Lewy body disease. In AD, the major neurodegenerative correlate is accumulation of phosphotau in neurofibrillary tangles. However, these neurons are less vulnerable to the tauopathy of FTLD. An intriguing finding is that the intracellular tau of AD causes destruction of the BFCN, whereas that of FTLD does not. This observation has profound implications for exploring the impact of different species of tauopathy on neuronal survival. The proteinopathy of FTLD-TDP shows virtually no abnormal inclusions within the BFCN. Thus, the BFCN are highly vulnerable to the neurodegenerative effects of tauopathy in AD, resilient to the neurodegenerative effect of tauopathy in FTLD and apparently resistant to the emergence of proteinopathy in FTLD-TDP and perhaps also in Pick's disease. Investigations are beginning to shed light on the potential mechanisms of this differential vulnerability and their implications for therapeutic intervention.

     

Age-related changes of hypocretin in basal forebrain of guinea pig

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) have been implicated in a wide variety of functions including sleep and wakefulness as well as related behaviors. Many of these functions of the hypocretins involve the activation of cholinergic neurons in the basal forebrain (BF). These neurons have been shown to exhibit age-related changes in a variety of species. In the present experiment, in adult and aged guinea pigs, we compared hypocretin immunoreactivity in regions of the BF that include the medial septal nucleus (MS), the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB) and the magocellular preoptic nucleus (MCPO). In adult guinea pigs (3–5 months of age), all of the preceding BF regions contained dense hypocretin fibers with varicosities. On the contrary, in old guinea pigs (27–28 months), although the MS exhibited a similar intensity of hypocretin immunoreactivity compared with the adult guinea pig, there was a significant decrease in the intensity of immunoreactivity of hypocretinergic fibers in the VDB, HDB and MCPO. These data indicate that the hypocretinergic innervation of specific nuclei of the BF is compromised during the aging process. We suggest that the reduction in hypocretinergic innervation of the BF nuclei may contribute to the age-related changes in the states of sleep and wakefulness as well as deficits in related systems that occur in old age.     

The pre‐synaptic Munc13‐1 binds alcohol and modulates alcohol self‐administration in Drosophila

Munc13‐1 is a pre‐synaptic active‐zone protein essential for neurotransmitter release and involved in pre‐synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13‐1 with EC₅₀s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13‐1 C1 exclusively at Glu‐582, which was identified by mass spectrometry. Mutation of Glu‐582 to alanine, leucine, and histidine reduced the alcohol binding two‐ to five‐fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild‐type Munc13‐1 compared with the mutants. If Munc13‐1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss‐of‐function mutation in the conserved Dunc‐13 in Drosophila melanogaster. The Dunc‐13^P84200/+ heterozygotes have 50% wild‐type levels of Dunc‐13 mRNA and display a very robust increase in ethanol self‐administration. This phenotype is reversed by the expression of the rat Munc13‐1 protein within the Drosophila nervous system. The present studies indicate that Munc13‐1 C1 has binding site(s) for alcohols and Munc13‐1 activity is sufficient to restore normal self‐administration to Drosophila mutants deficient in Dunc‐13 activity.

     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Conditional cell ablation via diphtheria toxin reveals distinct requirements for the basal plate in the regional identity of diencephalic subpopulations

The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1–p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen‐ induced, Cre‐mediated recombination of the ROSA^DTA allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate‐deficient embryos, the p1 alar plate exhibited reduced expression of subtype‐specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments. genesis 53:356–365, 2015. © 2015 Wiley Periodicals, Inc.