Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

Brain-Wide Maps of Synaptic Input to Cortical Interneurons.

Cortical inhibition is mediated by diverse inhibitory neuron types that can each play distinct roles in information processing by virtue of differences in their input sources, intrinsic properties, and innervation targets. Previous studies in brain slices have demonstrated considerable cell-type specificity in laminar sources of local inputs. In contrast, little is known about possible differences in distant inputs to different cortical interneuron types. We used the monosynaptic rabies virus system, in conjunction with mice expressing Cre recombinase in either parvalbumin-positive, somatostatin-positive (SST+), or vasoactive intestinal peptide-positive (VIP+) neurons, to map the brain-wide input to the three major nonoverlapping classes of interneurons in mouse somatosensory cortex. We discovered that all three classes of interneurons received considerable input from known cortical and thalamic input sources, as well as from probable cholinergic cells in the basal nucleus of Meynert. Despite their common input sources, these classes differed in the proportion of long-distance cortical inputs originating from deep versus superficial layers. Similar to their laminar differences in local input, VIP+ neurons received inputs predominantly from deep layers while SST+ neurons received mostly superficial inputs. These classes also differed in the amount of input they received. Cortical and thalamic inputs were greatest onto VIP+ interneurons and smallest onto SST+ neurons. SIGNIFICANCE STATEMENT: These results indicate that all three major interneuron classes in the barrel cortex integrate both feedforward and feedback information from throughout the brain to modulate the activity of the local cortical circuit. However, differences in laminar sources and magnitude of distant cortical input suggest differential contributions from cortical areas. More input to vasoactive intestinal peptide-positive (VIP+) neurons than to somatostatin-positive (SST+) neurons suggests that disinhibition of the cortex via VIP+ cells, which inhibit SST+ cells, might be a general feature of long-distance corticocortical and thalamocortical circuits.

Stress alters asenapine-induced Fos expression in the Meynert's nucleus: response of adjacent hypocretin and melanin-concentrating hormone neurons in rat.

OBJECTIVE: Asenapine (ASE), an atypical antipsychotic drug used in the treatment of schizophrenia, induces Fos expression in forebrain. Effect of ASE on activity of basal nucleus of Meynert (NBM) cells, a part of the striatal-cortical circuits, was studied. We were also interested to reveal whether a chronic unpredictable variable mild stress (CMS) preconditioning might affect the ASE impact. METHODS: Rats were divided into as follows: controls-vehicle, controls-ASE, stressed-vehicle and stressed-ASE groups. CMS included restrain, social isolation, crowding, swimming and cold applied for 21 days. On the 22nd day, rats were subcutaneously injected with ASE (0.3 mg/kg) or vehicle (saline 300 µl/rat), 90 min prior euthanizing. After transcardial fixation, brains were cut into 30 µm thick coronal sections. Fos protein presence, as indicator of cell activity, was detected by ABC immunohistochemistry. Hypocretin (Hcrt) and melanin-concentrating hormone (MCH) containing cells were visualized with fluorescent dyes. RESULTS: ASE induced significant increase in Fos expression in NBM in both controls and CMS preconditioned rats in comparison with the related vehicle-treated controls. CMS preconditioning, however, significantly lowered the Fos response to ASE in NBM. From Hrct and MCH cells, only Hcrt ones displayed Fos presence in response to ASE. DISCUSSION: This study demonstrates for the first time that ASE may target a special group of cells occupying NBM, which effect can be modulated by CMS preconditioning. This finding extends a view that ASE impact may extend beyond the classical forebrain target areas common for the action of all antipsychotics and might be helpful in the identification of sites and side effects of its therapeutic actions.

Distribution of osmoregulatory peptides and neuronal-glial configuration in the hypothalamic magnocellular nuclei of desert rodents.

The desert rodents Psammomys obesus and Gerbillus tarabuli live under extreme conditions and overcome food and water shortage by modes of food and fluid intake specific to each species. Using immunohistochemistry and electron microscopy, we found that the hypothalamic magnocellular nuclei, and in particular, their vasopressinergic component, is highly and similarly developed in Psammomys and Gerbillus. In comparison to other rodents, the hypothalamus in both species contains more magnocellular VP neurons that, together with oxytocin neurons, accumulate in distinct and extensive nuclei. As in dehydrated rodents, many magnocellular neurons contained both neuropeptides. A striking feature of the hypothalamic magnocellular system of Psammomys and Gerbillus was its display of ultrastructural properties related to heightened neurosecretion, namely, a significant reduction in glial coverage of neuronal somata and dendrites in the hypothalamic nuclei. There were many neuronal elements whose surfaces were directly juxtaposed and shared the same synapses. Their magnocellular nuclei also showed a high level of sialylated isoform of the Neural Cell Adhesion Molecule (PSA-NCAM) that underlies their capacity for neuronal and glial plasticity. These species thus offer striking models of structural neuronal and glial plasticity linked to natural conditions of heightened neurosecretion.

Induction of c-Fos expression by electrical stimulation of the nucleus basalis magnocellularis.

The present study examined the expression of the immediate-early gene c-fos in different brain regions following a single 20-min session of unilateral electrical stimulation of the nucleus basalis magnocellularis (NBM). Current findings confirm that NBM stimulation provides specific activation of several cortical and subcortical regions closely related to the NBM and involved in learning and memory processes, such as the cingulate, parietal, piriform and perirhinal cortices, dorsal subiculum, and the parafascicular, central lateral and central medial nuclei of the thalamus. In contrast, NBM stimulation did not increase c-Fos expression in some expected areas that receive direct NBM projections such as the entorhinal cortex or amygdala nuclei. Results are discussed in terms of the possibility that NBM electrical stimulation facilitates learning by inducing neural changes related to transcription factors such as c-Fos.

The influence of chronic lithium administration on deafferentation-induced cellular changes in the chick cochlear nucleus.

The avian brainstem serves as a useful model system to address the question of how afferent activity influences viability of target neurons. Approximately 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e. deafness produced by cochlea removal). Previous studies have identified cellular events that occur within hours following cochlea removal, which are thought to lead to the ultimate death of NM neurons. We have recently shown that chronic lithium treatment increases neuronal survival following deafferentation. To assess where in the cell death cascade lithium is having its effect, we evaluated some of the early deafferentation-induced cellular changes in NM neurons. Lithium did not affect deafferentation-induced changes that occur across the entire population of NM neurons. There were still deafferentation-induced increases in intracellular calcium concentrations and early changes in the ribosomes, as indicated by Y10b immunolabeling. Lithium did, however, affect changes that are believed to be indicative of the subpopulation of NM neurons that will eventually die. Ribosomes recovered in all of the deafferented NM neurons (as assessed by Y10b labeling) by 10 h following cochlea removal in subjects pretreated with lithium, while a subpopulation of the NM neurons in saline-treated subjects showed dramatic reduction in Y10b labeling at that time. Lithium treatment also prevented the robust upregulation of b cell leukemia/lymphoma-2 (Bcl-2) mRNA that is observed in a subpopulation of deafferented NM neurons 6 h following cochlea removal.

Estrogen enhances expression of the complement C5a receptor and the C5a-agonist evoked calcium influx in hormone secreting neurons of the hypothalamus.

In the present study we examined presence of the complement C5a receptor (C5aR) in hypothalamic neurosecretory neurons of the rodent brain and effect of estrogen on C5aR expression. Whole cell patch clamp measurements revealed that magnocellular neurons in the supraoptic and paraventricular nuclei of hypothalamic slices of the rats responded to the C5aR-agonist PL37-MAP peptide with calcium ion current pulses. Gonadotropin-releasing hormone (GnRH) producing neurons in slices of the preoptic area of the mice also reacted to the peptide treatment with inward calcium current. PL37-MAP was able to evoke the inward ion current of GnRH neurons in slices from ovariectomized animals. The amplitude of the inward pulses became higher in slices obtained from 17beta-estradiol (E2) substituted mice. Calcium imaging experiments demonstrated that PL37-MAP increased the intracellular calcium content in the culture of the GnRH-producing GT1-7 cell line in a concentration-dependent manner. Calcium imaging also showed that E2 pretreatment elevated the PL37-MAP evoked increase of the intracellular calcium content in the GT1-7 cells. The estrogen receptor blocker Faslodex in the medium prevented the E2-evoked increase of the PL37-MAP-triggered elevation of the intracellular calcium content in the GT1-7 cells demonstrating that the effect of E2 might be related to the presence of estrogen receptor. Real-time PCR experiments revealed that E2 increased the expression of C5aR mRNA in GT1-7 neurons, suggesting that an increased C5aR synthesis could be involved in the estrogenic modulation of calcium response. These data indicate that hypothalamic neuroendocrine neurons can integrate immune and neuroendocrine functions. Our results may serve a better understanding of the inflammatory and neurodegeneratory diseases of the hypothalamus and the related neuroendocrine and autonomic compensatory responses.

Activation of a G protein-coupled inwardly rectifying K+ current and suppression of Ih contribute to dexmedetomidine-induced inhibition of rat hypothalamic paraventricular nucleus neurons.

BACKGROUND: Alpha2-adrenoceptor agonist has been reported to produce inhibition of arginine vasopressin release, diuresis, and sympatholytic effects. However, its mechanisms of central action remain incompletely understood. Hypothalamic paraventricular nucleus (PVN) neurons, which are in direct contact with noradrenergic synapses and are controlled by the hyperpolarization-activated currents, are called Ih (H current). The effect of dexmedetomidine, a highly selective and potent agonist, at alpha2 adrenoceptors on Ih is unknown. The purpose of this study was to examine the effects of dexmedetomidine on the PVN neuron, which is involved in the arginine vasopressin release and autonomic regulation. METHODS: The authors investigated the effects of dexmedetomidine on the membrane properties in PVN magnocellular neurons and an Ih in PVN parvocellular neurons with a whole cell patch clamp technique using a rat brain slice preparation. RESULTS: Dexmedetomidine dose-dependently hyperpolarized PVN magnocellular neurons. In the voltage clamp mode, dexmedetomidine induced an outward current, with a reversal potential of -94 mV, and this was shown to depend on the external concentration of K. Pretreatment with Ba or peptide toxin tertiapin blocked hyperpolarization induced by dexmedetomidine. The effect of dexmedetomidine was blocked by an alpha2-adrenoceptor antagonist, yohimbine. Ih was suppressed dose dependently by dexmedetomidine in PVN parvocellular neurons. Pretreatment with Cs occluded the Ih suppression by dexmedetomidine. Yohimbine blocked the Ih suppression by dexmedetomidine. The Ih sensitive to dexmedetomidine was weakly modulated by intracellular cyclic adenosine monophosphate. CONCLUSIONS: Dexmedetomidine inhibited PVN magnocellular neurons by activation of the G protein-coupled inwardly rectifying K current and inhibited PVN parvocellular neurons by suppression of Ih.

Increased metabolic activity in nucleus basalis of Meynert neurons in elderly individuals with mild cognitive impairment as indicated by the size of the Golgi apparatus.

In this study, we examined the metabolic activity of nucleus basalis of Meynert (NBM) neurons in individuals clinically diagnosed with no cognitive impairment (NCI, n = 8), mild cognitive impairment (MCI, n = 9), and subjects with moderate Alzheimer disease (AD, n = 7). We used Golgi apparatus (GA) size as a measure of neuronal metabolic activity. Subjects with MCI showed increased NBM metabolic activity; they had significantly more neurons with larger GA size as compared with NCI and AD subjects. In contrast, more NBM neurons with extremely small GA sizes, indicating reduced metabolic activity, were seen in AD. When these cases were classified according to their AD pathology (Braak I-II, III-IV, or V-VI), Braak III-IV subjects showed significantly increased GA sizes, comparable with the increase in clinically diagnosed MCI, whereas in Braak V-VI, GA sizes were dramatically reduced. Of all MCI and NCI subjects with similar Braak III-IV pathology, the MCI subjects again had significantly larger GA sizes. The larger NBM neuronal GA size seen in MCI suggests increased metabolic activity, associated with both the clinical progression from NCI to MCI, and with the early stages of AD pathology.

Neuroanatomical approaches of the tectum-reticular pathways and immunohistochemical evidence for serotonin-positive perikarya on neuronal substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear-induced analgesia.

Deep layers of the superior colliculus, the dorsal periaqueductal gray matter and the inferior colliculus are midbrain structures involved in the generation of defensive behavior and fear-induced anti-nociception. Local injections of the GABA(A) antagonist bicuculline into these structures have been used to produce this defense reaction. Serotonin is thought to be the main neurotransmitter to modulate such defense reaction in mammals. This study is the first attempt to employ immunohistochemical techniques to locate serotonergic cells in the same midbrain sites from where defense reaction is evoked by chemical stimulation with bicuculline. The blockade of GABA(A) receptors in the neural substrates of the dorsal mesencephalon was followed by vigorous defensive reactions and increased nociceptive thresholds. Light microscopy immunocytochemistry with streptavidin method was used for the localization of the putative cells of defensive behavior with antibodies to serotonin in the rat's midbrain. Neurons positive to serotonin were found in the midbrain sites where defensive reactions were evoked by microinjection of bicuculline. Serotonin was localized to somata and projections of the neural networks of the mesencephalic tectum. Immunohistochemical studies showed that the sites in which neuronal perikarya positive to serotonin were identified in intermediate and deep layers of the superior colliculus, and in the dorsal and ventral columns of the periaqueductal gray matter are the same which were activated during the generation of defense behaviors, such as alertness, freezing, and escape reactions, induced by bicuculline. These findings support the contention that serotonin and GABAergic neurons may act in concert in the modulation of defense reaction in the midbrain tectum. Our neuroanatomical findings indicate a direct neural pathway connecting the dorsal midbrain and monoaminergic nuclei of the descending pain inhibitory system, with profuse synaptic terminals mainly in the pontine reticular formation, gigantocellularis nucleus, and nucleus raphe magnus. The midbrain tectum-gigantocellularis complex and midbrain tectum-nucleus raphe magnus neural pathways may provide an alternative output allowing the organization of the fear-induced anti-nociception by mesencephalic networks.

Agonist and hypertonic saline-induced trafficking of the NK3-receptors on vasopressin neurons within the paraventricular nucleus of the hypothalamus.

The neurokinin 3 receptor (NK3R) is colocalized with vasopressinergic neurons within the hypothalamic paraventricular nucleus (PVN) and intraventricular injections of NK3R agonists stimulate vasopressin (VP) release. Our objectives were to test the hypotheses that intraventricular injections of the selective NK3R agonist, succinyl-[Asp6, N-Me-Phe8] substance P (senktide), activate NK3R expressed by vasopressinergic neurons within the PVN, and see whether NK3R expressed by vasopressinergic neurons in the PVN are activated by hyperosmolarity. NK3R internalization was used as a marker of receptor activation. Immunohistochemistry revealed that NK3Rs were membrane-bound on VP immunoreactive neurons in control rats. Following senktide injection, there was a significant increase in the appearance of NK3R immunoreactivity within the cytoplasm and a morphological rearrangement of the dendrites, indicating receptor internalization, which was reversible. Furthermore, pretreatment with a selective NK3R antagonist, SB-222200, blocked the senktide-induced VP release and internalization of the NK3R in the PVN. These results show that the trafficking of the NK3R is due to ligand binding the NK3R. In a subsequent experiment, rats were administered intragastric loads of 2 or 0.15 M NaCl, and NK3R immunohistochemistry was used to track activation of the receptor. In contrast to control rats, 2 M NaCl significantly increased plasma VP levels and caused the internalization of the NK3R on VP neurons. Also, NK3R immunoreactivity was located in the nuclei of vasopressinergic neurons after senktide and 2 M NaCl treatment. These results show that hyperosmolarity stimulates the local release of an endogenous ligand in the PVN to bind to and activate NK3R on vasopressinergic neurons.

Sensitized attentional performance and Fos-immunoreactive cholinergic neurons in the basal forebrain of amphetamine-pretreated rats.

BACKGROUND: The consequences of repeated exposure to psychostimulants have been hypothesized to model aspects of schizophrenia. This experiment assessed the consequences of the administration of an escalating dosing regimen of amphetamine (AMPH) on attentional performance. Fos-like immunoreactivity (Fos-IR) in selected regions of these rats' brains was examined to test the hypothesis that AMPH-sensitized attentional impairments are associated with increased recruitment of basal forebrain cholinergic neurons. METHODS: Rats were trained in a sustained attention task and then treated with saline or in accordance with an escalating dosing regimen of AMPH (1-10 mg/kg). Performance was assessed during the pretreatment and withdrawal periods and following the subsequent administration of AMPH "challenges" (.5, 1.0 mg/kg). Brain sections were double-immunostained to visualize Fos-IR and cholinergic neurons. RESULTS: Compared with the acute effects of AMPH, AMPH "challenges," administered over 2 months after the pretreatment was initiated, resulted in significant impairments in attentional performance. In AMPH-pretreated and -challenged animals, an increased number of Fos-IR neurons was observed in the basal forebrain. The majority of these neurons were cholinergic. CONCLUSIONS: The evidence supports the hypothesis that abnormally regulated cortical cholinergic inputs represent an integral component of neuronal models of the attentional dysfunctions of schizophrenia.

The role of nerve growth factor receptors in cholinergic basal forebrain degeneration in prodromal Alzheimer disease.

Dysfunction of nerve growth factor (NGF) and its high (TrkA) and low (p75NTR) affinity receptors has been suggested to underlie the selective degeneration of the nucleus basalis (NB) cholinergic cortical projection neurons in end stage Alzheimer disease (AD). Whether the NGF system is dysfunctional during the prodromal stages of AD has only recently been evaluated. Surprisingly, the number of choline acetyltransferase-containing neurons remains stable despite a significant reduction in NGF receptor-positive cells in people with mild cognitive impairment (MCI), suggesting a phenotypic NGF receptor downregulation but not a frank loss of NB neurons during prodromal AD. Moreover, there is a loss of cortical TrkA in the face of stable p75NTR and increased proNGF levels, the precursor molecule of mature NGF, in early AD. Depending upon the cellular context these changes may result in increased pro-apoptotic signaling, cell survival, or a defect in retrograde transport mechanisms. Alterations in NGF and its receptors within the cholinotrophic NB system in early AD suggest that NGF-mediated cell signaling is required for the longterm survival of these neurons. Therapeutic neurotrophic intervention might delay or prevent NB neuron degeneration and preserve cholinergic cortical function during prodromal AD.

Urea enhances the nerve growth factor-induced neuroprotective effect on cholinergic neurons in organotypic rat brain slices.

Cholinergic neurons degenerate in Alzheimer's disease and dementia and neuroprotective substances are of high interest to counteract this cell death. The aim of the present study was to test the effect of urea and the nitric oxide synthetase inhibitor l-thiocitrulline on the survival of cholinergic neurons. Organotypic brain slices of the basal nucleus of Meynert were cultured for 2 weeks in the presence of 1-100 microM urea with or without NGF or other growth factors or with or without 1-10 microM of the NOS inhibitor L-thiocitrulline. A high number of cholinergic neurons survived in the presence of 0.1-100 ng/ml NGF. Urea or L-thiocitrulline alone did not exhibit neuroprotective activity; however, when brain slices were incubated with urea or L-thiocitrulline together with NGF there was a significant potentiating survival effect. Incubation of brain slices with NGF + urea + L-thiocitrulline did not further enhance the number of cholinergic neurons. NGF as well as urea did not stimulate expression of the enzyme choline acetyltransferase pointing to survival promoting effects. Urea did not modulate the NGF binding in PC12 cells indicating that this effect was indirect. It is concluded that urea may play a role as an indirect survival promoting molecule possibly involving the nitric oxide pathway.

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis.

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.

Effects of two years of estrogen loss or replacement on nucleus basalis cholinergic neurons and cholinergic fibers to the dorsolateral prefrontal and inferior parietal cortex of monkeys.

The present study examined the long-term (2 years) effects of estrogen loss or estrogen replacement therapy (ERT) on cholinergic neurons in the nucleus basalis of Meynert and on cholinergic fibers in the prefrontal and parietal cortex of adult female cynomolgus monkeys. Cholinergic fiber density in layer II of the prefrontal cortex was decreased in monkeys who were ovariectomized and treated with placebo for 2 years. In contrast, ovariectomized monkeys receiving ERT for 2 years had fiber densities that were comparable to those of intact controls. No differences in parietal cholinergic fiber density or nucleus basalis cholinergic neuron number or volume were found among intact, ovariectomized, or ERT monkeys. Our results suggest that ERT is effective in preventing region-specific changes in cortical cholinergic fibers that result from the loss of circulating ovarian hormones. These modest but appreciable effects on cholinergic neurobiology following long-term estrogen loss and ERT may contribute to changes in visuospatial attention function that is mediated by the prefrontal cortex.

The toxicity of tumor necrosis factor-alpha upon cholinergic neurons within the nucleus basalis and the role of norepinephrine in the regulation of inflammation: implications for Alzheimer's disease.

Inflammation and reduced forebrain norepinephrine are features of Alzheimer's disease that may interact to contribute to the degeneration of specific neural systems. We reproduced these conditions within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease. Tumor necrosis factor-alpha was infused into the basal forebrain of young mice pretreated with a norepinephrine neuronal toxin, N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine (DSP4), with the expectation that the loss of noradrenergic input would enhance the loss of cholinergic neurons. The results indicate that chronic infusion of tumor necrosis factor-alpha alone significantly decreased cortical choline acetyltransferase activity and increased the number of activated microglia and astrocytes within the basal forebrain. The loss of forebrain norepinephrine following systemic treatment with DSP4 did not alter the level of cortical choline acetyltransferase activity or activate microglia but significantly activated astrocytes within the basal forebrain. Infusion of tumor necrosis factor-alpha into DSP4-pretreated mice also reduced cortical choline acetyltransferase activity on the side of the infusion; however, the decline was not significantly greater than that produced by the infusion of tumor necrosis factor-alpha alone. The neurodegeneration seen may be indirect since a double-immunofluorescence investigation did not find evidence for the co-existence of tumor necrosis factor-alpha type I receptors on choline acetyltransferase-positive cells in the basal forebrain. The results suggest that noradrenergic cell loss in Alzheimer's disease does not augment the consequences of the chronic neuroinflammation and does not enhance neurodegeneration of forebrain cholinergic neurons.

NGF and NT-3 exert differential effects on the expression of neuropeptides in the suprachiasmatic nucleus of rats withdrawn from ethanol treatment.

Some neurotrophins have the capability of enhancing neuropeptide expression in several regions of the brain. It was also recently shown that NGF, infused over 1 month, offsets the decreased synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN) of rats submitted to chronic ethanol treatment and withdrawal. In the present study we examined the effectiveness of neutrotrophin-3 (NT-3) in promoting such effects, given that SCN neurons express both the high and the low affinity receptors for this neurotrophin. NT-3 was intraventricularly infused during 10 days to rats withdrawn from prolonged ethanol treatment. The total number, and the mean somatic volume, of VP- and VIP-immunoreactive neurons was compared with the estimates obtained from control rats and withdrawn rats treated with either NGF or cerebrospinal fluid during the same period. The infusion of cerebrospinal fluid and of NT-3 did not prevent the reduction in the number of peptide-producing neurons induced by withdrawal from ethanol treatment. Conversely, NGF infusion increased their number to control levels and led to neuronal hypertrophy. Our results show that, unlike NGF, NT-3 does not display the capacity of enhancing neuropeptide expression in the SCN. Because SCN neurons express the low affinity p75(NTR), which is equally activated by both neurotrophins, our results additionally indicate that the effects of NGF upon SCN neurons are not receptor-mediated. Taken together, our data suggest that indirect mechanisms, rather than direct neutrophin signaling, are likely to mediate the trophic effects exerted by NGF upon SCN neurons.

GABAB receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert.

The transduction mechanisms underlying presynaptic GABAB receptor-mediated inhibition of transmitter release have been characterized for a variety of synapses in the central nervous system (CNS). These studies have suggested a range of transduction mechanisms, including a role for second messengers such as protein kinases A (PKA) and C (PKC). In the present study, we have examined the intracellular signalling pathways underlying baclofen-induced inhibition of GABA release from terminals synapsing onto rat basalis of Meynert neurons using patch-clamp recordings. Baclofen, a selective GABAB receptor agonist, reversibly decreased both evoked and spontaneous, miniature, GABAergic inhibitory postsynaptic currents (eIPSCs and mIPSCs, respectively). Such baclofen actions were completely abolished by CGP55845A, a selective GABAB receptor antagonist, and by staurosporine, a non-selective PKA and PKC inhibitor. The mIPSC frequency was still decreased by baclofen even in the presence of 4 AP, a K+ channel blocker, and Cd2+, a voltage-dependent calcium channel blocker. Pharmacological activation or inhibition of PKC activity affected basal GABA release and mildly affected the response to baclofen. Inhibition of the cAMP/PKA cascade also affected basal GABA release and, in a subset of neurons, occluded the effects of baclofen, suggesting that the GABAB receptor-mediated inhibitory action on GABA release was mediated via decreases in PKA activity. In addition, PKA inhibition occluded the effects of PKC modulation on both basal GABA release and on the response to baclofen. Our results characterize the transduction pathway of baclofen at these nucleus basalis of Maynert (nBM) synapses and show, for the first time, some cross-talk between the cAMP/PKA and PKC pathways in mammalian presynaptic nerve terminals.

Effects of ageing and long-term hormone replacement on cholinergic neurones in the medial septum and nucleus basalis magnocellularis of ovariectomized rats.

Ovariectomized aged rats, some of which received long-term hormone replacement with oestrogen or oestrogen plus progesterone, were evaluated for the number and size of basal forebrain cholinergic neurones, as well as relative levels of choline acetyltransferase (ChAT) and trkA mRNA, in order to determine whether effects on basal forebrain cholinergic cell survival and function correspond with differences in cognitive performance previously described. The results show that ageing combined with long-term loss of ovarian function produced substantial reductions in the levels of ChAT and trkA mRNA in the medial septum and nucleus basalis magnocellularis, relative to much younger ovariectomized controls. In contrast, no significant effects on the number or size of the cholinergic cells were detected, indicating that loss of ovarian function does not cause a loss of cholinergic neurones with age. Long-term hormone replacement had no apparent effect on the number of ChAT-positive neurones detected, and did not prevent the reductions in ChAT and trkA mRNA associated with ovariectomy and ageing. Collectively, the data suggest that ageing combined with long-term loss of ovarian function has a severe negative impact on basal forebrain cholinergic function, but not on cholinergic cell survival per se.