Oestrogen inhibits salt-dependent hypertension by suppressing GABAergic excitation in magnocellular AVP neurons.

AIMS: Abundant evidence indicates that oestrogen (E2) plays a protective role against hypertension. Yet, the mechanism underlying the antihypertensive effect of E2 is poorly understood. In this study, we sought to determine the mechanism through which E2 inhibits salt-dependent hypertension. METHODS AND RESULTS: To this end, we performed a series of in vivo and in vitro experiments employing a rat model of hypertension that is produced by deoxycorticosterone acetate (DOCA)-salt treatment after uninephrectomy. We found that E2 prevented DOCA-salt treatment from inducing hypertension, raising plasma arginine-vasopressin (AVP) level, enhancing the depressor effect of the V1a receptor antagonist (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-vasopressin, and converting GABAergic inhibition to excitation in hypothalamic magnocellular AVP neurons. Moreover, we obtained results indicating that the E2 modulation of the activity and/or expression of NKCC1 (Cl- importer) and KCC2 (Cl- extruder) underpins the effect of E2 on the transition of GABAergic transmission in AVP neurons. Lastly, we discovered that, in DOCA-salt-treated hypertensive ovariectomized rats, CLP290 (prodrug of the KCC2 activator CLP257, intraperitoneal injections) lowered blood pressure, and plasma AVP level and hyperpolarized GABA equilibrium potential to prevent GABAergic excitation from emerging in the AVP neurons of these animals. CONCLUSION: Based on these results, we conclude that E2 inhibits salt-dependent hypertension by suppressing GABAergic excitation to decrease the hormonal output of AVP neurons.

Resting state activity and connectivity of the nucleus basalis of Meynert and globus pallidus in Lewy body dementia and Parkinson's disease dementia.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are two related diseases which can be difficult to distinguish. There is no objective biomarker which can reliably differentiate between them. The synergistic combination of electrophysiological and neuroimaging approaches is a powerful method for interrogation of functional brain networks in vivo. We recorded bilateral local field potentials (LFPs) from the nucleus basalis of Meynert (NBM) and the internal globus pallidus (GPi) with simultaneous cortical magnetoencephalography (MEG) in six PDD and five DLB patients undergoing surgery for deep brain stimulation (DBS) to look for differences in underlying resting-state network pathophysiology. In both patient groups we observed spectral peaks in the theta (2-8 Hz) band in both the NBM and the GPi. Furthermore, both the NBM and the GPi exhibited similar spatial and spectral patterns of coupling with the cortex in the two disease states. Specifically, we report two distinct coherent networks between the NBM/GPi and cortical regions: (1) a theta band (2-8 Hz) network linking the NBM/GPi to temporal cortical regions, and (2) a beta band (13-22 Hz) network coupling the NBM/GPi to sensorimotor areas. We also found differences between the two disease groups: oscillatory power in the low beta (13-22Hz) band was significantly higher in the globus pallidus in PDD patients compared to DLB, and coherence in the high beta (22-35Hz) band between the globus pallidus and lateral sensorimotor cortex was significantly higher in DLB patients compared to PDD. Overall, our findings reveal coherent networks of the NBM/GPi region that are common to both DLB and PDD. Although the neurophysiological differences between the two conditions in this study are confounded by systematic differences in DBS lead trajectories and motor symptom severity, they lend support to the hypothesis that DLB and PDD, though closely related, are distinguishable from a neurophysiological perspective.

Bilateral nucleus basalis of Meynert deep brain stimulation for dementia with Lewy bodies: A randomised clinical trial.

BACKGROUND: Dementia with Lewy bodies (DLB) is the second most common form of dementia. Current symptomatic treatment with medications remains inadequate. Deep brain stimulation of the nucleus basalis of Meynert (NBM DBS) has been proposed as a potential new treatment option in dementias. OBJECTIVE: To assess the safety and tolerability of low frequency (20 Hz) NBM DBS in DLB patients and explore its potential effects on both clinical symptoms and functional connectivity in underlying cognitive networks. METHODS: We conducted an exploratory randomised, double-blind, crossover trial of NBM DBS in six DLB patients recruited from two UK neuroscience centres. Patients were aged between 50 and 80 years, had mild-moderate dementia symptoms and were living with a carer-informant. Patients underwent image guided stereotactic implantation of bilateral DBS electrodes with the deepest contacts positioned in the Ch4i subsector of NBM. Patients were subsequently assigned to receive either active or sham stimulation for six weeks, followed by a two week washout period, then the opposite condition for six weeks. Safety and tolerability of both the surgery and stimulation were systematically evaluated throughout. Exploratory outcomes included the difference in scores on standardised measurements of cognitive, psychiatric and motor symptoms between the active and sham stimulation conditions, as well as differences in functional connectivity in discrete cognitive networks on resting state fMRI. RESULTS: Surgery and stimulation were well tolerated by all six patients (five male, mean age 71.33 years). One serious adverse event occurred: one patient developed antibiotic-associated colitis, prolonging his hospital stay by two weeks. No consistent improvements were observed in exploratory clinical outcome measures, but the severity of neuropsychiatric symptoms reduced with NBM DBS in 3/5 patients. Active stimulation was associated with functional connectivity changes in both the default mode network and the frontoparietal network. CONCLUSION: Low frequency NBM DBS can be safely conducted in DLB patients. This should encourage further exploration of the possible effects of stimulation on neuropsychiatric symptoms and corresponding changes in functional connectivity in cognitive networks. TRIAL REGISTRATION NUMBER: NCT02263937.

Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease.

Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer's disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD.

Gender differences in neurotrophin and glutamate receptor expression in cholinergic nucleus basalis neurons during the progression of Alzheimer's disease.

The higher incidence rate of Alzheimer's disease (AD) in elderly women indicates that gender plays a role in AD pathogenesis. Evidence from clinical and pharmacologic studies, neuropathological examinations, and models of hormone replacement therapy suggest that cholinergic basal forebrain (CBF) cortical projection neurons within the nucleus basalis (NB), which mediate memory and attention and degenerate in AD, may be preferentially vulnerable in elderly women compared to men. CBF neurons depend on nerve growth factor (NGF) and their cognate receptors (trkA and p75(NTR)) for their survival and maintenance. We recently demonstrated a shift in the balance of NGF and its receptors toward cell death mechanisms during the progression of AD. To address whether gender affects NGF signaling system expression within the CBF, we used single cell RNA amplification and custom microarray technologies to compare gene expression profiles of single cholinergic NB neurons in tissue specimens from male and female members of the Religious Orders Study who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. p75(NTR) expression within male cholinergic NB neurons was unchanged across clinical diagnosis, whereas p75(NTR) mRNA levels in female NB neurons exhibited a ∼40% reduction in AD compared to NCI. Male AD subjects displayed a ∼45% reduction in trkA mRNA levels within NB neurons compared to NCI and MCI. In contrast, NB neuronal trkA expression in females was reduced ∼50% in both MCI and AD compared to NCI. Reduced trkA mRNA levels were associated with poorer global cognitive performance and higher Braak scores in the female subjects. In addition, we found a female-selective reduction in GluR2 AMPA glutamate receptor subunit expression in NB neurons in AD. These data suggest that cholinergic NB neurons in females may be at greater risk for degeneration during the progression of AD and support the concept of gender-specific therapeutic interventions during the preclinical stages of the disease.

Lovastatin improves neurological outcome after nucleus basalis magnocellularis lesion in rats.

Increasing evidence indicates that statins, specific inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, exerts neuroprotective actions rather than simply lowering cholesterol. However, the underlying mechanism has not been elucidated clearly. Here, the effect of lovastatin on the neurological outcomes of nucleus basalis magnocellularis (NBM)-lesioned rats and the pathophysiological mechanisms were investigated. Sprague-Dawley rats were divided into three groups: (i) a sham group; (ii) a model group: bilateral NBM of rats were injured by infusion of ibotenic acid; and (iii) a lovastatin-treated group: lovastatin was administrated orally for 4 weeks before treated by ibotenic acid. We show that lovastatin significantly improves the neurological outcomes as well as the choline acetyltransferase (ChAT) activity and muscarinic/NMDA receptor binding activity impaired by NBM lesion, and that lovastatin prevents neuron loss and induces Akt whereas inhibits p38 phosphorylation. Overall, the neuro-restorative and -protective effect of lovastatin may be attributed to the regulation of Akt- and p38-mediated signaling pathway together with improvement of muscarinic/NMDA receptor functions. Statins may be useful in the treatment of neurological disorders.

Huperzine A reverses cholinergic and monoaminergic dysfunction induced by bilateral nucleus basalis magnocellularis injection of beta-amyloid peptide (1-40) in rats.

(1) Huperzine A, a promising therapeutic agent for Alzheimer's disease (AD), was tested for its effects on cholinergic and monoaminergic dysfunction induced by injecting beta-amyloid peptide-(1-40) into nucleus basalis magnocellularis of the rat. (2) Bilateral injection of 10 microg beta-amyloid peptide-(1-40) into nucleus basalis magnocellularis produced local deposits of amyloid plaque and functional abnormalities detected by microdialysis. In medial prefrontal cortex, reductions in the basal levels and stimulated release of acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine were observed. However, oral huperzine A (0.18 mg/kg, once daily for 21 consecutive days) markedly reduced morphologic abnormalities at the injection site in rats infused with beta-amyloid peptide-(1-40). Likewise, this treatment ameliorated the beta-amyloid peptide-(1-40)-induced deficits in extracellular acetylcholine, dopamine, and norepinephrine (though not 5-hydroxytryptamine) in medial prefrontal cortex, and lessened the reduction in nicotine or methoctramine-stimulated release of acetylcholine and K(+)-evoked releases of acetylcholine and dopamine. (3) The present results provide the first direct evidence that huperzine A acts to oppose neurotoxic effects of beta-amyloid peptide on cholinergic, dopaminergic, and noradrenergic systems of the rat forebrain.

Protracted effects of chronic oral haloperidol and risperidone on nerve growth factor, cholinergic neurons, and spatial reference learning in rats.

The primary therapeutic agents used for schizophrenia, antipsychotic drugs, ameliorate psychotic symptoms; however, their chronic effects on cognition (or the physiologic processes of the brain that support cognition) are largely unknown. The purpose of this rodent study was to extend our previous work on this subject by investigating persistent effects (i.e. during a 14 day drug-free washout period) of chronic treatment (i.e. ranging from 45 days to 6 months) with a representative first and second generation antipsychotic. Drug effects on learning and memory and important neurobiological substrates of memory, the neurotrophin, nerve growth factor (NGF) and its receptors, and certain components of the basal forebrain cholinergic system were investigated. Behavioral effects of oral haloperidol (2.0 mg/kg/day), or risperidone (2.5 mg/kg/day) were assessed in an open field, a water maze task, and a radial arm maze procedure and neurochemical effects in brain tissue were subsequently measured by enzyme-linked immunosorbent assays (ELISAs). The results indicated that both antipsychotics produced time-dependent and protracted deficits in the performance of a water maze procedure when compared with vehicle-treated controls, while neither drug was associated with significant alterations in radial arm maze performance. Interestingly, haloperidol, but not risperidone, was detectible in the rodent brain in appreciable levels for up to 2 weeks after drug discontinuation. Both antipsychotics were also associated with reduced levels of NGF protein in the basal forebrain and prefrontal cortex and significant (or nearly significant) decreases in phosphorylated tropomyosin-receptor kinase A (TrkA) protein and the vesicular acetylcholine transporter (depending on the brain region analyzed). Neither antipsychotic markedly affected TrkA or p75 neurotrophin receptor levels. These data in rats indicate that chronic treatment with either haloperidol or risperidone may be associated with protracted negative effects on cognitive function as well as important neurotrophin and neurotransmitter pathways that support cognition.

Behavioral and immunohistological effects of cholinergic damage in immunolesioned rats: alteration of c-Fos and polysialylated neural cell adhesion molecule expression.

The aim of this study was to determine the brain structures as well as the plasticity events associated with the behavioral effects of cholinergic damage. Rats were submitted to injection of 192 IgG-saporin in the medial septum/diagonal band of Broca complex and the nucleus basalis magnocellularis. The immunohistochemical expression of c-Fos protein and PSA-NCAM (polysialylated neural cell adhesion molecule) and the behavioral performances in the nonmatching-to-position task were assessed at various post-lesion times. Thus, 3 days after injection of the immunotoxin, increased c-Fos labeling was observed in the areas of infusion, indicating these cells were undergoing some plastic changes and/or apoptotic processes. A drastic increase was observed in the number of PSA-NCAM positive cells and in their dendritic arborization in the dentate gyrus. At 7 days post-lesion, no behavioral deficit was observed in immunolesioned rats despite the drastic loss of cholinergic neurons. These neurons showed decreased c-Fos protein expression in the piriform and entorhinal cortex and in the dentate gyrus. In the latter, PSA-NCAM induction was high, suggesting that remodeling occurred, which in turn might contribute to sustaining some mnemonic function in immunolesioned rats. At 1 month, cholinergic neurons totally disappeared and behavioral deficits were drastic. c-Fos expression showed no change. In contrast, the increased PSA-NCAM-labeling observed at short post-lesion times was maintained but the plastic changes due to this molecule could not compensate the behavioral deficit caused by the immunotoxin. Thus, as the post-lesion time increases, a gradual degeneration process should occur that may contribute to mnemonic impairments. This neuronal loss leads to molecular and cellular alterations, which in turn may aggravate cognitive deficits.

Lesions to the nucleus basalis magnocellularis lower performance but do not block the retention of a previously acquired learning set.

Rats were first trained to acquire an olfactory discrimination learning set (ODLS) on 40 olfactory-unique discrimination problems. Following acquisition of ODLS, animals were lesioned bilaterally in the nucleus basalis magnocellularis (nBM) using either quisqualic acid (QUIS) or 192 IgG-saporin (SAP). QUIS animals performed significantly worse than control animals following surgery and SAP animals performed transiently worse than control animals. Despite lowered performances, both QUIS and SAP animals performed significantly better than expected by chance on trial 2 indicating retention of the ODLS previously acquired. Implications for the role of the nBM in aspects of cognitive flexibility and its role in acquisition versus retention are discussed.

Age-dependent loss of NGF signaling in the rat basal forebrain is due to disrupted MAPK activation.

The loss of nerve growth factor (NGF) and its high affinity receptor TrkA has been implicated in the loss of cholinergic tone and function in Alzheimer's disease (AD) and normal aging. We employed an animal model of aging, the aged rat, which also exhibits memory loss and NGF alterations. Basal forebrain TrkA levels increased after injection of NGF in the hippocampus within 1h in young rats, but this response was diminished in aged animals as determined by Western blot analysis. Further, NGF activated MAPK pathways without changing total ERK levels and the activation of these pathways was also diminished in aged animals. The exogenous NGF injection did not appear to activate the PI-3K pathway or alter total levels of Akt significantly. These data shed light on mechanisms of NGF signaling in the CNS, and alterations in this signaling cascade associated with age and memory loss. These findings might lead to development of novel treatment therapies for the memory loss associated with AD and other age-associated neurodegenerative diseases.

Inhibition of Wnt signaling, modulation of Tau phosphorylation and induction of neuronal cell death by DKK1.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimer's Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Select estrogens within the complex formulation of conjugated equine estrogens (Premarin) are protective against neurodegenerative insults: implications for a composition of estrogen therapy to promote neuronal function and prevent Alzheimer's disease.

BACKGROUND: Results of the Women's Health Initiative Memory Study (WHIMS) raised concerns regarding the timing and formulation of hormone interventions. Conjugated equine estrogens (CEE), used as the estrogen therapy in the WHIMS trial, is a complex formulation containing multiple estrogens, including several not secreted by human ovaries, as well as other biologically active steroids. Although the full spectrum of estrogenic components present in CEE has not yet been resolved, 10 estrogens have been identified. In the present study, we sought to determine which estrogenic components, at concentrations commensurate with their plasma levels achieved following a single oral dose of 0.625 mg CEE (the dose used in the WHIMS trial) in women, are neuroprotective and whether combinations of those neuroprotective estrogens provide added benefit. Further, we sought, through computer-aided modeling analyses, to investigate the potential correlation of the molecular mechanisms that conferred estrogen neuroprotection with estrogen interactions with the estrogen receptor (ER). RESULTS: Cultured basal forebrain neurons were exposed to either beta-amyloid(25-35) or excitotoxic glutamate with or without pretreatment with estrogens followed by neuroprotection analyses. Three indicators of neuroprotection that rely on different aspects of neuronal damage and viability, LDH release, intracellular ATP level and MTT formazan formation, were used to assess neuroprotective efficacy. Results of these analyses indicate that the estrogens, 17alpha-estradiol, 17beta-estradiol, equilin, 17alpha-dihydroequilin, equilinen, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, and Delta8,9-dehydroestrone were each significantly neuroprotective in reducing neuronal plasma membrane damage induced by glutamate excitotoxicity. Of these estrogens, 17beta-estradiol and Delta8,9-dehydroestrone were effective in protecting neurons against beta-amyloid25-35-induced intracellular ATP decline. Coadministration of two out of three neuroprotective estrogens, 17beta-estradiol, equilin and Delta8,9-dehydroestrone, exerted greater neuroprotective efficacy than individual estrogens. Computer-aided analyses to determine structure/function relationships between the estrogenic structures and their neuroprotective activity revealed that the predicted intermolecular interactions of estrogen analogues with ER correlate to their overall neuroprotective efficacy. CONCLUSION: The present study provides the first documentation of the neuroprotective profile of individual estrogens contained within the complex formulation of CEE at concentrations commensurate with their plasma levels achieved after an oral administration of 0.625 mg CEE in women. Our analyses demonstrate that select estrogens within the complex formulation of CEE contribute to its neuroprotective efficacy. Moreover, our data predict that the magnitude of neuroprotection induced by individual estrogens at relatively low concentrations may be clinically undetectable and ineffective, whereas, a combination of select neuroprotective estrogens could provide an increased and clinically meaningful efficacy. More importantly, these data suggest a strategy for determining neurological efficacy and rational design and development of a composition of estrogen therapy to alleviate climacteric symptoms, promote neurological health, and prevent age-related neurodegeneration, such as AD, in postmenopausal women.

Time course of behavioral changes following basal forebrain cholinergic damage in rats: Environmental enrichment as a therapeutic intervention.

The present experiment was designed to study changes in behavior following immunolesioning of the basal forebrain cholinergic system. Rats were lesioned at 3 months of age by injection of the 192 IgG-saporin immunotoxin into the medial septum area and the nucleus basalis magnocellularis, and then tested at different times after surgery (from days 7-500) on a range of behavioral tests, administered in the following order: a nonmatching-to-position task in a T-maze, an object-recognition task, an object-location task, and an open-field activity test. The results revealed a two-way interaction between post-lesion behavioral testing time and memory demands. In the nonmatching-to-position task, memory deficits appeared quite rapidly after surgery, i.e. at a post-lesion time as short as 1 month. In the object-recognition test, memory impairments appeared only when rats were tested at late post-lesion times (starting at 15 months), whereas in the object-location task deficits were apparent at early post-lesion times (starting from 2 months). Taking the post-operative time into account, one can hypothesize that at the shortest post-lesion times, behavioral deficits are due to pure cholinergic depletion, while as the post-lesion time increases, one can speculate the occurrence of a non-cholinergic system decompensation process and/or a gradual degeneration process affecting other neuronal systems that may contribute to mnemonic impairments. Interestingly, when middle-aged rats were housed in an enriched environment, 192 IgG-saporin-lesioned rats performed better than standard-lesioned rats on both the nonmatching-to-position and the object-recognition tests. Environment enrichment had significant beneficial effects in 192 IgG-saporin-lesioned rats, suggesting that lesioned rats at late post-lesion times (over 1 year) still have appreciable cognitive plasticity.

Vascular endothelial growth factor counteracts NMDA-induced cell death of adult cholinergic neurons in rat basal nucleus of Meynert.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in the brain and has recently been shown to possess neuroprotective activity. The aim of the present study was to observe if VEGF can counteract the excitotoxic N-methyl-D-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert in vivo in adult rats. Immunohistochemistry was used to detect vascular structures (RECA-1 or laminin staining) and cholinergic neurons (choline-acetyltransferase). To compare the in vivo VEGF effects also in vitro, VEGF was applied to axotomized cholinergic neurons in organotypic brain slices with or without NMDA. Our data provide evidence that VEGF counteracted cell death in vivo in adult rats but did not protect cholinergic neurons in developing brain slices. Nerve growth factor protected cholinergic neurons in vivo as well as in vitro. In conclusion, VEGF exhibits neuroprotective activity on adult cholinergic neurons of the basal nucleus of Meynert.

Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA.

Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders.

The cognitive effects of ovariectomy and estrogen replacement are modulated by aging.

Recent experimental and clinical studies suggest that estrogen may be an important factor influencing neuronal function during normal and pathological aging. Using different behavioral paradigms in rodents, estrogen replacement was shown to enhance learning and memory as well as attenuate learning deficits associated with cholinergic impairment. The goal of this study was to determine whether cognitive sensitivity to estrogen manipulations (short-term ovariectomy and chronic estrogen replacement) is affected by aging. Middle-aged and old female Fischer-344 rats were used to estimate the effects of estrogen manipulations at two different stages of reproductive aging. At middle age, when the females underwent an initial stage of reproductive aging (irregular cyclicity), ovariectomy did not significantly affect the acquisition of the T-maze active avoidance as compared with Sham rats, while estrogen replacement decreased behavioral vulnerability to scopolamine. However, when tested at more advanced stage of aging (consistent diestrus), old ovariectomized rats were more sensitive to scopolamine as compared with the control rats. Moreover, estrogen treatment at this age did not produce any protective effect against scopolamine. Contrasting findings of the effects of estrogen replacement in middle-aged and old rats suggest that the ability of estrogen to enhance the basal forebrain cholinergic function declines with age. These data indicate that aging processes may substantially modulate the mechanisms of estrogen action. A "time window" during which hormone replacement must be initiated in order to be effective could be determined in terms of the stages of reproductive senescence. This study is the first to clearly demonstrate that the cognitive effects of estrogen replacement are still preserved during the initial stages of reproductive aging (irregular cyclicity) and dramatically limited as aging progresses (cessation of proestrus).

Tumor necrosis factor-alpha triggers cell death of sensitized potassium chloride-stimulated cholinergic neurons.

Cell death of cholinergic neurons of the basal forebrain plays an important role in neurodegenerative disorders, such as Alzheimer's disease. Inflammatory cytokines, such as, for example, tumor necrosis factor-alpha (TNF-alpha), may be involved in these neurodegenerative processes. The aim of this project was to study the role of TNF-alpha in the survival and nerve fiber growth of cholinergic neurons of the basal nucleus of Meynert in organotypic brain slices and in adult rats. Cholinergic neurons were visualized by immunohistochemistry for the enzyme choline acetyltransferase and nerve fibers by histochemistry for the enzyme acetylcholinesterase. When co-slices of basal nucleus of Meynert and neocortex were sensitized for 15 min with 30 mM potassium chloride and subsequently incubated for 1 week with 20 ng/ml TNF-alpha, cholinergic neurons and nerve fibers markedly degenerated. Incubation with different growth factors rescued the loss of cholinergic cell bodies and cholinergic nerve fibers. Injection of 30 mM potassium chloride and 50 ng TNF-alpha into four defined cortical regions of anesthetized adult rats resulted in predominant cell death of cholinergic neurons on the ipsilateral side. In conclusion, our data show that TNF-alpha potentiated cell death of cholinergic neurons possibly via retrograde axonal damage in vitro and in vivo. Cortical overactivation combined with an increased expression of pro-inflammatory cytokines may contribute to the cell death observed in Alzheimer's disease and ageing.

Selective lesions of basal forebrain cholinergic neurons produce anterograde and retrograde deficits in a social transmission of food preference task in rats.

We examined the performance of Long-Evans rats with 192 IgG-saporin lesions of the medial septum/vertical limb of the diagonal band (MS/VDB) or nucleus basalis magnocellularis/substantia innominata (NBM/SI), which removed cholinergic projections mainly to hippocampus or neocortex, respectively. We studied the effects of these lesions on anterograde and retrograde memory for a natural form of hippocampal-dependent associative memory, the social transmission of food preference. In a study of anterograde memory, MS/VDB lesions did not affect the immediate, 24-h or 3-week retention of the task. In contrast, NBM/SI lesions severely impaired immediate and 24-h retention. In a study of retrograde memory in which rats acquired the food preference 5 days or 1 day before surgery and they were tested 10-11 days after surgery, MS/VDB-lesioned rats showed striking memory deficits for the preference acquired at a long delay (5 days) before surgery, although all lesioned rats exhibited poorer retention on both retest sessions than on their pretest performance. Subsequent testing of new anterograde learning in these rats revealed no disrupting effects of lesions on a standard two-choice test. When rats were administered a three-choice test, in which the target food was presented along with two more options, NBM/SI-lesioned rats were somewhat impaired on a 24-h retention test. These results provide evidence that NBM/SI and MS/VDB cholinergic neurons are differentially involved in a social memory task that uses olfactory cues, suggesting a role for these neurons in acquisition and consolidation/retrieval of nonspatial declarative memory.

Effects of 192 IgG-saporin on acetylcholinesterase histochemistry in male and female rats.

Sex hormones may exert neuroprotective effects in various models of brain lesions. Male and female Long-Evans rats were subjected to intracerebroventricular injections of 2 microg 192 IgG-saporin or vehicle. Starting 2 days before surgery, half the male rats were treated with estradiol for 7 days. Three weeks after surgery, they were sacrificed for histochemical staining of acetylcholinesterase (AChE) and densitometric evaluations. The lesion induced a substantial to dramatic decrease of the AChE-positive fiber density in the cingulate, somatosensory, piriform, retrosplenial and perirhinal cortices, and in the hippocampus. Weak effects were found in the striatum. There was no significant decrease in the dorsal thalamus. Sex had no significant effect on AChE-positive staining in any brain area. In males, estradiol treatment did not alter the effects of 192 IgG-saporin. These results show that sex or estradiol treatment in male rats does not interfere with the immunotoxic effects of intracerebroventricular injections of 192 IgG-saporin on cholinergic projections from the basal forebrain.