Although controversial, estrogens remain one of the few agents purported to influence the incidence of Alzheimer's disease and one of their postulated mechanisms of action is their effects on basal forebrain cholinergic neurons. However, it is unclear whether the responses of cholinergic neurons to estrogens are direct or mediated via the retrograde influences of neurotrophins, known to be induced by estrogens in the hippocampus and neocortex. In the present study, we explore the issue of the primary site of action of estrogens by studying the regulation of expression of genes that characterize mature cholinergic neurons, i.e., choline acetyltransferase, trkA, and p75(NTR) in the medial septum and the nucleus basalis complex. In parallel, we study the hippocampal expression of NGF, BDNF, and NT-3, i.e., neurotrophins with known trophic roles on cholinergic neurons. Gene expression is studied by RT-PCR in ovariectomized female rats with and without estrogen supplementation within the physiological estradiol range and in rats with complete fimbria-fornix transactions treated with estrogen or vehicle. To clarify mechanisms of estrogen transduction in cholinergic neurons, we study the effects of estrogen treatment on fimbria-fornix-lesioned mice with genetic ablations of ER subtypes alpha and beta. The results of the present study suggest that, while estrogens do regulate BDNF expression in the hippocampus and neocortex, they also exert stimulatory non-trophic effects on basal forebrain cholinergic neurons, primarily on ChAT expression. Cholinergic neurons retain their ability to respond to estrogens after their complete separation from the hippocampus. The elimination of ERalpha alters significantly the phenotypic responsiveness of cholinergic neurons to estrogens, whereas elimination of ERbeta appears to have no effect. Our findings support the idea that estrogens directly enhance cholinergic neuron function and that ERalpha plays a significant role in transducing these regulatory effects.

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-beta deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.

The GDNF family ligands (GFLs) are a group of neurotrophic factors that influence the development, survival, and maintenance of specific populations of neurons in the central and peripheral nervous systems. The cholinergic neurons of the basal forebrain provide cholinergic innervation to cortical structures and their integrity is vital to normal cognitive function. GDNF, the original member of the GFL family promotes the survival of developing basal forebrain cholinergic neurons in vitro. We have now found that neurturin (NRTN) and persephin (PSPN) also promote the survival of basal forebrain neurons including both cholinergic neurons and a population of non-cholinergic neurons with an efficacy comparable to NGF. We also demonstrate that developing and mature basal forebrain cholinergic neurons (BFCN) express GFL receptors. Ret, the signaling component of the GFL-receptor complex, is expressed in most adult rat BFCN. In addition, Ret and the GFL co-receptors GFRalpha1 and GFRalpha2 are expressed in developing cholinergic neurons in cultures of embryonic basal forebrain. Our results suggest that the GFLs may be effective as neuroprotective agents for BFCNs in vivo.

This study investigated whether animals sustaining experimental damage to the basal forebrain cholinergic system would benefit from treatment with exogenous neurotrophic factors. Specifically, we set out to determine whether neurotrophic factors would rescue damaged cholinergic neurons and improve behavioral performance on a spatial learning and memory task. Adult rats received bilateral injections of either saline (controls) or 192 IgG-saporin to damage basal forebrain cholinergic neurons (BFCNs). Two weeks later, animals received implants of an Alzet mini-pump connected to cannulae implanted bilaterally in the lateral ventricles. Animals received infusions of nerve growth factor (NGF), neurotrophin 3 (NT3), a combination of NGF and NT3, or a saline control over a 4-week period. Compared to saline-treated controls, animals sustaining saporin-induced damage to BFCNs took significantly more trials to learn a delayed match to position task and also performed more poorly on subsequent tests, with increasing delays between test runs. In contrast, animals infused with neurotrophins after saporin treatment performed significantly better than animals receiving saline infusions; no differences were detected for performance scores among animals infused with NGF, NT3, or a combination of NGF and NT3. Studies of ChAT immunnocytochemical labeling of BFCNs revealed a reduction in the numbers of ChAT-positive neurons in septum, nucleus of diagonal band, and nucleus basalis in animals treated with saporin followed by saline infusions, whereas animals treated with infusions of NGF, NT3 or a combination of NGF and NT3 showed only modest reductions in ChAT-positive neurons. Together, these data support the notion that administration of neurotrophic factors can rescue basal forebrain cholinergic neurons and improve learning and memory performance in rats.

Treatment of degenerating basal forebrain cholinergic neurons with nerve growth factor (NGF) in Alzheimer's disease has long been contemplated, but an effective and safe delivery method has been lacking. Towards achieving this goal, we are currently developing CERE-110, an adeno-associated virus-based gene delivery vector that encodes for human NGF, for stereotactic surgical delivery to the human nucleus basalis of Meynert. Results indicate that NGF transgene delivery to the targeted brain region via CERE-110 is reliable and accurate, that NGF transgene distribution can be controlled by altering CERE-110 dose, and that it is possible to achieve restricted NGF expression limited to but covering the target brain region. Results from animals examined at longer time periods of 3, 6, 9 and 12 months after CERE-110 delivery indicate that NGF transgene expression is stable and sustained at all time points, with no loss or build-up of protein over the long-term. In addition, results from a series of experiments indicate that CERE-110 is neuroprotective and neurorestorative to basal forebrain cholinergic neurons in the rat fimbria-fornix lesion and aged rat models, and has bioactive effects on young rat basal forebrain cholinergic neurons. These findings, as well as those from several additional non-clinical experiments conducted in both rats and monkeys, led to the initiation of a Phase I clinical study to evaluate the safety and efficacy of CERE-110 in Alzheimer's disease subjects, which is currently ongoing.

It has been recently shown that the substantia nigra pars reticulata (SNr) of 6-hydroxydopamine (6-OHDA)-lesioned rats, under urethane anaesthesia, manifests a prominent low frequency oscillation (LFO) of around 1Hz, synchronized with cortical slow wave activity (SWA). Nevertheless, it is poorly understood whether these electrophysiological alterations are correlated only with severe dopamine depletion or may also play a relevant pathogenetic role in the early stages of the dopamine denervation. Hence, here we recorded SNr single units and electrocorticogram (ECoG) in two models of dopamine denervation: (i) acute dopamine denervated rats, obtained by injection of tetrodotoxin (TTX), (ii) chronic dopamine depleted rats, 2 weeks after 6-OHDA lesioning. Both TTX and 6-OHDA were infused into the medial forebrain bundle (MFB). The acute TTX-mediated dopamine depletion caused a fast developing occurrence of a SNr/ECoG coherence, peaking between 0.48 and 1.22 Hz, parallel with a consistent decrease of firing rate (from 22.61 ± 7.04 to 15.35 ± 9.04 Hz) homolateraly to the infusion. Strikingly, this abnormal 1 Hz synchronization, TTX-mediated was qualitatively similar to the ECoG/SNr synchronization detectable in the 6-OHDA lesioned hemisphere (LH). In addition, TTX infusion in the un-lesioned hemispheres (UH) of 6-OHDA treated rats, produced ECoG/SNr synchronization qualitatively similar to that recordable in the LH. Hence, our data support the proposition that LFO, is tightly correlated to cortex, and represent a critical hallmark of a basal ganglia (BG) failure from the early stages of dopamine denervation.

Down syndrome (DS) individuals develop several neuropathological hallmarks seen in Alzheimer's disease, including cognitive decline and the early loss of cholinergic markers in the basal forebrain. These deficits are replicated in the Ts65Dn mouse, which contains a partial trisomy of murine chromosome 16, the orthologous genetic segment to human chromosome 21. Oxidative stress levels are elevated early in DS, and may contribute to the neurodegeneration seen in these individuals. We evaluated oxidative stress in Ts65Dn mice, and assessed the efficacy of long-term antioxidant supplementation on memory and basal forebrain pathology. We report that oxidative stress was elevated in the adult Ts65Dn brain, and that supplementation with the antioxidant vitamin E effectively reduced these markers. Also, Ts65Dn mice receiving vitamin E exhibited improved performance on a spatial working memory task and showed an attenuation of cholinergic neuron pathology in the basal forebrain. This study provides evidence that vitamin E delays onset of cognitive and morphological abnormalities in a mouse model of DS, and may represent a safe and effective treatment early in the progression of DS neuropathology.

LIS1 is one of principal genes related with Type I lissencephaly, a severe human brain malformation characterized by abnormal neuronal migration in the cortex. The LIS1 gene encodes a brain-specific 45kDa non-catalytic subunit of platelet-activating factor (PAF) acetylhydrolase-1b (PAFAH1b), an enzyme that inactivates the PAF. We have studied the role of Lis1 using a Lis1/sLis1 murine model, which has deleted the first coding exon from Lis1 gene. Homozygous mice are not viable but heterozygous have shown a delayed corticogenesis and neuronal dysplasia, with enhanced cortical excitability. Lis1/sLis1 embryos also exhibited a delay of cortical innervation by the thalamocortical fibers. We have explored in Lis1/sLis1 mice anomalies in forebrain cholinergic neuron development, which migrate from pallium to subpallium, and functionally represent the main cholinergic input to the cerebral cortex, modulating cortical activity and facilitating attention, learning, and memory. We hypothesized that primary migration anomalies and/or disorganized cortex could affect cholinergic projections from the basal forebrain and septum in Lis1/sLis1 mouse. To accomplish our objective we have first studied basal forebrain neurons in Lis1/sLis1 mice during development, and described structural and hodological differences between wild-type and Lis1/sLis1 embryos. In addition, septohippocampal projections showed altered development in mutant embryos. Basal forebrain abnormalities could contribute to hippocampal excitability anomalies secondary to Lis1 mutations and may explain the cognitive symptoms associated to cortical displasia-related mental diseases and epileptogenic syndromes.

To study the role of neurotrophin-responsive neurons in brain growth and developmental resistance to seizure-induced injury, we infused saporin-conjugated 192-IgG (192 IgG-saporin), a monoclonal antibody directed at the P75 neurotrophin receptors (p75(NTR)), into the ventricles of postnatal day 8 (P8) rat pups. 7-10 days after immunotoxin treatment, loss of p75(NTR) immunoreactivity was associated with depletion of basal forebrain cholinergic projection to the neocortex and hippocampus. Kainic acid (KA)-induced seizures on P15 resulted in hippocampal neuronal injury in the majority of toxin-treated animals (13/16), but only rarely in saline-injected controls (2/25) (P < 0.001). In addition, widespread cerebral atrophy and a significant decrease in brain weight with preserved body weight were observed. Volumetric analysis of the hippocampal hilar region revealed a 2-fold reduction in perikaryal size and a 1.7-fold increase in cell packing density after 192 IgG-saporin injection. These observations indicate that neurotrophin-responsive neurons including basal forebrain magnocellular cholinergic neurons may be critical for normal brain growth and play a protective role in preventing excitotoxic neuronal injury during development.

Cholinergic neurons of the basal forebrain degenerate in Alzheimer's disease. Nerve growth factor (NGF) is so far the most potent molecule to counteract this neurodegeneration; however, the delivery of NGF into the brain is very difficult. The aim of the present study was to observe, if transplanted primary monocytes secreting NGF may counteract N-methyl-D-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert (nBM) in vivo. Monocytes were purified by indirect magnetic separation from rat blood. Recombinant NGF was introduced into cells using the novel protein-delivery reagent BioPORTERtrade mark and secretion of NGF was measured by ELISA. Monocytes secreted approximately 4000 pg NGF/day/1 x 10(6) cells. Injection of monocytes onto organotypic brain slices of the nBM in vitro protected cholinergic neurons against cell death. When monocytes were transplanted in vivo into the lateral ventricle, the cells survived for up to 7 days and counteracted the NMDA-induced cell death of cholinergic neurons. In conclusion, primary monocytes secreting recombinant NGF are useful to deliver NGF directly into the brain.

The pedunculopontine nucleus (PPN) is a new deep brain stimulation (DBS) target for Parkinson's disease (PD), but little is known about PPN firing pattern alterations in PD. The anesthetized rat is a useful model for investigating the effects of dopamine loss on the transmission of oscillatory cortical activity through basal ganglia structures. After dopamine loss, synchronous oscillatory activity emerges in the subthalamic nucleus and substantia nigra pars reticulata in phase with cortical slow oscillations. To investigate the impact of dopamine cell lesion-induced changes in basal ganglia output on activity in the PPN, this study examines PPN spike timing with reference to motor cortex (MCx) local field potential (LFP) activity in urethane- or ketamine-anesthetized rats. Seven to ten days after unilateral 6-hydroxydopamine lesion of the medial forebrain bundle, spectral power in PPN spike trains and coherence between PPN spiking and PPN LFP activity increased in the approximately 1 Hz range in urethane-anesthetized rats. PPN spike timing also changed from firing predominantly in phase with MCx slow oscillations in the intact urethane-anesthetized rat to firing predominantly antiphase to MCx oscillations in the hemi-parkinsonian rat. These changes were not observed in the ketamine-anesthetized preparation. These observations suggest that dopamine loss alters PPN spike timing by increasing inhibitory oscillatory input to the PPN from basal ganglia output nuclei, a phenomenon that may be relevant to motor dysfunction and PPN DBS efficacy in PD patients.

Excessive beta activity has been shown in local field potential recordings from the cortico-basal ganglia loop of Parkinson's disease patients and in its various animal models. Recent evidence suggests that enhanced beta oscillations may play a central role in the pathophysiology of the disorder and that beta activity may be directly linked to the motor impairment. However, the temporal evolution of exaggerated beta oscillations during the ongoing dopaminergic neurodegeneration and its relation to the motor impairment and histological changes are still unknown. We investigated motor behavioral, in-vivo electrophysiological (subthalamic nucleus, motor cortex) and histological changes (striatum, substantia nigra compacta) 2, 5, 10 and 20-30 days after a 6-hydroxydopamine injection into the medial forebrain bundle in Wistar rats. We found strong correlations between subthalamic beta power and motor impairment. No correlation was found for beta power in the primary motor cortex. Only subthalamic but not cortical beta power was strongly correlated with the histological markers of the dopaminergic neurodegeneration. Significantly increased subthalamic beta oscillations could be detected before this increase was found in primary motor cortex. At the latest observation time point, a significantly higher percentage of long beta bursts was found. Our study is the first to show a strong relation between subthalamic beta power and the dopaminergic neurodegeneration. Thus, we provide additional evidence for an important pathophysiological role of subthalamic beta oscillations and prolonged beta bursts in Parkinson's disease.

An important aspect of aging and Alzheimer's disease (AD) pathology includes the degeneration of basal forebrain cholinergic neurons (BFCNs), possibly due to disrupted nerve growth factor (NGF) signaling. Previous studies on disrupted NGF signaling have focused on changes in retrograde transport. This study focuses on two other possible mechanisms for loss of trophic support: diminished release of NGF from hippocampal neurons or diminished TrkA receptor response of BFCNs to NGF. We measured NGF levels in the effluent of hippocampal slices from young and aged rats in response to potassium chloride and glutamate. We found that release of NGF was not altered in aged hippocampal slices compared to slices from young controls. To measure the in situ response of the BFCNs to NGF, we injected NGF intraparenchymally into the right hippocampus of young and aged rats. Injections of cytochrome C served as controls. Fifteen minutes post-administration, a dramatic increase in TrkA immunoreactivity was found in the cell bodies of medial septal neurons. We found that this rapid response was blunted in aged rats compared to young adult controls. To determine whether retrograde transport was necessary for this rapid response, we injected colchicine prior to NGF injection. The NGF-induced upregulation was not blocked by colchicine, suggesting that this acute response was not dependent on classical retrograde transport. Since cholinergic degeneration coupled with altered levels of NGF and TrkA receptors are also seen in human aging and AD, the loss of acute responsivity to NGF in the BFCNs may also play a role in these processes.