Basal Forebrain Neurons Research Articles

Low voltage-activated calcium and fast tetrodotoxin-resistant sodium currents define subtypes of cholinergic and noncholinergic neurons in rat basal forebrain

Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT−/GAD− cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca 2+ channel subunits. Ca v3.2 was detected in 31/34 cholinergic neurons and Ca v3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Ca v3.3 mRNA expression, although BF tissue expression was observed. In young rats (2–4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT−/GAD−) possessed fast TTX-R sodium currents resembling those mediated by Na + channel subunit Na v1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT−/GAD− neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function.

Acetylcholine release is elicited in the visual cortex, but not in the prefrontal cortex, by patterned visual stimulation: A dual in vivo microdialysis study with functional correlates in the rat brain

By its projections to the primary visual and the prefrontal cortices, the basal forebrain cholinergic system is involved in cognitive processing of sensory stimuli. It has been suggested that visual stimulus-induced cholinergic activation of the visual cortex may exert a permissive role on thalamocortical inputs. However, it is not known if visual stimulation elicits cholinergic activation of high-order brain areas in the absence of attentional need. In the present study, we measured the effects of patterned visual stimulation (horizontal grating) on the release of acetylcholine with dual-probe in vivo microdialysis in the visual and the prefrontal cortices of anesthetized rats. We also used retrograde tracing to determine the anatomical relationships of cholinergic neurons with neurons of the visual system and the prefrontal cortex. Finally, we evaluated a functional correlate of this stimulation, namely c-fos immunolabeling. Patterned visual stimulation elicited significant increases in acetylcholine release in the visual cortex, accompanied by an increased number of c-fos immunoreactive neurons in this brain area. In contrast, in the prefrontal cortex, neither the level of acetylcholine release nor the number of c-fos immunoreactive neurons was significantly changed because of the stimulation. Cholinergic basal forebrain neurons projecting to the visual or the prefrontal cortices were both localized within the horizontal limb of the diagonal band of Broca but were not immunoreactive for c-fos during visual stimulation. No parts of the visual system were found to directly project to these basal forebrain neurons. These results suggest the differential involvement of cholinergic projections in the integration of sensory stimuli, depending on the level of activity of the targeted cortical area.

Trking Down an Inside Job

Nerve growth factor (NGF) binds to the TrkA receptor tyrosine kinase at the cell surface, leading to receptor autophosphorylation and activation of intracellular signaling pathways. However, ligand binding to some G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs), such as the adenosine A 2A receptor or the pituitary adenylate cyclase-activating polypeptide (PACAP) PAC1 receptor, can independently stimulate TrkA phosphorylation and activation of TrkA signaling pathways. Rajagopal et al. used phosphotyrosine-specific antibodies on pan-Trk immunoprecipitates of PC12-615 cells to investigate TrkA phosphorylation in response to adenosine and observed that forms of the TrkA receptor that are not targeted to the plasma membrane were phosphorylated first. Inhibitors of transcription or translation blocked phosphorylation in response to the adenosine agonist CGS 21680 but not phosphorylation induced by NGF; reverse transcription polymerase chain reaction (RT-PCR) indicated that CGS 21680 treatment did not elicit NGF production. Cell surface biotinylation experiments indicated that CGS 21680 activated an intracellular pool of Trk receptors. Immunofluorescence analysis in combination with confocal microscopy revealed that, whereas NGF treatment stimulated TrkA phosphorylation at the plasma membrane, CGS 21680 activated perinuclear TrkA associated with the Golgi apparatus. Moreover, pharmacological inhibition of Golgi vesicle fusion abolished perinuclear TrkA immunoreactivity and blocked CGS 21680-dependent TrkA phosphorylation and the downstream phosphorylation of Akt. PACAP stimulated a similar pattern of perinuclear immunofluorescence in primary cultures of basal forebrain neurons. Thus, GPCR-dependent activation of TrkA appears to take place in an intracellular compartment, a finding the authors discuss in the context of retrograde signaling from internalized TrkA receptors. R. Rajagopal, Z.-Y. Chen, F. S. Lee, M. V. Chao, Transactivation of Trk neurotrophin receptors by G-protein-coupled receptor ligands occurs on intracellular membranes. J. Neurosci . 24 , 6650-6658 (2004). [Abstract] [Full Text]

Antagonist of the amylin receptor blocks beta-amyloid toxicity in rat cholinergic basal forebrain neurons.

Salvage of cholinergic neurons in the brain through a blockade of the neurotoxic effects of amyloidbeta protein (Abeta) is one of the major, but still elusive, therapeutic goals of current research in Alzheimer's disease (AD). To date, no receptor has been unequivocally identified for Abeta. Human amylin, which acts via a receptor composed of the calcitonin receptor-like receptor and a receptor-associated membrane protein, possesses amyloidogenic properties and has a profile of neurotoxicity that is strikingly similar to Abeta. In this study, using primary cultures of rat cholinergic basal forebrain neurons, we show that acetyl-[Asn30, Tyr32] sCT(8-37) (AC187), an amylin receptor antagonist, blocks Abeta-induced neurotoxicity. Treatment of cultures with AC187 before exposure to Abeta results in significantly improved neuronal survival as judged by MTT and live-dead cell assays. Quantitative measures of Abeta-evoked apoptotic cell death, using Hoechst and phosphotidylserine staining, confirm neuroprotective effects of AC187. We also demonstrate that AC187 attenuates the activation of initiator and effector caspases that mediate Abeta-induced apoptotic cell death. These data are the first to show that expression of Abeta toxicity may occur through the amylin receptor and suggest a novel therapeutic target for the treatment of AD.

Decreased neurogenesis after cholinergic forebrain lesion in the adult rat.

Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival-promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.

Muscarinic cholinergic contribution to memory consolidation: with attention to involvement of the basolateral amygdala.

The central cholinergic system and muscarinic cholinergic receptor (mR) activation have long been associated with cognitive function. And degeneration of the cholinergic basal forebrain (CBF) neurons is a pronounced hallmark of Alzheimer's Disease (AD). However, CBF immunolesions as animal models of AD cholinergic degeneration have not replicated the robust memory deficits of nonselective excitotoxic lesions. The less studied cholinergic projections to the amygdala, which are affected in AD but unaffected by immunolesions, may be more important in memory storage than previously suspected. The sparing of these amygdalopetal projections may help explain the dissociation between excitotoxic and immunotoxic CBF lesions. The CBF projections to cortex have since been shown to be important for attentional processes, which may contribute indirectly to memory. Nonetheless, there are conditions under which their selective ablation produces clear memory deficits. For example, memory enhancement induced by posttraining basolateral amygdalar activation is ineffective when corticopetal cholinergic projections are lesioned. Moreover, posttraining cholinergic agonism enhances long-term memory. Such findings suggest that cholinergic innervation of the cortex may be particularly important during modulation of memory storage for stressful and/or arousing events. In concordance, mR agonism facilitates neuronal plasticity and can induce expression of memory-associated immediate early genes. The present article reviews the behavior, physiology and inducible genetic expression literatures which together suggest that the early CBF lesion data were not a red herring but rather that CBF projections not only to cortex but also to the amygdala may in fact have important neuromodulatory functions in memory consolidation processes.

Sleep-wake related discharge properties of basal forebrain neurons recorded with micropipettes in head-fixed rats.

The basal forebrain has been shown to play an important role in cortical activation of wake and paradoxical sleep (PS), yet has also been posited to play a role in slow wave sleep (SWS). In an effort to determine whether these different roles may be fulfilled by different cell groups, including cholinergic and GABAergic cells, we recorded from 123 units in waking-sleeping, head-fixed rats using micropipettes to allow juxtacellular labeling. Functional sets of intermingled cell groups emerged as units whose discharge was as follows: 1) maximum in active wake (aW) and positively or not correlated with EEG gamma activity, while positively correlated with nuchal EMG activity, and thus potentially facilitatory for waking and behavioral arousal (12%); 2) maximum in SWS or SWS-PS and positively correlated with delta EEG activity, while not or negatively correlated with EMG activity, and thus potentially promotive for sleep with cortical slow wave activity and/or accompanying behavioral changes (16%); 3) maximum in PS or PS and aW and positively correlated with gamma and theta EEG activity, while negatively or not correlated with EMG activity, and thus potentially promotive for cortical activation during PS or PS and W (62%); and 4) equivalent across all states and thus not involved in state regulation ( approximately 10%). Units of each group also manifested different firing patterns typified as slow tonic (19.5%), fast tonic (32.5%), or fast phasic (48%), including rhythmic bursting (6%). Through these diverse cell groups, the basal forebrain has the capacity to modulate cortical activity, behavior, and/or related physiological processes across the sleep-waking cycle and thereby regulate the sleep-wake state of the animal.

Impact of the selective estrogen receptor modulator, tamoxifen, on neuronal outgrowth and survival following toxic insults associated with aging and Alzheimer's disease

We investigated the estrogen agonist/antagonist properties of the selective estrogen receptor modulators (SERMs), tamoxifen (TMX) and 4-hydroxy-tamoxifen (OHT), using an in vitro neuron model system to determine the impact of the neuroprotective and neurotrophic properties of these SERMs. Low concentrations of TMX or OHT were without effect on a marker of neuronal viability, basal release of lactate dehydrogenase (LDH), whereas high concentrations of both SERMs (2500 ng/ml) induced a significant increase in LDH, indicating the potential toxicity of both SERMs at high concentrations. Subsequent experiments revealed that subtoxic concentrations of both TMX and OHT induced significant neuroprotection against β-amyloid 25–35-induced toxicity; 15–20% and 10–15% ( P < 0.05), respectively and also against glutamate-induced toxicity; 25–30% and 20–40% ( P < 0.05 and P < 0.01), respectively. Additional in vitro experiments included analysis of neuron survival to determine whether the SERM, OHT, acted competitively or synergistically with the endogenous estrogen, 17 β-estradiol (E 2). These revealed that neuron survival following exposure to the neurotoxins β-amyloid and excitotoxic glutamate was significantly increased in cultures treated with OHT (50 ng/ml) (10%, P < 0.01) and that the magnitude of survival was equivalent to E 2 (10 ng/ml). The combined presence of OHT and E 2 significantly protected against both β-amyloid 25–35 and excitotoxic glutamate-induced neuron death (10%, P < 0.01) but was not significantly different from either OHT or E 2 alone. To assess neurotrophic effects of these same SERMs, cultured neurons from brain regions involved in memory function and Alzheimer's disease were evaluated by morphological analysis of individual neurons. Results of these analyses demonstrated that TMX treatment did not significantly increase the process outgrowth or morphological complexity of cortical, hippocampal, or basal forebrain neurons. Similar analyses showed that OHT also failed to significantly increase the neuronal outgrowth of either cortical or hippocampal neurons. Results of these studies predict that TMX and OHT could exert a neuroprotective function but would not promote estrogen-dependent memory function.

Differential regulation of neurotrophin expression in basal forebrain astrocytes by neuronal signals.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3) promote the function and/or survival of basal forebrain (BF) cholinergic neurons in vivo and in culture. The neurotrophin source is commonly thought to be targets of cholinergic neurons and the possibility that local glial sources support cholinergic neurons has not been well examined. These sources, however, may be critical to BF neurons before or even after they reach their targets. We investigated neurotrophin expression in BF astrocytes and its regulation by neural signals. Solution hybridization and immunocytochemical assays revealed that NGF, BDNF, and NT(3) mRNA and proteins were expressed in cultured BF astrocytes. To investigate roles of neuronal signals in neurotrophin regulation, effects of K(+), glutamate, and the cholinergic agonist carbachol were examined. These stimuli affected neurotrophin expression differentially. KCl increased BDNF mRNA but did not alter NGF or NT(3) mRNA. The effect was blocked by nifedipine, suggesting that it was mediated by L-type voltage-dependent calcium currents. Carbachol also increased BDNF mRNA levels without changing NGF or NT(3). Effects were blocked by the muscarinic antagonist, atropine. In contrast, glutamate increased both NGF and BDNF mRNA. NT(3) mRNA again was unaffected. The metabotropic agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) reproduced glutamate effects, whereas kainate or N-methyl-D-aspartate (NMDA) plus glycine did not. Lack of antagonism by ionotropic antagonists and blockade of glutamate effects by metabotropic antagonists confirmed metabotropic mediation. We suggest that BF astrocytes are local sources of neurotrophins for BF cholinergic neurons during development and are regulated differentially by specific neuronal signals. Critical neuronal-glial interactions may underlie basal forebrain function.

Reinforcement-related neurons in the primate basal forebrain respond to the learned significance of task events rather than to the hedonic attributes of reward

The objective of this study was to determine if the responses of basal forebrain neurons are related to the cognitive processes necessary for the performance of behavioural tasks, or to the hedonic attributes of the reinforcers delivered to the monkey as a consequence of task performance. In all cases, it was found that the primary neuronal responses were to visual stimuli that required analysis important for the selection of a behavioural response and not to the delivery of reinforcement per se. Indeed, reinforcement-related neurons that responded only to the delivery of juice or of saline were never encountered. In additional experiments, it was found that abstract visual cues—specific gestures of the experimenter—that signaled the impending delivery of reinforcement were able to activate these neurons. These data are consistent with the view that reinforcement-related basal forebrain neurons influence the sensory and motor processes in the cerebral cortex, providing control signals that optimise the processing of complex sensory stimuli and/or the generation of appropriate behavioural responses.

Reduced mitochondrial buffering of voltage-gated calcium influx in aged rat basal forebrain neurons

Alterations of neuronal Ca 2+ homeostatic mechanisms could be responsible for many of the cognitive deficits associated with aging in mammals. Mitochondrial participation in Ca 2+ signaling is now recognized as a prominent feature in neuronal physiology. We combined voltage-clamp electrophysiology with Ca 2+-sensitive ratiometric microfluorimetry and laser scanning confocal microscopy to investigate the participation in Ca 2+ buffering of in situ mitochondria in acutely dissociated basal forebrain neurons from young and aged F344 rats. By pharmacologically blocking mitochondrial Ca 2+ uptake, we determined that mitochondria were not involved in rapid buffering of small Ca 2+ influx through voltage-gated Ca 2+ channels (VGCCs) in the somatic compartment. For larger Ca 2+ influx, aged mitochondria showed a significant buffering deficit. Evidence obtained with the potentiometric indicator, JC-1, suggests a significantly reduced mitochondrial membrane potential in aged neurons. These results support the interpretation that there is a fundamental difference in the way young and aged neurons buffer Ca 2+, and a corresponding difference in the quality of the Ca 2+ signal experienced by young and aged neurons for different intensities of cytoplasmic Ca 2+ influx.

Modulation of the excitability of cholinergic basal forebrain neurones by KATP channels.

The expression of ATP-sensitive K(+) (K(ATP)) channels by magnocellular cholinergic basal forebrain (BF) neurones was investigated in thin brain slice and dissociated cell culture preparations using a combination of whole-cell, perforated-patch and single-channel recording techniques. Greater than 95% of BF neurones expressed functional K(ATP) channels whose activation resulted in membrane hyperpolarization and a profound fall in excitability. The whole-cell K(ATP) conductance was 14.0 +/- 1.5 nS and had a reversal potential of -91.4 +/- 0.9 mV that shifted by 59.6 mV with a tenfold increase in [K(+)](o). I(KATP) was inhibited reversibly by tolbutamide (IC(50) of 34.1 microM) and irreversibly by glibenclamide (0.3-3 nM) and had a low affinity for [ATP](i) (67% reduction with 6 mm[MgATP](i)). Using perforated-patch recording, a small proportion of the conductance was found to be tonically active. This was weakly potentiated by diazoxide (0.1 mm extracellular glucose) but insensitive to pinacidil (< or =500 microM). Single-channel K(ATP) currents recorded in symmetrical 140 mm K(+)-containing solutions exhibited weak inward rectification with a mean conductance of 66.2 +/- 1.9 pS. Channel activity was inhibited by MgATP (>50 microM) and activated by MgADP (200 microM). The K(+) channels opener diazoxide (200-500 microM) increased channel opening probability (NP(o)) by 486 +/- 120% whereas pinacidil (500 microM) had no effect. In conclusion, the characteristics of the K(ATP) channels expressed by BF neurones are very similar to channels composed of SUR1 and Kir6.2 subunits. In the native cell, their affinity for ATP is close to the resting [ATP](i), potentially allowing them to be modulated by physiologically relevant changes in [ATP](i). The effect of these channels on the level of ascending cholinergic excitation of the cortex and hippocampus is discussed.

Cholinergic modulation of the cortical microvascular bed

Cortical microvessels receive a cholinergic input that originates primarily from basal forebrain neurons which, upon stimulation, induce significant increases in cortical perfusion together with a dilation of intracortical microvessels. Heterogeneous mAChRs have been detected in cortical microvessels with expression of M2 and M5 subtypes in endothelial cells, while M1 and M3, and possibly M5 mAChR subtypes, were expressed in smooth muscle cells. Application of ACh to isolated and pressurized microarterioles, whether at basal tone or pharmacologically preconstricted, elicited only a dilation. This response was dependent on NO production, and was mediated by a mAChR, the pharmacology of which correlated best with the M5 receptor subtype. ACh afferents also project to intracortical neurons that synthesize NO and VIP. These correspond to distinct sub-populations of GABA interneurons which were found to send numerous projections to local microvessels. Preliminary results suggest expression of the VPAC1 receptor in the smooth muscle cells of intracortical arterioles, where it could mediate dilation as it does in cerebral arteries. Together these results indicate that basal forebrain ACh fibers can directly affect the cortical microvascular bed, but further suggest that specific populations of GABA interneurons could serve as a functional relay to adapt perfusion to locally increased neuronal activity. In confirmed cases of Alzheimer's disease, we found a severe ACh denervation of both cortical microvessels and NO neurons, suggesting that two important regulators of cortical perfusion are dysfunctional in this pathology.

Activity, modulation and role of basal forebrain cholinergic neurons innervating the cerebral cortex

The basal forebrain constitutes the ventral extra-thalamic relay from the brainstem activating system to the cerebral cortex. Cholinergic neurons form an important contingent of this relay, yet represent only a portion of the cortically projecting and other basal forebrain neurons, which include GABAergic neurons. By recording, labeling and identifying neurons by their neurotransmitter first in vitro and then in vivo, we have determined that the cholinergic neurons have different physiological and pharmacological properties than other codistributed neurons. Cholinergic neurons discharge at higher rates during cortical activation than during cortical slow wave activity, and are excited by transmitters released from brainstem afferent neurons, including glutamate from reticular formation, noradrenaline (NA) from locus coeruleus, and orexin and histamine from posterior hypothalamus. In contrast, particular GABAergic neurons discharge at higher rates during cortical slow wave activity and are inhibited by NA. When NA is administered into the basal forebrain in naturally sleeping-waking rats, it elicits an increase in fast gamma EEG activity and diminution of slow delta EEG activity while promoting waking and eliminating slow wave sleep (SWS). Cholinergic neurons also have the capacity to discharge in rhythmic bursts when activated by particular agonists, notably neurotensin (NT). When NT is administered into the basal forebrain, it stimulates theta activity in addition to gamma while promoting waking and paradoxical sleep (PS). By increasing discharge and firing in rhythmic bursts in response to transmitters of the activating systems, cholinergic neurons can thus stimulate cortical activation with gamma and theta activity along with the states of waking and PS. Colocalized GABAergic basal forebrain neurons which are inhibited by transmitters of the arousal systems can oppose these actions and promote delta activity and SWS.

Netrin 1-mediated chemoattraction regulates the migratory pathway of LHRH neurons.

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the vomeronasal organ (VNO) to the forebrain in all mammals studied. In mice, the direction of LHRH neuron migration is dependent upon axons that originate in the VNO, but bypass the olfactory bulb and project caudally into the basal forebrain. Thus, factors that guide this unique subset of vomeronasal axons that comprise the caudal vomeronasal nerve (cVNN) are candidates for regulating the migration of LHRH neurons. We previously showed that deleted in colorectal cancer (DCC) is expressed by neurons that migrate out of the VNO during development [Schwarting et al. (2001) J. Neurosci., 21, 911-919]. We examined LHRH neuron migration in Dcc-/- mice and found that trajectories of the cVNN and positions of LHRH neurons are abnormal. Here we extend these studies to show that cVNN trajectories and LHRH cell migration in netrin 1 (Ntn1) mutant mice are also abnormal. Substantially reduced numbers of LHRH neurons are found in the basal forebrain and many LHRH neurons migrate into the cerebral cortex of Ntn1 knockout mice. In contrast, migration of LHRH cells is normal in Unc5h3rcm mutant mice. These results are consistent with the idea that the chemoattraction of DCC+ vomeronasal axons by a gradient of netrin 1 protein in the ventral forebrain guides the cVNN, which, in turn, determines the direction of LHRH neuron migration in the forebrain. Loss of function through a genetic deletion in either Dcc or Ntn1 results in the migration of many LHRH neurons to inappropriate destinations.

GABAergic basal forebrain neurons which express receptor for neurokinin B and send axons to the cerebral cortex.

GABAergic basal forebrain neurons which express receptor for neurokinin B and send axons to the cerebral cortex.

The presence of the app swe mutation in mice does not increase the vulnerability of cholinergic basal forebrain neurons to neuroinflammation

Neuroinflammation, and elevated levels of inflammatory proteins, such as tumor necrosis factor-α, and the deposition of β-amyloid may interact to contribute to the pathogenesis of Alzheimer's disease. We reproduced a component of the neuroinflammatory state within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease, of transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein (APPswe). We have previously shown that basal forebrain cholinergic neurons are selectively vulnerable to the consequences of neuroinflammation. In the current study, tumor necrosis factor-α was infused into the basal forebrain region of APPswe and nontransgenic control mice for 20 days with the expectation that the presence of the transgene would enhance the loss of cholinergic neurons. Chronic infusion of tumor necrosis factor-α significantly decreased cortical choline acetyltransferase activity, reduced the number of choline acetyltransferase-immunoreactive cells and increased the number of activated astrocytes and microglia within the basal forebrain. The presence of the APPswe gene did not enhance the vulnerability of forebrain cholinergic neurons to the chronic neuroinflammation. Furthermore, combined treatment of these mice with memantine demonstrated that the neurotoxic effects of tumor necrosis factor-α upon cholinergic cells did not require the activation of the N-methyl- d-aspartate receptors. In contrast, we have previously shown that memantine was able to provide neuroprotection to cholinergic forebrain neurons from the consequences of exposure to the inflammogen lipopolysaccharide. These results provide insight into the mechanism by which neuroinflammation may selectively target specific neural systems during the progression of Alzheimer's disease.

Basal forebrain neurons modulate the synthesis and expression of neuropeptides in the rat suprachiasmatic nucleus

We tested the hypothesis that efferents from the nucleus basalis magnocellularis (NBM) play a direct role in the regulation of neuropeptide synthesis and expression by neurons of the rat suprachiasmatic nucleus (SCN). Adult male rats in which the NBM was destroyed with quinolinic acid, either unilaterally or bilaterally, were compared with rats injected with physiological saline and with control rats. The estimators used to assess the effects of cholinergic deafferentation on the neuroanatomy and neurochemistry of the SCN were the total number of SCN neurons, the total number and somatic size of SCN neurons producing vasopressin (VP) and vasoactive intestinal polypeptide (VIP), and the respective mRNA levels. Bilateral destruction of the NBM did not produce cell death in the SCN, but caused a marked reduction in the number and somatic size of SCN neurons expressing VP and VIP, and in the mRNA levels of these peptides. The decrease in the number of VP- and VIP-producing neurons provoked by unilateral lesions was less striking than that resulting from bilateral lesions. It was, however, statistically significant in the ipsilateral hemisphere, but not in the contralateral hemisphere. The results show that the reduction of cholinergic inputs to the SCN impairs the synthesis, and thereby decreases the expression of neuropeptides by SCN neurons, and that the extent of the decline correlates with the amount of cholinergic afferents destroyed. This supports the notion that acetylcholine plays an important, and direct role in the regulation of the metabolic activity of SCN neurons.

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone [J Neurochem 66 (1996) 804]. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.

Phylogenetic and ontogenetic aspects of the basal forebrain cholinergic neurons and their innervation of the cerebral cortex

This chapter discusses the phylogenetic and ontogenetic aspects of the basal forebrain (BF) cholinergic neurons. The BF cholinergic system, consisting of a cluster of cholinergic neurons in the ventral telencephalon and their projections to the cortex or pallium, is present in tetrapods (mammals, birds, reptiles, and amphibians) and bony fishes. In contrast, the cholinergic BF system appears to be absent in selected orders of fishes that were derived earlier from the common ancestral lineage of vertebrates. These include chondrosteans as represented by sturgeon, elasmobranchs as represented by dogfish, and cyclostomes as represented by lampreys. Despite this conservation, variation exists among terrestrial vertebrates. First, the size and complexity of the cholinergic BF system exhibit a phylogenetic graduation, in parallel with general evolution of the brain. Specifically, the number of cholinergic BF neurons appears to be relatively small in fishes, whereas it is the highest in humans, and possibly in cetaceans. Similarly, the area of innervation, that is, the cortex, increases in size, and also in complexity as reflected by the formation of multiple layers. Both phylogenetic and ontogenetic approaches have been used in the past to understand the formation and organization of the brain. The phylogenetic approach aims at understanding how the brains of living animals evolved through interaction of both genetic and environmental factors during phylogeny. The ontogenetic approach uncovers cellular and molecular processes that underlie the formation of the brain.