The organization of cholinergic projections in the visual thalamus of the mouse.

Cholinergic projections from the brainstem serve as important modulators of activity in visual thalamic nuclei such as the dorsal lateral geniculate nucleus (dLGN). While these projections have been studied in several mammals, a comprehensive examination of their organization in the mouse is lacking. We used the retrograde transport of viruses or cholera toxin subunit B (CTB) injected in the dLGN, immunocytochemical labeling with antibodies against choline acetyltransferase (ChAT), brain nitric oxide synthase (BNOS), and vesicular acetylcholine transporter (VAChT), ChAT-Cre mice crossed with a reporter line (Ai9), as well as brainstem virus injections in ChAT-Cre mice to examine the pattern of thalamic innervation from cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus (LDTg), and the parabigeminal nucleus (PBG). Retrograde tracing demonstrated that the dLGN receives input from the PPTg, LDTg, and PBG. Viral tracing in ChAT-Cre mice and retrograde tracing combined with immunocytochemistry revealed that many of these inputs originate from cholinergic neurons in the PBG and PPTg. Most notable was an extensive cholinergic projection from the PBG which innervated most of the contralateral dLGN, with an especially dense concentration in the dorsolateral shell, as well as a small region in the dorsomedial pole of the ipsilateral dLGN. The PPTg was found to provide a sparse somewhat diffuse innervation of the ipsilateral dLGN. Neurons in the PPTg co-expressed ChAT, BNOS, and VAChT, whereas PBG neurons expressed ChAT, but not BNOS or VAChT. These results highlight the presence of distinct cholinergic populations that innervate the mouse dLGN.

Elaeagnus glabra f. oxyphylla Attenuates Scopolamine-Induced Learning and Memory Impairments in Mice by Improving Cholinergic Transmission via Activation of CREB/NGF Signaling.

We aimed to investigate the therapeutic effects of an Elaeagnus glabra f. oxyphylla (EGFO) ethanol extract in mice with scopolamine-induced memory dysfunction. Fifty male mice were randomly divided into a normal control group, a scopolamine-treated group, a scopolamine and EGFO extract-treated group, and a scopolamine and tacrine-treated group. EGFO (50 or 100 mg/kg/day) was received for 21 days. Step-through passive avoidance and Y-maze tests were performed to examine the effects of treatment on learning and memory impairments. Acetylcholine (Ach) levels and acetylcholinesterase (AchE) activity were measured via an enzyme-linked immunosorbent assay (ELISA). Levels of choline acetyltransferase (ChAT), nerve growth factor (NGF), cAMP response element-binding protein (CREB), and apoptosis-related protein expression were determined via Western blot analysis. EGFO pretreatment significantly attenuated scopolamine-induced memory impairments, relative to findings observed in the scopolamine-treated group. Levels of cholinergic factors in the brain tissues were markedly attenuated in the scopolamine-treated group. EGFO treatment also attenuated neural apoptosis in scopolamine-treated mice by decreasing the expression of apoptosis-related proteins such as Bax, Bcl2, cleaved caspase-3, and TUNEL staining. These results suggest that EGFO improves memory and cognition in a mouse model of memory impairment by restoring cholinergic and anti-apoptotic activity, possibly via activation of CREB/NGF signaling.

Bushen-Yizhi formula ameliorates cognitive dysfunction through SIRT1/ER stress pathway in SAMP8 mice.

The Chinese formula Bushen-Yizhi (BSYZ) has been reported to ameliorate cognitive dysfunction. However the mechanism is still unclear. In this study, we employ an aging model, SAMP8 mice, to explore whether BSYZ could protect dementia through SIRT1/endoplasmic reticulum (ER) stress pathway. Morris water maze and the fearing condition test results show that oral administration of BSYZ (1.46 g/kg/d, 2.92 g/kg/d and 5.84 g/kg/d) and donepezil (3 mg/kg/d) shorten the escape latency, increase the crossing times of the original position of the platform and the time spent in the target quadrant, and increase the freezing time. BSYZ decreases the activity of acetylcholinesterase (AChE), and increases the activity of choline acetyltransferase (ChAT) and the concentration of acetylcholine (Ach) in both hippocampus and cortex. In addition, western blot results (Bcl-2, Bax and Caspase-3) and TUNEL staining show that BSYZ prevents neuron from apoptosis, and elevates the expression of neurotrophic factors, including nerve growth factor (NGF), postsynapticdensity 95 (PSD95) and synaptophysin (SYN), in both hippocampus and cortex. BSYZ also increases the protein expression of SIRT1 and alleviates ER stress-associated proteins (PERK, IRE-1α, eIF-2α, BIP, PDI and CHOP). These results indicate that the neuroprotective mechanism of BSYZ might be related with SIRT1/ER stress pathway.

Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation.

Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2(+) progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling(+) apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction of cholinergic inputs into granule cell lineages and/or GABAergic interneurons as indicated by decreased transcript levels of Chrnb2 and numbers of Chrnb2(+) interneurons caused by myelin vacuolation in the septal-hippocampal pathway.

Alleviating effects of Bushen-Yizhi formula on ibotenic acid-induced cholinergic impairments in rat.

This study explored the curative effect and underlying mechanisms of a traditional Chinese medicine compound prescription, Bushen-Yizhi formula (BSYZ), in ibotenic acid (IBO)-induced rats. Morris water maze and novel object recognition tests showed that BSYZ significantly improved spatial and object memory. Brain immunohistochemistry staining showed that BSYZ significantly up-regulated expression of choline acetyltransferase (ChAT) and nerve growth factor (NGF) in the hippocampus and cortex. The protein tyrosine kinase high-affinity receptor TrkA was slightly increased in the hippocampus and cortex, and significantly enhanced in the nucleus basalis of Meynert (NBM) after BSYZ intervention. The immunoreactivity of the p75 low-affinity receptor in BSYZ-treated rats was significantly strengthened in the cortex. Similar expression trends of nerve growth factor (NGF), TrkA, and p75 mRNA were observed in the hippocampus and cortex. Additionally, BSYZ reversed IBO-induced disorders of acetylcholine (ACh) levels, ChAT, and cholinesterase (ChE) in the cortex, which was consistent with the changes in mRNA levels of ChAT and acetylcholinesterase (AChE). Expression of ChAT and AChE proteins and mRNA in the hippocampus was up-regulated, whereas the apoptosis-relative protein cleaved caspase-3 was decreased after administration of BSYZ. Moreover, changes in cell death were confirmed by histological morphology. Thus, the results indicated that the BSYZ formula could ameliorate memory impairments in IBO-induced rats, and it exerted its therapeutic action probably by modulating cholinergic pathways, NGF signaling, and anti-apoptosis. Overall, it is suggested that the BSYZ formula might be a potential therapeutic approach for the treatment of Alzheimer's disease (AD) and other cholinergic impairment-related diseases.

Extracts of Cordyceps militaris lower blood glucose via the stimulation of cholinergic activation and insulin secretion in normal rats.

Previous studies have shown that Cordyceps militaris (CM) has a hypoglycemic effect, but the actual mechanism remains unclear. This study explored the hypoglycemic mechanism of aqueous extracts of CM in normal Wistar rats. First, the optimal dose of CM for lowering plasma glucose and insulin secretion was tested. Further, atropine and hemicholinium-3 (HC-3) were injected and a western blot was used to investigate insulin signaling. It was found that 10 mg/kg CM extracts had a stronger hypoglycemic effect than a higher dose (100 mg/kg); therefore, a dose of 10 mg/kg was used in subsequent experiments. In normal rats, CM extracts decreased plasma glucose by 21.0% and induced additional insulin secretion by 54.5% after 30 min. When atropine or HC-3 was injected, CM induced a hypoglycemic effect, but the enhancement of insulin secretion was blocked. By western blotting, significant increases in the insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) were observed after CM feeding. However, the elevation of these signaling proteins was abolished by atropine or HC-3. Taken together, these findings indicate that CM can lower plasma glucose via the stimulation of insulin secretion and cholinergic activation involved in the hypoglycemic mechanism of normal Wistar rats.

Optogenetic release of ACh induces rhythmic bursts of perisomatic IPSCs in hippocampus.

Acetylcholine (ACh) influences a vast array of phenomena in cortical systems. It alters many ionic conductances and neuronal firing behavior, often by regulating membrane potential oscillations in populations of cells. Synaptic inhibition has crucial roles in many forms of oscillation, and cholinergic mechanisms regulate both oscillations and synaptic inhibition. In vitro investigations using bath-application of cholinergic receptor agonists, or bulk tissue electrical stimulation to release endogenous ACh, have led to insights into cholinergic function, but questions remain because of the relative lack of selectivity of these forms of stimulation. To investigate the effects of selective release of ACh on interneurons and oscillations, we used an optogenetic approach in which the light-sensitive non-selective cation channel, Channelrhodopsin2 (ChR2), was virally delivered to cholinergic projection neurons in the medial septum/diagonal band of Broca (MS/DBB) of adult mice expressing Cre-recombinase under the control of the choline-acetyltransferase (ChAT) promoter. Acute hippocampal slices obtained from these animals weeks later revealed ChR2 expression in cholinergic axons. Brief trains of blue light pulses delivered to untreated slices initiated bursts of ACh-evoked, inhibitory post-synaptic currents (L-IPSCs) in CA1 pyramidal cells that lasted for 10's of seconds after the light stimulation ceased. L-IPSC occurred more reliably in slices treated with eserine and a very low concentration of 4-AP, which were therefore used in most experiments. The rhythmic, L-IPSCs were driven primarily by muscarinic ACh receptors (mAChRs), and could be suppressed by endocannabinoid release from pyramidal cells. Finally, low-frequency oscillations (LFOs) of local field potentials (LFPs) were significantly cross-correlated with the L-IPSCs, and reversal of the LFPs near s. pyramidale confirmed that the LFPs were driven by perisomatic inhibition. This optogenetic approach may be a useful complementary technique in future investigations of endogenous ACh effects.

Determination of high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in the same population of cultured cells.

Cholinergic neurons are a major constituent of the mammalian central nervous system. Acetylcholine, the neurotransmitter used by cholinergic neurons, is synthesized from choline and acetyl CoA by the enzymatic action of choline acetyltransferase (ChAT). The transport of choline into the cholinergic neurons, which results in synthesis of ACh, is hemicholinium-sensitive and is referred to as high-affinity choline uptake (HACU). Thus, the formation of acetylcholine in cholinergic neurons largely depends on both the levels of choline being transported into the cells from the extracellular space and the activity of ChAT. Several methods were described previously to measure HACU and ChAT simultaneously in synaptosomes, but the same for cultured cells is lacking. We describe a procedure to measure HACU and ChAT at the same time in cultured cells by simple techniques employing radionuclides. In this procedure, we determined quantitatively hemicholinium-sensitive choline uptake and ChAT enzyme activity in a small number of differentiated human neuroblastoma (SK-N-SH) cells. We also determined the kinetics of choline uptake in the SK-N-SH cells. We believe that these simple methods can be used for neurochemical and drug discovery studies in several models of neurodegenerative disorders including Alzheimer's disease.

Neonatal stress affects vulnerability of cholinergic neurons and cognition in the rat: involvement of the HPA axis.

Adverse experiences early in life may sensitize specific neurocircuits to subsequent stressors. We have evaluated in maternal separation (MS) rats, an animal paradigm of early-life stress, the effects of a selective cholinergic lesion on cognitive function as well as susceptibility of cholinergic neurons to the lesion. MS rats subjected to a cholinergic lesion by administration of the immunotoxin 192 IgG-saporin, showed significant decreases in both choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity compared to control lesioned rats. Morris water maze results revealed a significant impairment in learning and memory function in MS adult rats and further cognitive deficits after the lesion. The lesion of cholinergic neurons induced a significant decrease in glucocorticoid receptor density in MS rats, accompanied by increases in CRF mRNA expression. Decreases in NGF and increases in NGF-p75NTR expression have also been found in MS rats. Our results suggest that vulnerability of basal forebrain cholinergic nerve cells might be affected by the HPA axis. The present data are discussed not only in terms of conditions that occur during ageing or Alzheimer disease, but also regarding a purported involvement of the cholinergic system in the regulation of HPA axis activity.

Age-related responses of the rat cerebral cortex: influence of vitamin E and exercise on the cholinergic system.

In this study, we have assessed the impact of vitamin E and exercise on acquisition and retention of spatial memory for a given task in aging rats, using a T-maze. Acetylcholine esterase (AChE) and cholineacetyl transferase (ChAT) activities and acetylcholine (ACh) were measured in the cerebral cortex (CC) of male Wistar rats of 4- (adult), 12- (middle-aged) and 18-months (old) of age. Animals were categorized into sedentary [(SEC (N)], sedentary supplemented [SEC (+E)], swim trained [SWT (N)] and swim trained supplemented [SWT (+E)]. In the old, ChAT activity increased in the SEC (+E). AChE activity was highest in the adults, irrespective of training or supplementation. By contrast, ACh concentration remained unaltered with age, exercise and supplementation. Middle-aged and old rats were benefited in terms of a better acquisition and retention in the case of those that were trained and supplemented with Vitamin E. Adults showed better retention in all the groups after 7 and 15 days, while in the middle-aged, training was beneficial after 15 days. We observed decreased AChE activity when old rats were trained with the supplement. Our results also suggest that this regimen may be analogous to the AChE inhibitors that are widely advocated to derive positive benefits in up-regulating the possible reduction in ACh and in turn age-associated memory deficits.

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.

Acetylcholine innervation of the adult rat thalamus: distribution and ultrastructural features in dorsolateral geniculate, parafascicular, and reticular thalamic nuclei.

The acetylcholine (ACh) innervation of thalamus arises mainly from the brainstem pedunculopontine and laterodorsal tegmental nuclei. By using immunocytochemistry with a monoclonal antibody against whole rat choline acetyltransferase (ChAT), we quantified the distribution and characterized the ultrastructural features of these nerve terminals (axon varicosities) in the dorsolateral geniculate (DLG), parafascicular (PF), and reticular thalamic (Rt) nuclei of adult rat. The regional density of ACh innervation was the highest in PF (2.1 x 10(6) varicosities/mm(3)), followed by Rt (1.7 x 10(6)) and DLG (1.3 x 10(6)). In single thin sections, ChAT-immunostained varicosity profiles appeared comparable in shape and content in the three nuclei, but significantly larger in PF than in DLG and Rt. The number of these profiles displaying a synaptic junction was also much higher in PF than in DLG and Rt, indicating that all ChAT-immunostained varicosities in PF were synaptic, but only 39% in DLG and 33% in Rt. The hypothesis that glutamate corelease might account for the maintenance of the entirely synaptic ACh innervation in PF was refuted by the lack of colocalization of ChAT and vesicular glutamate transporter 2 (VGLUT2) in PF axon varicosities after dual immunolabeling. These data suggest that diffuse as well as synaptic transmission convey modulatory effects of the ACh input from brainstem to DLG and Rt during waking. In contrast, the entirely synaptic ACh input to PF should allow for a direct relaying of the information from brainstem, affecting basal ganglia function as well as perceptual awareness, including attention and pain perception.

[Effect of Gengnianchun Recipe on learning memory function and hippocampal cholinergic system in ovariectomized rats].

OBJECTIVE: To evaluate the effect of Gengnianchun Recipe (GNC) on learning memory function and its regulatory effect on hippocampal cholinergic system in ovariectomized rats. METHODS: Female rats 10-12 months old were randomized into 5 groups, the sham-operation group, the model group treated with normal saline, the positive control group treated with Nilestriol, and the two GNC groups treated with high and low dose GNC respectively. A little fat around ovary was cut in the sham-operation group. The treatment lasted for 12 weeks after ovariectomy. Changes of learning memory function were tested by Morris water maze; serum level of estradiol (E2) was measured by chemical fluorescent method; hippocampal acetylcholinesterase (AChE) mRNA was determined with Real-time PCR; and the activities of acetylcholine (ACh), AChE and choline acetyltransferase (ChAT) in hippocampus were detected by immunohistochemistry respectively. RESULTS: Twelve weeks after ovariectomy, serum E2 and learning memory function markedly decreased in the ovariectomized rats (P < 0.01, P < 0.05). Nilestriol and high dose GNC showed an effect in improving the symptoms of learning memory functional deprivation and elevating the activities of hippocampal ACh, AChE and ChAT (all P < 0.05). CONCLUSION: GNC can improve learning memory function of ovariectomized rats, and its mechanism might be realized by regulating the cholinergic system in hippocampus.

Changes in neural activity associated with a surprising change in the predictive validity of a conditioned stimulus.

Changes in how well a conditioned stimulus (CS) predicts future events can alter the amount of attention paid to that cue. For example, the unexpected violation of a previously established relationship between a CS and another stimulus can increase attentional processing and subsequent conditioning to that cue [J.M. Pearce & G. Hall (1980)Psych. Rev., 106, 532-552]. Previous lesion studies have implicated the central nucleus of the amygdala (CN) and basal forebrain corticopetal cholinergic system in mediating surprise-induced changes in attention. Here, expression of the immediate-early gene c-fos was used to determine which cortical targets of the basal forebrain cholinergic system are activated during an increase in attentional processing. Consistent with previous studies, increased Fos expression was observed in the posterior parietal cortex (PPC) when a visual stimulus no longer reliably predicted occurrence of a tone. Similar results were observed in the secondary auditory cortex; however, there were no significant changes in Fos expression in other auditory or visual cortices or in other cortical association areas that have been implicated in attentional function (frontal, cingulate or retrosplenial cortex). These findings support the notion that the PPC is the primary cortical component of a neural system mediating incremental changes in attention. In addition, an increase in Fos-positive cells was detected in the substantia innominata/nucleus basalis and the CN at the time of surprise. An opposite pattern of results was observed in the basal lateral nucleus of the amygdala, providing evidence for different stimulus-processing mechanisms in regions of the amygdala.

The pharmacological effects of Daikenchuto, a traditional herbal medicine, on delayed gastrointestinal transit in rat postoperative ileus.

The effect of Daikenchuto, a traditional herbal medicine, on gastrointestinal hypoperistalsis in postoperative ileus (POI) was investigated. POI was induced by laparotomy with manipulation of the gastrointestine under anesthesia, and gastrointestinal transit was calculated by migration of Evans blue. Daikenchuto (270 - 2,700 mg/kg, p.o.) dose-dependently improved the delayed gastrointestinal transit in POI. This effect of Daikenchuto was partially inhibited by SB204070 (1 mg/kg, s.c.), a 5-hydroxytriptamine(4) (5-HT(4))-receptor antagonist and completely abolished by atropine (1 mg/kg, s.c.), a muscarine-receptor antagonist. Among the constituents of Daikenchuto, the medical herb zanthoxylum fruit (60 mg/kg, p.o.) and maltose syrup (2,400 mg/kg, p.o.) significantly ameliorated the delayed gastrointestinal transit, but ginseng and processed ginger did not affect the gastrointestinal transit in the rat POI. The improvement induced by zanthoxylum fruit was also inhibited by atropine or SB204070. In addition, the high osmotic pressure of the maltose syrup (2400 mg/10 mL per kg) was related to the improvement of delayed gastrointestinal transit. These results demonstrated that Daikenchuto ameliorates postoperative hypoperistalsis via cholinergic nerves and 5-HT(4) receptors and that osmotic pressure also may be involved in this action. Moreover, zanthoxylum fruit and maltose syrup were crucial medical herbs contributing to the ability of Daikenchuto.

Neurobiology of REM and NREM sleep.

This paper presents an overview of the current knowledge of the neurophysiology and cellular pharmacology of sleep mechanisms. It is written from the perspective that recent years have seen a remarkable development of knowledge about sleep mechanisms, due to the capability of current cellular neurophysiological, pharmacological and molecular techniques to provide focused, detailed, and replicable studies that have enriched and informed the knowledge of sleep phenomenology and pathology derived from electroencephalographic (EEG) analysis. This chapter has a cellular and neurophysiological/neuropharmacological focus, with an emphasis on rapid eye movement (REM) sleep mechanisms and non-REM (NREM) sleep phenomena attributable to adenosine. The survey of neuronal and neurotransmitter-related brainstem mechanisms of REM includes monoamines, acetylcholine, the reticular formation, a new emphasis on GABAergic mechanisms and a discussion of the role of orexin/hypcretin in diurnal consolidation of REM sleep. The focus of the NREM sleep discussion is on the basal forebrain and adenosine as a mediator of homeostatic control. Control is through basal forebrain extracellular adenosine accumulation during wakefulness and inhibition of wakefulness-active neurons. Over longer periods of sleep loss, there is a second mechanism of homeostatic control through transcriptional modification. Adenosine acting at the A1 receptor produces an up-regulation of A1 receptors, which increases inhibition for a given level of adenosine, effectively increasing the gain of the sleep homeostat. This second mechanism likely occurs in widespread cortical areas as well as in the basal forebrain. Finally, the results of a new series of experimental paradigms in rodents to measure the neurocognitive effects of sleep loss and sleep interruption (modeling sleep apnea) provide animal model data congruent with those in humans.

Differential expression of molecular motors in the motor cortex of sporadic ALS.

The molecular mechanisms underlying the selective neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) are inadequately understood. Recent breakthroughs have implicated impaired axonal transport, mediated by molecular motors, as a key element for disease onset and progression. The current work identifies the expression of 15 kinesin-like motors in healthy human motor cortex, including three novel isoforms. Our comprehensive quantitative mRNA analysis in control and sporadic ALS (SALS) motor cortex specimens detects SALS-specific down-regulation of KIF1Bbeta and novel KIF3Abeta, two isoforms we show to be enriched in the brain, and also of SOD1, a key enzyme linked to familial ALS. This is accompanied by a marked reduction of KIF3Abeta protein levels. In the motor cortex KIF3Abeta localizes in cholinergic neurons, including upper motor neurons. No mutations causing splicing defects or altering protein-coding sequences were identified in the genes of the three proteins. The present study implicates two motor proteins as possible candidates in SALS pathology.

Cholinergic neuronal deficits in CADASIL.

BACKGROUND AND PURPOSE: Previous evidence from MRI and acetylcholinesterase histochemistry suggests cholinergic fibers are affected in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: As a measure of cholinergic function, we assessed choline acetyltransferase (ChAT) activities in the frontal and temporal neocortices and the immunocytochemical distribution of ChAT and p75 neurotrophin receptor (P75(NTR)) by in vitro imaging in the nucleus basalis of Meynert of CADASIL subjects. RESULTS: ChAT activities were significantly reduced by 60% to 70% in frontal and temporal cortices of CADASIL cases, as were ChAT and P75(NTR) immunoreactivities in the nucleus basalis. CONCLUSIONS: Our findings suggest cholinergic neuronal impairment in CADASIL and implicate cholinomimetic therapy for subcortical vascular dementias.

Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels.

It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-d-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the nucleus basalis has not yet been investigated. Here, by means of choline acetyl transferase and NR2B or NR2C double staining, we demonstrate that mice express both the NR2C and NR2B subunits in nucleus basalis cholinergic cells. We generated NR2C-2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C-2B mutant was used to examine whether a subunit exchange in cholinergic neurons would affect acetylcholine (ACh) content in several brain structures. We found increased ACh levels in the frontal cortex and amygdala in the brains of NR2C-2B mutant mice. Brain ACh has been implicated in neuroplasticity, novelty-induced arousal and encoding of novel stimuli. We therefore assessed behavioral habituation to novel environments and objects as well as object recognition in NR2C-2B subunit exchange mice. The behavioral analysis did not indicate any gross behavioral alteration in the mutant mice compared with the wildtype mice. Our results show that the NR2C by NR2B subunit exchange in mice affects ACh content in two target areas of the nucleus basalis.

Effect of N-methyl-d-aspartate receptor blockade on plasticity of frontal cortex after cholinergic deafferentation in rat.

Cholinergic projections from the nucleus basalis play a critical role in cortical plasticity. For instance, cholinergic deafferentation increases dendritic spine density and expression of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor in frontal cortex. Acetylcholine modulates glutamatergic activity in cortex, and the N-methyl-d-aspartate subtype of glutamate receptor plays a role in many forms of synaptic plasticity. To assess whether N-methyl-d-aspartate receptors mediate the increase in GluR1 and spine density resulting from cholinergic deafferentation, we examined the effect of N-methyl-d-aspartate receptor blockade on nucleus basalis lesion-induced upregulation of GluR1 and dendritic spines. Rats received unilateral sham or 192 IgG saporin lesions of the nucleus basalis. Half of the rats in each group were treated with the N-methyl-d-aspartate antagonist MK-801 or phosphate-buffered saline. Two weeks later, brains were processed for either immunohistochemical staining of the GluR1 subunit or Golgi histology. In layer II-III of frontal cortex, neuronal GluR1 expression was assessed using an unbiased stereological technique, and spine density was assessed on basilar branches of pyramidal neurons. GluR1 expression was increased after nucleus basalis lesion, but this increase was prevented with MK-801. Similarly, nucleus basalis-lesioned animals had significantly higher spine densities, and this effect was also prevented by treatment with MK-801. Thus, N-methyl-d-aspartate receptor blockade prevented both GluR1 and spine density upregulation following cholinergic deafferentation, suggesting that these effects are N-methyl-d-aspartate receptor-mediated.