Rat Cholinergic Neurons Research Articles

Basal Forebrain Cholinergic Deficits Reduce Glucose Metabolism and Function of Cholinergic and GABAergic Systems in the Cingulate Cortex.

PurposeReduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits.Materials and MethodsWe lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography.ResultsDuring water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex.ConclusionOur results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.

SLC10A4 is a protease-activated transporter that transports bile acids

SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. We performed multiple amino acid alignments and examined the relationships between the SLC10 proteins. The extracellular N-terminus of SLC10A4 was predicted to be relatively longer at the amino acid level than those of SLC10A1, SLC10A2 and SLC10A6. We examined the relationship between the N-terminus and transport activity of SLC10A4. Rat Slc10a4 is predominantly expressed in rat cholinergic neurons; therefore, TE671 cells expressing the acetylcholine receptor and acetylcholinesterase were used. After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. Substrates were added to the cells, and their uptake was quantified by liquid chromatography tandem mass spectrometry. Lithocholic acid (LCA) and taurocholic acid (TCA) uptake and cell death effects of LCA were increased by thrombin treatment. After RNA interference treatment for SLC10A4, bile acid uptake was also quantified. In consequence, increases in the LCA and TCA uptake did not occur. Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. We have demonstrated that SLC10A4 appears to be a protease-activated transporter and transports bile acids.

Neuroendocrine Mechanisms of Acupuncture

Neuroendocrine Mechanisms of Acupuncture

Acupuncture Stimulation Alleviates Corticosterone-Induced Impairments of Spatial Memory and Cholinergic Neurons in Rats

The purpose of this study was to examine whether acupuncture improves spatial cognitive impairment induced by repeated corticosterone (CORT) administration in rats. The effect of acupuncture on the acetylcholinergic system was also investigated in the hippocampus. Male rats were subcutaneously injected with CORT (5 mg/kg) once daily for 21 days. Acupuncture stimulation was performed at the HT7 (Sinmun) acupoint for 5 min before CORT injection. HT7 acupoint is located at the end of transverse crease of ulnar wrist of forepaw. In CORT-treated rats, reduced spatial cognitive function was associated with significant increases in plasma CORT level (+36%) and hippocampal CORT level (+204%) compared with saline-treated rats. Acupuncture stimulation improved the escape latency for finding the platform in the Morris water maze. Consistently, the acupuncture significantly alleviated memory-associated decreases in cholinergic immunoreactivity and mRNA expression of BDNF and CREB in the hippocampus. These findings demonstrate that stimulation of HT7 acupoint produced significant neuroprotective activity against the neuronal impairment and memory dysfunction.

S100b Counteracts Neurodegeneration of Rat Cholinergic Neurons in Brain Slices after Oxygen-Glucose Deprivation

Alzheimer's disease is a severe chronic neurodegenerative disorder characterized by beta-amyloid plaques, tau pathology, cerebrovascular damage, inflammation, reactive gliosis, and cell death of cholinergic neurons. The aim of the present study is to test whether the glia-derived molecule S100b can counteract neurodegeneration of cholinergic neurons after oxygen-glucose deprivation (OGD) in organotypic brain slices of basal nucleus of Meynert. Our data showed that 3 days of OGD induced a marked decrease of cholinergic neurons (60% of control), which could be counteracted by 50 μg/mL recombinant S100b. The effect was dose and time dependent. Application of nerve growth factor or fibroblast growth factor-2 was less protective. C-fos-like immunoreactivity was enhanced 3 hours after OGD indicating metabolic stress. We conclude that S100b is a potent neuroprotective factor for cholinergic neurons during ischemic events.

Selective lesion of septal cholinergic neurons in rats impairs acquisition of a delayed matching to position T-maze task by delaying the shift from a response to a place strategy

This study tested the hypothesis that septal cholinergic lesions impair acquisition of a delayed matching to position (DMP) T-maze task in male rats by affecting learning strategy. Rats received either the selective cholinergic immunotoxin, 192 IgG-saporin (SAP) or artificial cerebrospinal fluid directly into the medial septum. Two weeks later, animals were trained to acquire the DMP task. SAP-treated rats took significantly longer to acquire the task than corresponding controls. Both SAP-treated and control rats adopted a persistent turn and utilized a response strategy during early periods of training. By the time rats reached criterion the persistent turn was no longer evident, and all rats had shifted to an allocentric strategy, i.e., were relying on extramaze cues to a significant degree. During the acquisition period, SAP-treated rats spent significantly more days showing a persistent turn and using a response strategy than corresponding controls. The added time spent using a response strategy accounted entirely for the added days required to reach criterion among the SAP-treated rats. This suggests that the principal mechanism by which septal cholinergic lesions impair DMP acquisition in male rats is by increasing the predisposition to use a response vs. a place strategy, thereby affecting the ability to switch from one strategy to another.

Expression and regulation of the two cyclooxygenase isoforms in rhesus macaque brain during lentivirus infection: differentiation of reversible inflammatory and irreversible neuronal sequelae

The regulation of cyclooxygenase type-1 and type-2 (COX-1, COX-2), key enzymes in the conversion of arachidonic acid to prostanoids and mediators of inflammation, was investigated in the brain of simian immunodeficiency virus (SIV)-infected rhesus macaques. COX-1 and COX-2 were constitutively expressed in hippocampal and cortical neurons. COX-1 was additionally found to be expressed in cholinergic basal forebrain neurons (septum, diagonal band of Broca and nucleus basalis of Meynert). This expression was highly species-specific, as COX-1 was coexpressed with specific cholinergic markers choline acetyl transeferase, vesicular acetyl choline transporter (VAChT) and high affinity choline transporter in these neurons, in both the rhesus macaque and human, but was absent from basal forebrain cholinergic neurons in rat, mouse and guinea pig brain. COX-1 immunoreactivity was decreased more than 50% in cholinergic projection neurons of the basal forebrain in AIDS-diseased monkeys. Decreased COX-1 expression was accompanied by a decrease of VAChT-positive fibers and terminals in parietal and frontal cortex, and hippocampus. Additionally, COX-1 and COX-2 were downregulated in frontal cortex neurons in AIDS. Nonneuronally, only COX-1 was expressed constitutively in microglia and endothelial cells, and was not changed in early SIV infection. Late stage of disease was characterized by upregulation of COX-1 in scattered microglia/macrophages, macrophage nodules and multinucleated giant cells. Endothelial COX-1 expression was unchanged during SIV infection. In AIDS-symptomatic monkey brains COX-2 was induced focally in nodule and syncytium forming macrophages and in endothelial cells in areas of viral burden and inflammatory cell infiltrates. Treatment with the antiretroviral brain-permeant 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) substantially reduced focal expression of COX-1 and COX-2 in AIDS, but diffuse COX-1 microgliosis was not. 6-Cl-ddG did not decrease COX-1 immunoreactivity in cortical and cholinergic basal forebrain neurons and cortical cholinergic fiber loss. We conclude that COX-1 coexpression is a feature of the cholinergic basal forebrain telecephalic projection system specific to primates, and may be important in primate-specific functions of this system, and in the cholinergic dysregulation that accompanies primate lentiviral encephalitis. Nonneuronal COX-2 induction is associated with reversible overt inflammatory events to areas of active viral replication. Treatment with cyclooxygenase-inhibitors and central cholinomimetica might be beneficial beside highly active antiretroviral therapy in immunodeficiency virus-induced encephalopathy and encephalitis.

Subsequent exposure to the choline uptake enhancer MKC-231 antagonizes phencyclidine-induced behavioral deficits and reduction in septal cholinergic neurons in rats

This study examined the effects of subsequent, subchronic, treatment with choline uptake enhancer MKC-231 on the behavioral and cellular deficits induced by repeated PCP exposure in rats. Prior subchronic PCP exposure resulted in increased locomotion following an acute PCP or cocaine challenge, but resulted in decreased locomotor activity in response to a carbachol-challenge. MKC-231 significantly antagonized the alterations in the locomotor responses to cocaine and carbachol, but not to PCP. In the novel object recognition test, repeated PCP exposure caused cognitive deficits in rats, and the PCP-induced cognitive deficits were antagonized by MKC-231. In contrast, no effects of PCP exposure were shown in the repeated passive avoidance test. Furthermore, repeated PCP exposure decreased a number of choline acetyltransferase (ChAT)-positive cells in the medial septum and increased dynorphin A expression in the ventral striatum. Moreover, MKC-231 significantly antagonized the changes in septal ChAT-positive cells, but not the changes in ventrostriatal dynorphin A expression. These results suggest that MKC-231 could be a therapeutic drug for the treatment of schizophrenia.

Neuroprotective effect of electroacupuncture therapy on damaged cholinergic neurons in rats with Alzheimer's disease

To explore the neuroprotective effect of electroacupuncture therapy on damaged cholinergic neurons in hippocampus in aged rats with Alzheimer's disease (AD). Thirty six aged male rats were randomly divided into normal control group, sham-operated group, untreated group and electroacupuncture group. Animal model of AD was established with fimbria-fornix transection. The rats in the electroacupuncture group received electroacupuncture on Baihui (DU 20), Yongquan (KI 1), Taixi (KI 3) and Xuehai (SP10). The activity of choline acetyltransferase (ChAT) in septal area of brain was detected by radioimmunoassay, and the protein expressions of nerve growth factor (NGF) and c-fos in CA3 region of hippocampus were detected by immunohistochemical assay. The ChAT activity and the expression levels of NGF and c-fos proteins in the electroacupuncture group were significantly higher than those in the untreated group. Electroacupuncture therapy can protect cholinergic neurons in hippocampus in aged rats with AD by means of promoting synthesis of c-fos protein and increasing the expression level of NGF.

Nerve growth factor prevents cell death and induces hypertrophy of basal forebrain cholinergic neurons in rats withdrawn from prolonged ethanol intake

We have previously reported that the hippocampal cholinergic fiber network is severely damaged in animals withdrawn from ethanol, and that a remarkable recovery in fiber density occurs following hippocampal grafting, a finding that we suggested to be underpinned by the graft production of neurotrophic factors, which are known to be decreased after ethanol exposure. It is widely accepted that nerve growth factor (NGF) signals the neurons of the brain cholinergic system, including those of the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) nuclei, from which the septohippocampal projection arises. Because neurons in these nuclei are vulnerable to ethanol consumption and withdrawal we thought of interest to investigate, in withdrawn rats previously submitted to a prolonged period of ethanol intake, the effects of intraventricular delivery of NGF upon the MS/VDB cholinergic neurons. Stereological methods were applied to estimate neuron numbers and neuronal volumes in choline acetyltransferase (ChAT)-immunostained and Nissl-stained material. We have found that in ethanol-fed rats there was a significant reduction in the total number of Nissl-stained and cholinergic neurons in the MS/VDB, and that the suppression of ethanol intake further decreased neuron numbers. In addition, the somatic size of ChAT-IR neurons was reduced by ethanol intake, and withdrawal further aggravated neuronal atrophy. NGF treatment prevented the withdrawal-associated loss, and induced hypertrophy, of cholinergic neurons. These findings show that exogenous NGF protects the phenotype and prevents the withdrawal-induced degeneration of cholinergic neurons in the MS/VDB. These effects might be due to the trophic action of NGF upon the basal forebrain cholinergic neurons, including the hippocampal fiber network that conveys this neurotrophin retrogradely to the MS/VDB, and/or upon their targets, that is, the hippocampal formation neurons.

Influence of tooth-loss and concomitant masticatory alterations on cholinergic neurons in rats: immunohistochemical and biochemical studies.

The influence of tooth loss on the viability of cholinergic neurons was examined in rats. At 25th postnatal week, rats were divided into the three groups; a control group fed a solid diet, a soft diet group fed a powder diet and a molar crown-less group in which all molar crowns were removed and the powder diet was given. At 15 and 35 weeks post-treatment, the number of choline acetyltransferase (ChAT)-positive neurons in the nucleus of the diagonal band/medial septal nucleus (NDB/MS) was significantly smaller in the molar crown-less group than in the control group (P < 0.01). This was not the case in the pedunculopontine tegmental nucleus or (PPT) or in the trigeminal motor nucleus. Biochemical assay showed no statistically significant differences in choline concentrations in the hippocampus between the control and the molar crown-less group both at 15 and at 35 weeks post-treatment. Nevertheless, acetylcholine (ACh) concentration in the hippocampus of the molar crown-less group was significantly lower than that of the control group at 15 weeks post-treatment (P < 0.05). Taken together, a decrease of oral sensory information may have caused a reduction in the number of ChAT-positive neurons selectively in NDB/MS, which in turn caused a decline of ACh concentrations in the hippocampus.

Increased susceptibility to generalized seizures after immunolesions of the basal forebrain cholinergic neurons in rats

We investigated whether basal forebrain cholinergic neurons influence the expression of generalized seizures. Animals received intracerebroventricular injections of saporin (lesioned) or saline (controls) and were tested for susceptibility to flurothyl- or pentylenetetrazole-induced seizures. Lesioned rats had significantly shorter latencies to onset of generalized tonic-clonic seizures than controls. Our findings suggest that basal forebrain cholinergic neurons may participate in the modulation of generalized seizures.

Estrogen binding and estrogen receptor characterization (ERα and ERβ) in the cholinergic neurons of the rat basal forebrain

Estrogen is thought to enhance cognitive functions by modulating the production of acetylcholine in basal forebrain neurons; a system that projects to the cerebral cortex and hippocampus and plays a central role in learning and memory. To elucidate the mechanism of estrogen action in the cholinergic system, we utilized a combined in vivo autoradiography/immunocytochemistry technique to evaluate the distribution of estrogen binding sites in cholinergic neurons of the rat basal forebrain. The results of these studies revealed that a portion of the cholinergic neurons in the medial septum (41%), vertical (32%) and horizontal (29%) limbs of the diagonal band and in the substantia innominata/nucleus basalis (4%) contained estrogen receptors. Through the use of a double-label in situ hybridization/immunocytochemistry technique we have shown that estrogen receptor-α is the predominant estrogen receptor in the cholinergic neurons, with only a few cells containing estrogen receptor-β. The results of these studies provide evidence that biologically active estrogen receptors are present in the basal forebrain cholinergic neurons of the adult rat brain, with estrogen receptor-α being the predominant receptor subtype. The demonstration that cholinergic neurons contain estrogen receptors is consistent with the possibility that estrogen directly modulates the activity of cholinergic neurons in rats and may provide insight as to how estrogen improves cognitive functions in women.

In vivo [ 125I]-iodobenzovesamicol binding reflects cortical cholinergic deficiency induced by specific immunolesion of rat basal forebrain cholinergic system

In this study, radiolabeled iodobenzovesamicol (IBVM), which is known to bind with high affinity to the vesicular acetylcholine transporter, was tested for its usefulness in imaging cortical cholinergic deficits in vivo. To induce reductions in cortical cholinergic input, the cholinergic immunotoxin 192IgG-saporin was employed. This has been shown to selectively and efficiently destroy basal forebrain cholinergic neurons in rats. The efficiency of the immunolesion was verified by histochemical acetylcholinesterase staining. [ 125I]-IBVM binding before and after lesioning was measured using autoradiography. Basal forebrain cholinergic cell loss resulted in a considerable reduction in [ 125I]-IBVM binding in the cholinoceptive target regions, but not in the striatum and cerebellum, brain regions that do not receive a cholinergic input by the basal forebrain cholinergic nuclei, suggesting that [ 123I]-IBVM has potential in imaging cortical cholinergic deficits in vivo, at least in animals.

Sustained effect of metrifonate on cerebral glucose metabolism after immunolesion of basal forebrain cholinergic neurons in rats

To evaluate the influence of cholinergic projections from the basal forebrain on brain metabolism, we measured the cerebral metabolic rate of glucose (CMR(glu)) after unilateral lesioning of cholinergic basal forebrain neurons with the immunotoxin 192 IgG-saporin. CMR(glu) was determined in 24 cortical and 13 sub-cortical regions using the [ 14C]2-deoxy- d-glucose technique of Sokoloff. Average hemispheric CMR(glu) decreased by 7% ( P<0.02) and 5% ( P<0.05), 7 and 21 days after lesion, respectively. Regional effects were restricted to parietal and retrosplenial cortices, lateral habenula and the basal forebrain. We have previously shown that metrifonate increased CMR(glu) in intact rats. In lesioned rats, metrifonate (80 mg/kg, i.p.) was still active but the metabolic activation was reduced in terms of both the average hemispheric CMR(glu) and the number of regions significantly affected. Although it is reduced, the sustained effect of metrifonate in lesioned rats makes an argument for the use of this compound as treatment of cholinergic deficit in Alzheimer's disease.

Regulation of Galanin in Memory Pathways

Based on early immunocytochemical findings, galanin (GAL) was postulated to function as an inhibitory cotransmitter in rat cholinergic memory pathways. However, recent studies indicate that in the basal state GAL is not widely expressed by forebrain cholinergic neurons in rats. Inhibition of cholinergic transmission by cosecreted GAL may be enhanced under certain conditions, because GAL gene expression in the cholinergic basal forebrain is significantly increased prior to puberty and following nerve growth factor treatment. Other sources of GAL in rat septohippocampus that could interact with cholinergic pathways include noradrenergic neurons in the locus ceruleus and vasopressinergic neurons in the bed nucleus of the stria terminalis (BST) and medial amygdala (Me). GAL is extensively colocalized within these steroid-sensitive cell groups where its expression is upregulated by gonadal hormones. GAL, acting via the GALR1 receptor subtype, does not appear to directly regulate the activity of cholinergic neurons, but it may regulate the release of vasopressin and GAL into septohippocampus from BST/Me neurons.

Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling

Intraventricular 192 IgG-saporin was used to induce a selective lesion of basal forebrain cholinergic neurons in rats. When subjected to 40 rapid hippocampal kindling stimulations with 5-min intervals, these animals exhibited increased number of generalized seizures and a higher mean seizure grade in response to the first five stimulations, and required fewer stimuli to develop focal behavioural seizures, as compared to non-lesioned rats. In contrast, both groups showed similarly enhanced responsiveness when test stimulated four weeks later. Using in situ hybridization, cholinergic denervation was found to cause a significant decrease of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex, whereas gene expression for nerve growth factor, neurotrophin-3, and TrkB and TrkC was unchanged. Four weeks after rapid kindling stimulations, basal levels of brain-derived neurotrophic factor messenger RNA in the dentate granule cells were restored to normal in the lesioned rats, whereas neurotrophin-3 messenger RNA levels were decreased. No differences in the seizure-evoked levels of neurotrophin and Trk messenger RNAs were detected, except in the dentate granule cell layer, which had significantly higher brain-derived neurotrophic factor messenger RNA expression in the lesioned animals at 2 h. In conclusion, the basal forebrain cholinergic system (i) dampens the severity of recurring seizures induced by rapid hippocampal kindling stimulations, but has no effect on the subsequent delayed phase of epileptogenesis; and (ii) exerts a tonic stimulation of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex. The findings also indicate that the cholinergic lesion does not affect neurotrophin and Trk gene expression after recurring seizures, and that the kindling process leads to long-term changes in basal brain-derived neurotrophic factor and neurotrophin-3 messenger RNA levels in the denervated animals.

SDZ ENA 713 facilitates central cholinergic function and ameliorates spatial memory impairment in rats

We have clarified the effects of SDZ ENA 713 (ENA), a new phenyl-carbamate derivative, on the spatial learning impairment and neurochemical indices of central cholinergic neurons in rats. Basal forebrain (BF) lesioning with ibotenic acid markedly impaired acquisition ability in the water maze task without changing swimming rates and decreased choline acetyltransferase (ChAT) activity in the frontal cortex of rats. ENA (0.1, 0.2 mg/kg, p.o.) significantly ameliorated the impairment in acquisition ability in a dose-dependent manner. At 0.2 mg/kg, ENA prevented the reduction in ChAT activity. In normal rats, ENA (1 mg/kg, p.o.) increased extracellular ACh concentration of the prefrontal cortex. On the other hand, tissue concentrations of norepinephrine, serotonin, dopamine and their metabolites were not changed in the frontal cortex, hippocampus and striatum of normal rats. These results suggest that ENA ameliorates spatial learning disability by not only facilitating the cholinergic transmission, but normalizing impaired ChAT activity in the learning-impaired rat model.

Neuroleptic-induced motor abnormalities and cholinergic cell loss in rats

We performed a study of the effects of fluphenazine decanoate and antioxidants on head movements striatal cholinergic neurons in rats. Male Sprague-Dawley rats (aged 13 months) received 10 mg/kg fluphenazine decanoate or sesame seed oil IM q 2 weeks for 4 months. The animals were divided into four groups, with sesame oil or fluphenazine injections and basal or antioxidant diet (with supplemented vitamin E, C, and betacarotene). At baseline and after treatment, head movements were quantified by accelerometry. Striatal cholinergic neurons were quantified by counting ChAT-stained cells using the disector technique. The results of the quantitative motor testing revealed a significant main effect for diet (F 13.01, df(1,25), p < 0.002) and for treatment (F = 4.3, dr(1,25), p < 0.05). The results of the cholinergic neuron analysis revealed a significant main effect for diet (F 20.1, df(1,28), p < 0.0002) and for treatment (F ~ 9.0, df(l,28), p < 0.006). There was a significant correlation (r = -0.50, p < 0.01 ) between the motor change scores and the density of cholinergic neurons in the striatum. Our findings suggest that (1) long-term neuroleptic treatment causes a reduction in striatal cholinergic neurons and an increase in head movements, (2) the increase in head movements relates to the density of cholinergic neurons, and (3) these abnormalities are reduced by antioxidants. The increase in head movements observed in rats treated long-term with neuroleptic drugs suggests that this technique has potential to serve as an animal model for TD.

Synthesis of radioiodinated analogs of 2-(4-Phenylpiperidino)cyclohexanol (vesamicol) as vesamicol-like agent

Three iodovesamicol analogs, iodinated at the ortho, meta, and para positions of the 4-phenylpiperidine moiety, were synthesized and labeled with 125I by isotopic exchange reaction. Their potencies as a vesamicol-like drug were evaluated with competitive inhibition studies using (⊃[ 3H]vesamicol. The radiochemical yields were 40–85%, the radiochemical purities exceeded 95% and their specific activities were 370–740 GBq/mmol. The descending order of binding affinity of the tested compounds against the vesamicol receptor was m-iodovesamicol > o-iodovesamicol > p-iodovesamicol. The receptor binding affinity of m-iodovesamicol (IC 50 = 133 nM) was comparable with that of vesamicol (IC 50 = 109 nM). Therefore, the meta position of the 4-phenylpiperidinyl fragment of vesamicol was the optimum site for iodination, and radioiodinated m-iodovesamicol may serve as a useful radiopharmaceutical for in vitro and in vivo studies of presynaptic cholinergic neurons in rats.