Cortical Cholinergic Neurons Research Articles

Nootropic and Free Radical Scavenging Activity of Plumbago zeylanica Linn. By Different In-Vivo & In-Vitro Methods

AIM- The aim of the present investigation is to evaluate Plumbago zeylanica L. (Plumbaginaceae) for evaluation of memory enhancing activity. MATERIAL & METHODS- All the plant materials were dried under shade and subjected to coarse powder for extraction process. Accurately weighed quantity of roots of Plumbago zeylanica Linn. extracted using petroleum ether, chloroform, methanol, butanol and finally water by soxhlet apparatus for 72 h. Qualitative chemical tests of chloroform, methanol, butanol and water extracts were subjected to various chemical tests to detect various Phytoconstituents. Briefly, 6.0 mg ß-carotene was dissolved in 10 ml of chloroform, and then 1 ml of solution pipette to glass filled of 20 mg linoleic acid. 5 ml of mixture then pipette to reaction tube filled of extract in a range of concentration, mixed homogenously. Briefly, 1 mL of 0.3 mM of DPPH solution was added to 1 mL each of the test solutions, and was incubated in the dark at room temperature for 30 min. Escape latency (EL) was recorded 120 min after drug administration from 11th day to 14th day. On 15th day, time spent in target quadrant (TSTQ) was noted 120 min after the drug administration. In case of animals administered with physostigmine, EL and TSTQ was noted after 30 min of drug administration. 0.4 ml of brain homogenate was added into a test tube containing 2.6 ml of phosphate buffer. 5,5-dithiobis-2-nitrobenzoic acid reagent (0.1 ml) was added to the above mixture and absorbance was noted at 412 nm. RESULTS- The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds. Vitamin E as standard was used in this assay and 84% inhibition was found to be at 30 minutes. Chloroform extract also showed 70% inhibition at 30 minutes which was reduced to 45% at the time of 120 minutes. Hydroxyl radical scavenging ability calculated as IC50 reveals that chloroform; methanol, butanol and water extracts have IC50 values of 47 μg/mL, 100μg/mL, 200 μg/mL and 150μg/mL, respectively. Among all the extracts, chloroform extract showed a highly significant effect on EL and TSTQ. Chloroform extracts significantly decreased (P<0.001) EL and significantly increased TSTQ as compared to vehicle treated control. CONCLUSION- The deterioration and dysfunction of cortical cholinergic neurons is closely associated with cognitive deficits of Alzheimer’s disease. Keywords: Alzheimer’s disease, Vitamin E, DPPH solution, Plumbago zeylanica Linn., Plumbaginaceae, EL and TSTQ

The Effect of Valproic Acid on the Transcriptional Activity of Ngf and Bdnf Genes of in Vitro Cultured Neurons Under Oxidative Stress Conditions

Brain-derived neurotrophic factor (BDNF) is a secretory molecule that promotes peripheral neurons synaptic transmission and plasticity by TrkB receptor activation. This is shown in cultured central nervous system (CNS) neurons, including hippocampal and cortical cholinergic, dopaminergic and serotonergic neurons. Hypotheses suggesting that BDNF may play a potential role in the pathophysiology of schizophrenia are based on the key role of BDNF in the synaptic plasticity and, consequently, regulation of cognitive functions. In the schizophrenia treatment valproic acid is used in complex combined therapy regimens. Treatment of schizophrenia patients with valproate increases the BDNF level. Since it is not yet clear whether the BDNF protein levels measured in serum samples and in the brain correlate, we investigated valproate effects on the cultured neurons Bdnf transcription level. The primary neuron-glia culture was obtained from the cerebellum of 8-9-day-old Wistar rats. Valproic acid was added to the neurons (at a concentration of 50 µg/ml), oxidative stress was stimulated by 40 µMof H2O2, and injury was caused by mechanical damage to the neuron culture. It was shown that valproic acid in 3-24 hours increases the transcriptional activity of the Bdnf and Ngf (nerve growth factor) genes 2–2.5-fold (p<0.01) and approximately 1.5-fold (p<0.01), respectively. Mechanical trauma, unlike oxidative stress, activates the transcriptional activity of the Ngf and Bdnf genes (p<0.01). However, under oxidative stress and mechanical damage to neurons, the effect of valproic acid on the Ngf and Bdnf genes expression was insignificant. Fluorescence microscopy analysis using specific antibodies to neurons (anti-Map-2) showed that in the presence of valproic acid, the number of neuronal processes and contacts between them significantly increased. Evidently, valproate addition to antipsychotics can be effective for the overall clinical response. Relatively little research has been done on the signaling pathways in neurons that are activated by the valproic acid. However, we have obtained evidence of activation of the Ngf and Bdnf genes transcription in cultured neurons in vitro. We also found that in the presence of valproic acid, the number of neuronal processes and contacts between them significantly increased. However, we have also found that the oxidative stress accompanying the schizophrenia can significantly reduce the valproic acid effect on the Ngf and Bdnf genes expression. The results of the study may be potentially useful for new schizophrenia therapy strategies development.

Cholinergic inputs to cortical cholinergic neurons facilitate disinhibition of excitatory neurons in the rat cerebral cortex

Cholinergic inputs to cortical cholinergic neurons facilitate disinhibition of excitatory neurons in the rat cerebral cortex

Cholinergic anti-inflammatory pathway involves in the neuroprotective effect of huperzine A on sepsis-associated encephalopathy

To explore whether the cholinergic anti-inflammatory pathway is involved in the neuroprotective effect of acetylcholinesterase inhibitor huperzine A (HupA) on sepsis-associated encephalopathy (SAE) by observing the effect of HupA on the expressions of choline acetyltransferase (CHAT) and cholinergic muscarinic receptor M1 (CHRM1) of sepsis rats. Fifty-four male Wistar rats, 8 weeks old, were divided into three experimental groups according to random number table:control group,sepsis group, and HupA group, with 18 rats in each group. The rat model of sepsis was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS,1 mL),and the rats in control group were given the same volume of normal saline. The rats in HupA group were intraperitoneally administered with HupA 0.04 mg/kg (1 mL) at 30 minutes before model reproduction, while the rats in control group and sepsis group were treated with the same volume of saline instead. At 3,12,and 24 hours after model reproduction,6 rats in each group were sacrificed after clinical manifestation observation, and cerebral cortex tissue was collected. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1 β) in cerebral cortex were determined with enzyme linked immunosorbent assay (ELISA),along with the measurement of neuronal apoptosis by caspase-3 and neuronal nuclear antigen (NeuN) immune double standard staining. The mRNA expressions and positive expressions of ChAT and CHRM1 were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunofluorescence methods. Clinical manifestations of sepsis rats were present at 3 hours and reached a peak at 12 hours, including lethargy,vertical hair and lazy to move. Over-expression of pro-inflammatory cytokines TNF-α and IL-1β was found in sepsis group, and apoptotic neurons marked by two fluorescences were significantly increased in sepsis rats ,in comparison with deficient ChAT and CHRM1 proteins marked by red fluorescence, and low-expressed ChAT and CHRM1 mRNA as well. The differences between sepsis group and control group were statistically significant [12-hour TNF-α (ng/L):84.97±31.84 vs.40.31 ± 10.37,12-hour IL-1 β (ng/L):1 095.98± 127.09 vs.622.62± 117.25,12-hour ChAT mRNA (2-△△Ct):1.34 (0.67,1.86) vs.1.92 (1.12,2.87),12-hour CHRM1 mRNA (2-△△Ct):0.65±0.12 vs.1.16±0.42,all P ＜ 0.05].The septic symptoms were relieved after HupA administration,as well as the reduction of pro-inflammatory cytokines and the neuronal apoptosis, which might attribute to the increased expressions of ChAT and CHRM1.The differences between HupA group and sepsis group were statistically significant [12-hour TNF-α (ng/L):48.38 ± 12.62 vs.84.97 ± 31.84,12-hour IL-1 β (ng/L):718.13 ± 163.33 vs.1 095.98 ± 127.09,12-hour ChAT mRNA (2-△△Ct):18.04 (17.22,19.23) vs.1.34 (0.67,1.86),12-hour CHRM1 mRNA (2-△ △Ct):1.46 ± 0.69 vs 0.65 ± 0.12,all P ＜ 0.05].The clinical manifestations and neuroinflammation mainly recovered at 24 hours. The cortical cholinergic neurons dysfunction and the abnormal inflammatory response are involved in the onset of SAE process. HupA plays a protective role in SAE through recovering the function of cholinergic neurons and cholinergic anti-inflammatory pathway.

Variety of Neuronal Pathways to Achieve the Same Hypoxic Preconditioning Effect

The relevance of hypoxic preconditioning is due to its ability to increase the body's resistance to hypoxic/ischemic stress. A single session of moderate hypoxia eliminates the differences in endurance under severe hypoxia in intact rats and those pre-tested under severe hypoxia with high and low innate resistance to it. In these rat groups, the same preconditioning effect is achieved by different synaptic plastic tools (biochemical data). Thus, the synaptic mechanisms of preconditioning are dependent on the prior hypoxic experience. This conclusion confirmed our pharmacological experiments. Antagonists of alpha7 and non-alpha7 subtypes of the nicotinic receptors methyllycaconitine and mecamylamine in single IP injections selectively influenced the resistance to hypoxia of the low-resistant rats and were ineffective against the high-resistant and intact rats. Moreover, in the low-resistant rats, both drugs had ambiguous effects on the resistance to hypoxia after the preconditioning and without it. Based on the data, we substantiated in our review that the following cholinergic neuronal populations and networks were involved in mechanisms of the hypoxic preconditioning: 1) In the high-resistant rats, cholinergic projections from the pedunculopontine and/or laterodorsal tegmental nuclei into the nuclei of the ventrolateral medulla of the medulla oblongata as well as into the subcortical forebrain nuclei and, linked with them, cholinergic projections from these forebrain nuclei into the cortex. 2) In the low-resistant rats, cholinergic C-fibres into the nucleus tractus solitary of the medulla oblongata and influences of unidentified cholinergic neurons through the alpha7 nicotinic receptors in the caudal brainstem areas outside the nucleus tractus solitary. 3) In intact rats, cortical cholinergic interneurons and unidentified cholinergic neurons of the brainstem structures. In conclusion, the variety of neuronal pathways to put off apnea indicates a great adaptive potential of brain, and the specific mechanisms of its realization may be a promising therapeutic targets.

Effects of Abeta1–42 on the current of KATP channels in cultured cholinergic neurons

Objective: This study mainly determined the effect of beta-amyloid peptides (Abeta1–42) on the ATP-sensitive potassium (KATP) channels of the cultured cortical and hippocampal cholinergic neurons, and further confirmed the protective effect of Diazoxide, which was the specific opener of KATP channels, to the neurons.Methods: The primary rat cortical and hippocampal cholinergic neurons were cultured. Membrane currents were measured using whole-cell patch-clamp technique.Results: The data showed that the outward current of neurons significantly decreased after using Abeta1–42 (P<0·05), and that the outward current of decreased did not change after given Diazoxide again (P>0·05) compared with control. However, after being pretreated with Diazoxide for 1 hour before using Abeta1–42, this phenomenon did not exist; that is, the membrane current of the neurons did not change when we give Abeta1–42 after pretreatment with Diazoxide (P>0·05).Conclusions: It is concluded that Abeta1–42 inhibits the openings of the KATP channels, while this inhibitory action can be removed by pretreatment with Diazoxide. It is indicated that the opening of KATP channels may play a potential neuroprotective role in antineurotoxicity of Abeta1–42, and the application of Diazoxide in small dose may be helpful in the treatment of Abeta1–42 neurotoxicity.

Nuclear organization of cholinergic, putative catecholaminergic, serotonergic and orexinergic systems in the brain of the African pygmy mouse (Mus minutoides): Organizational complexity is preserved in small brains

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brain of the African pygmy mouse (Mus minutoides). The African pygmy mice studied had a brain mass of around 275mg, making these the smallest rodent brains to date in which these neural systems have been investigated. In contrast to the assumption that in this small brain there would be fewer subdivisions of these neural systems, we found that all nuclei generally observed for these systems in other rodent brains were also present in the brain of the African pygmy mouse. As with other rodents previously studied in the subfamily Murinae, we observed the presence of cortical cholinergic neurons and a compactly organized locus coeruleus. These two features of these systems have not been observed in the non-Murinae rodents studied to date. Thus, the African pygmy mouse displays what might be considered a typical Murinae brain organization, and despite its small size, the brain does not appear to be any less complexly organized than other rodent brains, even those that are over 100 times larger such as the Cape porcupine brain. The results are consistent with the notion that changes in brain size do not affect the evolution of nuclear organization of complex neural systems. Thus, species belonging to the same order generally have the same number and complement of the subdivisions, or nuclei, of specific neural systems despite differences in brain size, phenotype or time since evolutionary divergence.

Choline acetyltransferase-containing neurons in the human parietal neocortex.

A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.

Transduced PTD-BDNF fusion protein protects against beta amyloid peptide-induced learning and memory deficits in mice

Brain-derived neurotrophic factor (BDNF) promotes the survival and differentiation of hippocampal, cortical, and basal forebrain cholinergic neurons. However, the efficacy of BDNF via peripheral (i.v.) administration is limited by the lack of transport of the neurotrophin through the blood–brain barrier (BBB) in vivo. The present study describes that the i.v. administered recombinant human BDNF (rhBDNF) conjugated with a protein transduction domain (PTD ) is able to survive and promote the growth of hippocampal neurons impaired by Aβ25–35 (10 μM) in vitro and transport through the BBB in vivo. The Morris water maze test indicated that the i.v. PTD-rhBDNF improved the spatial learning and memory of mice impaired by the aggregated Aβ25–35. The peripherally administered PTD-rhBDNF exhibited neuroprotective effects in brain and raise the possibility of delivery of the exogenous rhBDNF in treatment of the brain diseases.

Pro-brain-derived neurotrophic factor is decreased in parietal cortex in Alzheimer’s disease

Brain-derived neurotrophic factor (BDNF) promotes the function and survival of the major neuronal types affected in Alzheimer disease, such as hippocampal, cortical and basal forebrain cholinergic neurons. We and others have demonstrated a reduction in BDNF mRNA expression in Alzheimer’s disease hippocampus and cortex, which may help to explain the selective vulnerability of these neurons. Several studies have also shown decreased BDNF protein in Alzheimer’s disease. BDNF protein is synthesized as a precursor, proBDNF, which is cleaved to the mature 14-kDa form. We demonstrate here that BDNF exists as a mixture of proBDNF and mature BDNF in all regions tested of human brain. Using Western blotting, we observe a 40% reduction in proBDNF levels in Alzheimer’s disease parietal cortex compared to controls. Thus, decreased BDNF protein measured by ELISA and immunohistochemistry likely represents a mixture of the two BDNF forms, and previously reported decreases in BDNF protein may be due, at least in part, to a significant reduction in proBDNF levels. Although the biological activity of proBDNF is unknown, reduced proBDNF may have functional consequences for the selective neuronal degeneration in Alzheimer’s disease brain.

A new brain-derived neurotrophic factor transcript and decrease in brain-derived neurotrophic factor transcripts 1, 2 and 3 in Alzheimer's disease parietal cortex.

Brain-derived neurotrophic factor (BDNF) supports hippocampal, cortical and basal forebrain cholinergic neurons, which lose function in Alzheimer's disease. In Alzheimer's tissues such as hippocampus and parietal cortex, brain- derived neurotrophic factor mRNA is decreased three- to four-fold compared with controls. However, the molecular mechanism of the down-regulation of BDNF in Alzheimer's disease is unknown. The human brain-derived neurotrophic factor gene has multiple promoters governing six non-coding upstream exons that are spliced to one downstream coding exon, leading to six different transcripts. Here we report an alternate human splice variant within exon 4I for a total of seven transcripts. Previous brain-derived neurotrophic factor mRNA measurements in Alzheimer's disease tissue were done using the downstream coding exon present in all transcripts. Using RT-PCR primers specific for each upstream exon, we observe a significant decrease in three human brain-derived neurotrophic factor mRNA transcripts in Alzheimer's disease samples compared with controls. Transcripts 1 and 3 each exhibit a two-fold decrease, and transcript 2 shows a five-fold decrease. There are no significant differences between control and Alzheimer's disease samples for the other transcripts, including the new splice variant. In rat, both transcripts 1 and 3 are regulated through the transcription factor cAMP response element binding protein, whose phosphorylation is decreased in the Alzheimer's disease brain. This could lead to specific down-regulation of the brain-derived neurotrophic factor transcripts shown here.

Rivastigmine antagonizes deficits in prepulse inhibition induced by selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis

Impairments of cortical cholinergic inputs from the nucleus basalis magnocellularis fundamentally alter information processing and attentional function, thereby advancing the severity of psychopathology in major neuropsychiatric disorders. It was previously shown in adult rats that bilateral 192 IgG saporin-induced selective immunolesioning of the cholinergic neurons in the nucleus basalis produces pronounced and long-lasting deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex. This behavioral paradigm is considered a valid model of sensorimotor gating deficits in the psychotic spectrum and efforts to analyze the significance of the cholinergic basal forebrain in this context are of great interest. In the present study the predictive value of the selective cholinergic immunolesioning model was tested by examining the ability of the cholinesterase inhibitor rivastigmine to restore prepulse inhibition in immunolesioned rats. We report here a pronounced restoring effect of acute (0.75 or 1.5 mg/kg s.c.) as well as repeated (0.75 mg/kg s.c. b.i.d., for 10 days) treatment with rivastigmine in this model of disrupted prepulse inhibition. Intra-nucleus basalis magnocellularis infusions of 192 IgG saporin resulted in extensive loss of basal–cortical cholinergic neurons as shown by the marked decrease in basal telencephalic choline acetyltransferase immunopositive neurons and cortical choline acetyltransferase activity. In this condition, rivastigmine was found to significantly increase cortical acetylcholine extracellular levels in lesioned animals measured by in vivo microdialysis. Taken together, our results strengthen the proposal that the nucleus basalis represents a critical station of the startle gating circuitry. In addition, our findings strongly indicate that even after dramatic decrease of cholinergic neurons, inhibition of acetylcholinesterase restores the cholinergic synaptic function to a point approaching normalization of experimentally induced psychopathology.

The ABC of Alzheimer's disease: cognitive changes and their management in Alzheimer's disease and related dementias.

Cognitive decline, commonly first recognized as memory impairment, is a typical feature of Alzheimer's disease (AD). Neuropathological changes in the cerebral cortex and limbic system lead to deficits in learning, memory, language, and visuospatial skills. The precise nature of cognitive dysfunction reflects the distribution of pathological changes in AD. These will vary along the disease severity continuum and may also depend on where the disease sits in the spectrum of dementia. For example, AD-related disorders such as Lewy body dementia (LBD) and Parkinson's disease dementia (PDD) also show symptoms of cognitive decline and share several pathological features, including degeneration of cortical cholinergic and striatal dopaminergic neurons. In vascular dementia (VaD), there is often an unequal distribution of cognitive deficit, with severe impairment in some functions and relative sparing of others. Cholinesterase (ChE) inhibitors, which help restore acetylcholine levels in the brain, are licensed for the symptomatic treatment of AD and have shown additional benefit in related dementias. Physiological correlates of cholinergic function/dysfunction in the brain include regional cerebral blood flow, glucose metabolism, and cerebrospinal fluid levels of ChE enzymes. These variables represent valuable markers of the clinical efficacy of ChE inhibitors. However, direct assessment of cognitive improvement, stabilization or decline is usually considered the key efficacy parameter in clinical studies of ChE inhibitors in AD and related dementias. Large-scale, placebo-controlled clinical studies of ChE inhibitors have demonstrated efficacy in treating the cognitive impairments associated with AD. Randomized comparative studies of ChE inhibitors are now under way to directly compare symptomatic efficacy and effects on disease progression. Clinical trial data of the cognitive effects of ChE inhibitors in AD, LBD, PDD, and VaD are discussed in detail in this article. The benefits of long-term treatment on symptomatic improvement in cognition and further potential disease-modifying effects are highlighted.

Long-term treatment with antidepressant drugs reduces the sensitivity of cortical cholinergic neurons to the activating actions of stress and the anxiogenic drug FG 7142

Certain antidepressant drugs exert an anxiolytic action in both humans and rodents. The effects of long-term treatment with imipramine or mirtazapine, two antidepressant drugs with different mechanisms of action, on the response of cortical cholinergic neurons to foot-shock stress or to the anxiogenic drug FG 7142 were investigated in freely moving rats. Chronic treatment with imipramine or mirtazapine reduced the increase in cortical acetylcholine output induced by foot-shock stress by ∼50%. The same treatment also reduced the sensitivity of cortical cholinergic neurons to the stimulatory effect of acute administration of FG 7142. In contrast, the administration of a single dose of either antidepressant 40 min before foot shock or FG 7142 injection failed to increase the threshold of excitability of cortical cholinergic neurons. These results demonstrate that long-term treatment with either imipramine or mirtazapine reduces the sensitivity of cortical cholinergic neurons to stress or to an anxiogenic drug with an efficacy similar to that of acute administration of benzodiazepines. The neurochemical mechanism responsible for regulation of cholinergic neuron sensitivity might contribute to the modulation of cognitive function associated with emotional and affective disorders.

Quantitation of BDNF mRNA in human parietal cortex by competitive reverse transcription-polymerase chain reaction: decreased levels in Alzheimer's disease

Alzheimer's disease is a progressive neurodegenerative disorder of the central nervous system. One pathological characteristic is excessive neuronal loss in specific regions of the brain. Among the areas most severely affected are the basal forebrain cholinergic neurons and their projection regions, the hippocampus and cortex. Neurotrophic factors, particularly the neurotrophins nerve growth factor and brain-derived neurotrophic factor, play an important role in the development, regulation and survival of basal forebrain cholinergic neurons. Furthermore, brain-derived neurotrophic factor regulates the function of hippocampal and cortical neurons. Neurotrophins are synthesized in hippocampus and cortex and retrogradely transported to the basal forebrain. Decreased levels of neurotrophic factors are suspected to be involved in the neurodegenerative changes observed in Alzheimer's disease. We examined autopsied parietal cortex samples from age- and gender-matched Alzheimer's diseased and neurologically non-impaired individuals using the quantitative technique of competitive RT-PCR. We demonstrate a 3.4-fold decrease in brain-derived neurotrophic factor mRNA levels in the parietal cortex of patients with Alzheimer's disease compared to controls ( p<0.004). A decrease in brain-derived neurotrophic factor synthesis could have detrimental effects on hippocampal, cortical and basal forebrain cholinergic neurons and may account for their selective vulnerability in Alzheimer's disease.

Somatomotor neuron-specific expression of the human cholinergic gene locus in transgenic mice

We examined the expression pattern of the vesicular acetylcholine transporter in the mouse nervous system, using rodent-specific riboprobes and antibodies, prior to comparing it with the distribution of vesicular acetylcholine transporter expressed from a human transgene in the mouse, using riboprobes and antibodies specific for human. Endogenous vesicular acetylcholine transporter expression was high in spinal and brainstem somatomotor neurons, vagal visceromotor neurons, and postganglionic parasympathetic neurons, moderate in basal forebrain and brainstem projection neurons and striatal interneurons, and low in intestinal intrinsic neurons. Vesicular acetylcholine transporter expression in intrinsic cortical neurons was restricted to the entorhinal cortex. The sequence of the mouse cholinergic gene locus to 5.1 kb upstream of the start of transcription of the vesicular acetylcholine transporter gene was determined and compared with the corresponding region of the human gene. Cis-regulatory domains implicated previously in human or rat cholinergic gene regulation are highly conserved in mouse, indicating their probable relevance to the regulation of the mammalian cholinergic gene locus in vivo. Mouse lines were established containing a human transgene that included the vesicular acetylcholine transporter gene and sequences spanning 5 kb upstream and 1.8 kb downstream of the vesicular acetylcholine transporter open reading frame. In this transgene, the intact human vesicular acetylcholine transporter was able to act as its own reporter. This allowed elements within the vesicular acetylcholine transporter open reading frame itself, shown previously to affect transcription in vitro, to be assessed in vivo with antibodies and riboprobes that reliably distinguished between human and mouse vesicular acetylcholine transporters and their messenger RNAs. Expression of the human vesicular acetylcholine transporter was restricted to mouse cholinergic somatomotor neurons in the spinal cord and brainstem, but absent from other central and peripheral cholinergic neurons. The mouse appears to be an appropriate model for the study of the genetic regulation of the cholinergic gene locus, and the physiology and neurochemistry of the mammalian cholinergic nervous system, although differences exist in the distribution of cortical cholinergic neurons between the mouse and other mammals. The somatomotor neuron-specific expression pattern of the transgenic human vesicular acetylcholine transporter suggests a mosaic model for cholinergic gene locus regulation in separate subdivisions of the mammalian cholinergic nervous system.

Failure in learning task and loss of cortical cholingergic fibers in microsphere-embolized rats.

The present study was undertaken to elucidate the pathological changes in learning and memory functions and in the metabolism of cortical cholinergic neurons following microsphere embolism in the rat. Microspheres (48 microm) were injected into the right internal carotid artery of rats. Learning and memory functions were measured 7 or more days after the embolism by active and passive avoidance, and water maze tasks. In the biochemical study, cortical acetylcholine and choline contents, and choline acetyltransferase activity were measured. Cortical acetylcholinesterase-containing fibers were quantitatively estimated in the embolized rat. The active and passive avoidance, and water maze tasks were impaired in the microsphere-embolized rat. In the histochemical study, the density of cortical acetylcholinesterase-containing fibers of the ipsilateral hemisphere of the microsphere-embolized rat was decreased, but cell density was unchanged. Furthermore, microsphere embolism decreased the cortical acetylcholine concentration and choline acetyltransferase activity and increased the choline concentration. The results suggest that microsphere embolism causes severe damage to cortical cholinergic neurons, which may be, at least in part, related to the impairment of learning and memory functions in the sustained brain ischemia.

Possible role of the cholinergic system and disease models.

Memory impairment associated with the loss of cortical cholinergic neurons in AD has stimulated the development of animal models based on blockade or destruction of these systems. Strategies include mechanical lesions, local injection of excitotoxic amino acids or ethylcholine aziridinium (AF 64A), which disrupt reference and working memory in rats, but lack specificity for cholinergic systems. Other models involving, reduction in cerebral blood flow and interference with oxidative metabolism of glucose, mimic those found in AD, and also interfere with working and long-term memory in the rat. Memory impairments can be reversed by acetylcholinesterase inhibitors and cholinergic agonists but beneficial effects of these agents in AD patients are small and inconsistent. This may be partly due to unfavorable pharmacokinetics and dose-limiting side effects of existing drugs. Newer, brain specific acetylcholinesterase inhibitors and M1 muscarinic agonists with a lower incidence of unwanted effects are currently being evaluated.

Focal Cortical Blood Flow Activation Is Regulated by Intrinsic Cortical Cholinergic Neurons

We evaluated the cholinergic mechanism underlying focal cortical vascular response to neuronal activation, using positron emission tomography for use on animals to measure cerebral blood flow and glucose metabolism activation upon vibrotactile stimulation in cats. Bromopyruvate, which blocks acetylcholine synthesis through inhibition of the production of acetyl CoA, was injected into the cerebral cortex and basal forebrain as well as the sphenopalatine ganglion, all of which have been confirmed to supply cholinergic terminals to the cerebral cortex. Although glucose metabolism was preserved, indicating that the neuronal activities were enhanced, cerebral blood flow increase during cortical neuronal activation was abolished by bromopyruvate injection into only the cerebral cortex and not other cholinergic systems. We conclude that the cholinergic intrinsic neurons control the focal cerebral blood flow increase in response to neuronal activation.

The 1993 Upjohn Award Lecture. Quinolinic acid induced brain neurotransmitter deficits: modulation by endogenous excitotoxin antagonists

Excitotoxins constitute a group of agents that are capable of activating excitatory amino acid receptors and producing axonsparing neuronal lesions. Focal injections of the exogenous excitotoxins kainic acid and ibotenic acid result in depletion of neurotransmitter markers in neuronal cell bodies located in areas of injection or in terminal zones of their projections. The discovery of endogenous agents that behave as excitotoxins has generated interest in the idea that excitotoxicity may contribute to the neuronal degeneration associated with a number of neurological diseases (Alzheimer's disease, Huntington's disease, Parkinson's disease) which involve selective neurotransmitter deficits. Quinolinic acid (QUIN), a pyridine dicarboxylic acid and metabolite of tryptophan, which has been detected in the central nervous system (CNS), behaves as an excitotoxin. In the mammalian brain QUIN has been localized to glial and immune cells, and its content increases with age. The neuro-excitatory and neurotoxic actions of QUIN are mediated via the Mg(2+)-sensitive N-methyl-D-aspartate (NMDA) receptor. The toxicity of QUIN, like that of kainate, but not ibotenate, is dependent on the presence of an intact glutamate-aspartate afferent input to the target area. Focal injections of QUIN into the nucleus basalis magnocellularis (nbM), a major source of cholinergic innervation to diencephalic areas, produce sustained loss of cholinergic neuron markers in the neocortex and amygdala. The neurotoxic action of QUIN on nbM results in an impairment of performance on memory-related tasks. Cortical and amygdaloid projecting cholinergic neurons show differential sensitivity to QUIN and other excitotoxic agents. This factor may partly explain the reported discrepancy between mnemonic deficits and the loss of cholinergic markers in the cerebral cortex induced by intra-nbM injections of certain excitotoxins. Cortical muscarinic receptor function is not significantly influenced by QUIN injections into the nbM producing loss of cortical cholinergic neurons. In the striatum, focal QUIN injections have been found to largely replicate the neurotransmitter deficits prevailing in Huntington's disease, an inherited movement disorder. Intrastriatal QUIN produces a profound loss of the NADPH diaphorase staining neurons in the area of injection but relatively spares these in the adjacent transition zone. QUIN is also highly damaging to the striatopallidal enkephalinergic neurons. However, at doses that are neurotoxic to striatal neurons, QUIN and several other excitotoxins produce significant elevations in enkephalin levels both in the striatum and globus pallidus. This elevation reflects the presence of a plasticity in the striatal enkephalinergic neuron population. The metabolic pathway yielding QUIN produces a number of intermediates that act as excitotoxin antagonists.(ABSTRACT TRUNCATED AT 400 WORDS)