RESUMEN
Nerve agents are highly toxic organophosphorus compounds that inhibit acetylcholinesterase resulting in rapid accumulation of the neurotransmitter acetylcholine (ACh) causing a cholinergic syndrome including respiratory failure. In the present study, respiratory responses and antimuscarinic treatment efficacy was evaluated ex vivo using rat precision-cut lung slices (PCLS) exposed to the nerve agent VX. The respiratory effects were evaluated either by adding exogenous ACh directly to the culture medium or by applying electric-field stimulation (EFS) to the PCLS to achieve a release of endogenous ACh from neurons in the lung tissue. The airway contraction induced by both methods was enhanced by VX and resulted in lingering airway recovery, in particular when airways were exposed to a high VX-dose. Both contractions induced by EFS and exogenously added ACh were significantly reduced by administration of the antimuscarinic drugs atropine or scopolamine. Two additions of atropine or scopolamine after maximal ACh-induced airway response was demonstrated effective to reverse the contraction. By adding consecutive doubled doses of antimuscarinics, high efficiency to reduce the cholinergic airway response was observed. However, the airways were not completely recovered by atropine or scopolamine, indicating that non-muscarinic mechanisms were involved in the smooth muscle contractions. In conclusion, it was demonstrated that antimuscarinic treatment reversed airway contraction induced by VX but supplemental pharmacological interventions are needed to fully recover the airways. Further studies should therefore clarify the mechanisms of physiological responses in lung tissue following nerve agent exposures to improve the medical management of poisoned individuals.
Asunto(s)
Atropina/farmacología , Fibras Colinérgicas/efectos de los fármacos , Inhibidores de la Colinesterasa/toxicidad , Pulmón/inervación , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/inervación , Compuestos Organotiofosforados/toxicidad , Escopolamina/farmacología , Acetilcolina/metabolismo , Acetilcolina/farmacología , Acetilcolinesterasa/metabolismo , Animales , Fibras Colinérgicas/enzimología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Femenino , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Ratas Sprague-DawleyRESUMEN
This study examines the role of cholinergic projections from the basal forebrain on development of the rodent barrel cortex. Pups were administered the immunotoxin IgG192-saporin (0.1 microg) intraventricularly at postnatal day (P) 0 and sacrificed at P1-P7. One ventricle was injected with saporin while the other side received saline, allowing comparison between the two sides of the same animal, as well as with controls receiving saline only. Compared to control animals, neuronal loss in the basal forebrain was present on both sides of saporin-treated pups but was significantly greater on the toxin-treated side, in all age groups and regions sampled. Depletion of acetylcholine did not prevent the formation of the barrel pattern, however it delayed its emergence by approximately 1 day. At P4, the thickness of layer IV barrel cortex was also significantly reduced; this reduction was undetectable by P7. From P3 to P5, the ratios of intensity of staining for acetylcholinesterase between the barrel centres and septa on the toxin-treated side were significantly lower than those on the saline side, although normal densities were present by P7. Thus, the depletion of cholinergic innervation at birth causes a transient delay in the development of the barrel pattern during the first postnatal week.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Colinérgicos/farmacología , Inmunotoxinas/farmacología , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/patología , Acetilcolinesterasa/metabolismo , Animales , Diferenciación Celular , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Fibras Colinérgicas/enzimología , Complejo IV de Transporte de Electrones/metabolismo , N-Glicosil Hidrolasas , Vías Nerviosas , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Tálamo/citología , Tálamo/crecimiento & desarrollo , Vibrisas/inervaciónRESUMEN
In Alzheimer's disease (AD), loss of cortical and hippocampal choline acetyltransferase (ChAT) activity has been correlated with dementia severity and disease duration, and it forms the basis for current therapies. However, the extent to which reductions in ChAT activity are associated with early cognitive decline has not been well established. We quantified ChAT activity in the hippocampus and four cortical regions (superior frontal, inferior parietal, superior temporal, and anterior cingulate) of 58 individuals diagnosed with no cognitive impairment (NCI; n = 26; mean age 81.4 +/- 7.3 years), mild cognitive impairment (MCI; n = 18; mean age 84.5 +/- 5.7), or mild AD (n =14; mean age 86.3 +/- 6.6). Inferior parietal cortex ChAT activity was also assessed in 12 subjects with end-stage AD (mean age 81.4 +/- 4.3 years) and compared to inferior parietal cortex ChAT levels of the other three groups. Only the end-stage AD group had ChAT levels reduced below normal. In individuals with MCI and mild AD, ChAT activity was unchanged in the inferior parietal, superior temporal, and anterior cingulate cortices compared to NCI. In contrast, ChAT activity in the superior frontal cortex was significantly elevated above normal controls in MCI subjects, whereas the mild AD group was not different from NCI or MCI. Hippocampal ChAT activity was significantly higher in MCI subjects than in either NCI or AD. Our results suggest that cognitive deficits in MCI and early AD are not associated with the loss of ChAT and occur despite regionally specific upregulation. Thus, the earliest cognitive deficits in AD involve brain changes other than simply cholinergic system loss. Of importance, the cholinergic system is capable of compensatory responses during the early stage of dementia. The upregulation in frontal cortex and hippocampal ChAT activity could be an important factor in preventing the transition of MCI subjects to AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Colina O-Acetiltransferasa/metabolismo , Trastornos del Conocimiento/metabolismo , Lóbulo Frontal/enzimología , Hipocampo/enzimología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Fibras Colinérgicas/enzimología , Fibras Colinérgicas/patología , Trastornos del Conocimiento/patología , Femenino , Lóbulo Frontal/patología , Hipocampo/patología , Humanos , Masculino , Pruebas Neuropsicológicas , Lóbulo Parietal/enzimología , Lóbulo Parietal/patología , Lóbulo Temporal/enzimología , Lóbulo Temporal/patologíaRESUMEN
A recently developed method for determining the length of cholinergic axons and number of cholinergic axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase was used to estimate the areal and laminar densities of the cholinergic innervation in rat frontal (motor), parietal (somatosensory) and occipital (visual) cortex at different postnatal ages. This cortical innervation showed an early beginning, a few immunostained fibers being already present in the cortical subplate at birth. In the first two postnatal weeks, it developed rapidly along three parameters: a progressive increase in the number of varicosities per unit length of axon, and a lengthening and branching of the axons. Between postnatal days 4 and 16, the number of varicosities increased steadily from two to four per 10 microm of cholinergic axon. The mean densities of cholinergic axons increased from 1.4 to 9.6, 1.7 to 9.3 and 0.7 to 7.2 m/mm(3), and the corresponding densities of varicosities from 0.4 to 3.9, 0.4 to 3.5, and 0.2 to 2.6x10(6)/mm(3) in the frontal, parietal and occipital areas, respectively. The rate of growth was maximal during these first two weeks, after which the laminar pattern characteristic of each area appeared to be established. Adult values were almost reached by postnatal day 16 in the parietal cortex, but maturation proceeded further in the frontal and particularly in the occipital cortex. These quantitative data on the ingrowth and maturation of the cholinergic innervation in postnatal rat cerebral cortex substantiate a role for acetylcholine in the development of this brain region and emphasize the striking growth capacity of individual cholinergic neurons.
Asunto(s)
Acetilcolina/análisis , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Fibras Colinérgicas/química , Factores de Edad , Animales , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/enzimología , Lóbulo Frontal/citología , Lóbulo Frontal/crecimiento & desarrollo , Inmunohistoquímica , Masculino , Vías Nerviosas , Lóbulo Occipital/citología , Lóbulo Occipital/crecimiento & desarrollo , Lóbulo Parietal/citología , Lóbulo Parietal/crecimiento & desarrollo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate the relationship between apolipoprotein E (APOE) genotype and both cholinergic dysfunction and synapse loss in AD. BACKGROUND: A reduction in neocortical synapses and marked losses in the cholinergic system occur in AD. It has been suggested that the number of APOE epsilon4 alleles is inversely related to choline acetyltransferase (ChAT) activity, thereby influencing cholinergic function. Whether APOE genotype may influence neocortical synapse loss remains unclear. METHODS: An autopsy series of 182 patients with AD (National Institute on Aging and Consortium to Establish a Registry for Alzheimer's Disease criteria) and 16 normal controls (NC). APOE genotype was determined in blood samples or in postmortem brain tissue. Midfrontal synapse counts (AU/microg) were quantified by a dot-immunobinding assay for synaptophysin (Syn). Midfrontal ChAT activity (nmol/h/100 mg) was assessed using standard assays. RESULTS: Mean midfrontal ChAT activity and Syn were both significantly reduced in patients with AD compared with NC. The relationship between ChAT activity and number of epsilon4 allele copies in AD was complex, with ChAT activity lower in patients with either two or no epsilon4 alleles compared with those with one epsilon4 allele. There was no relationship between APOE genotype and synapse loss in AD. Syn density was almost identical across the three genotypes. CONCLUSIONS: Unlike other studies, we failed to detect a linear relationship between ChAT activity and number of epsilon4 allele copies in the midfrontal cortex of this large sample of patients with AD. Our data also show that the presence of epsilon4 allele does not influence midfrontal synapse loss in AD. This suggests that factors other than APOE genotype may be operative in the decline in midfrontal cholinergic function and synapses seen in AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Apolipoproteínas E/genética , Fibras Colinérgicas/patología , Sinapsis/patología , Anciano , Anciano de 80 o más Años , Alelos , Apolipoproteína E4 , Autopsia , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/enzimología , Estudios de Cohortes , Lóbulo Frontal/patología , Dosificación de Gen , Genotipo , Humanos , Modelos Lineales , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sinapsis/enzimologíaRESUMEN
OBJECTIVE: To evaluate cholinergic activity in diseases with Lewy bodies (LB; LB variant of AD [LBV], diffuse LB disease [DLBD], and Parkinson's disease [PD]) to determine if 1) AD changes are requisite to cholinergic dysfunction, 2) cholinergic activity declines to the same extent in neocortical and archicortical areas, and 3) cholinergic loss is influenced by APOE genotype. BACKGROUND: Like AD, diseases with LB are associated with decreased choline acetyltransferase (ChAT) activity. Increased APOE epsilon4 allele frequency has been reported in LBV. Whether APOE genotype affects cholinergic function in LBV remains unclear. METHODS: An autopsy series of 182 AD (National Institute on Aging and Consortium to Establish a Registry for Alzheimer's Disease criteria), 49 LBV, 11 PD, 6 DLBD, and 16 normal control (NC) subjects. APOE genotype and ChAT activity (nmol/h/100 mg) in the midfrontal and hippocampal cortices were determined. RESULTS: Mean midfrontal ChAT activity was markedly reduced in diseases with LB (LBV: 53.3 +/- 39.0; PD: 54.8 +/- 35.7; DLBD: 41.3 +/- 24.8) compared to NC (255.4 +/- 134.6; p < 0.001) and AD (122.6 +/- 78.9; p < 0.05). Among diseases with LB, midfrontal ChAT activity was decreased to a similar extent in patients with (LBV) and without (DLBD and PD) AD pathology. Although mean ChAT activity for LBV was less than half that for AD in the midfrontal cortex, it was similar to that for AD in the hippocampus (LBV: 243.5 +/- 189.7; AD: 322.8 +/- 265.6; p > 0.05). However, hippocampal ChAT activity for both AD and LBV was lower than that for NC (666.5 +/- 360.3; p < 0.001). The epsilon4 allele dosage did not influence midfrontal ChAT activity in LBV. CONCLUSION: Marked losses in midfrontal ChAT activity occur in diseases with LB, independent of coexistent AD changes. A greater midfrontal, as opposed to hippocampal, cholinergic deficit may differentiate LBV from AD. The lack of a relationship between epsilon4 allele dosage and midfrontal ChAT activity suggests that other factors may play a role in its decline in LBV.
Asunto(s)
Fibras Colinérgicas/enzimología , Enfermedad por Cuerpos de Lewy/fisiopatología , Anciano , Anciano de 80 o más Años , Envejecimiento , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Apolipoproteína E4 , Apolipoproteínas E/genética , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/patología , Lóbulo Frontal/enzimología , Lóbulo Frontal/patología , Lóbulo Frontal/fisiopatología , Genotipo , Hipocampo/enzimología , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Enfermedad por Cuerpos de Lewy/genética , Persona de Mediana EdadRESUMEN
We investigated the effect of the noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) (1 or 3 x 50 mg/kg, intraperitoneally), on hippocampal, cortical and cerebellar noradrenaline levels after recovery of one, five and 11 months in control and apolipoprotein E-deficient mice. Apolipoprotein E-deficient mice had lower hippocampal noradrenaline levels than control mice. DSP-4-lesioned control mice had a more extensive recovery of hippocampal and cortical noradrenaline levels than DSP-4-lesioned apoE-deficient mice after five months' survival. Furthermore, the hippocampal noradrenaline levels after five and 11 months and cortical noradrenaline levels after five months of recovery had slightly recovered in control but not in apolipoprotein E-deficient mice treated with a single dose of DSP-4 compared with mice treated with three doses of DSP-4. These results show that apolipoprotein E-deficient mice have impaired recovery capacity in their locus coeruleus neurons.
Asunto(s)
Apolipoproteínas E/genética , Degeneración Nerviosa/metabolismo , Norepinefrina/sangre , Enfermedad de Alzheimer/metabolismo , Análisis de Varianza , Animales , Bencilaminas , Cerebelo/metabolismo , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Lóbulo Frontal/metabolismo , Hipocampo/metabolismo , Ratones , Ratones Mutantes , Degeneración Nerviosa/inducido químicamente , Serotonina/metabolismo , SimpatomiméticosRESUMEN
Degeneration of the cholinergic basal forebrain (CBF) and changes in cortical neuropeptide levels have been reported in Alzheimer's disease. In the present study, we sought to determine if a selective cholinergic lesion of nucleus basalis magnocellularis (Nbm) could affect the number and distribution of neuropeptide Y (NPY) and somatostatin (SS) immunoreactive neurons in the frontoparietal and occipital cortices of rats. Brain sections were evaluated at survival times of 1, 2, 4, 8, 12, 24, 48, 78 and 100 weeks after intraventricular injection of 192-saporin, an immunotoxin directed at the low affinity neurotrophin receptor (p75NGFr), that selectively destroys the CBF. Following the immunotoxin lesion of the Nbm, the number of NPY-labeled neurons decreased 33% in the frontoparietal cortex and 60% in the occipital cortex compared to age-matched normal controls at most survival time points. A significant loss of SS-labeled neurons in both cortical regions was seen 12 weeks after 192-saporin injection with no further change up to 100-week survival time. The effect of age on neuropeptidergic populations was evaluated in normal control rats. The number of NPY and SS immunoreactive neurons in aged rats (21-26 months) decreased by 42% in the frontoparietal cortex and 27% in the occipital cortex when compared with young (3-6 months) and middle-age (9-14 months) rats. When both non-lesioned and lesioned animals with different ages were pooled for linear regression, a significant correlation was found between the number of cortical NPY- and SS-labeled neurons and cortical acetylcholinesterase (AChE) histochemical staining intensity. These findings indicate that: (1) cholinergic denervation of the Nbm is associated with an irreversible loss of neocortical NPY and SS immunoreactive neurons analogous to that observed in Alzheimer's disease and aging; (2) the degree of the loss of cortical NPY and SS immunoreactive neurons seems to be related to the extent of the reduction of cortical AChE intensity in both toxin-injected and normal aged rats. These findings may reflect a trophic dependence of NPY and SS neurons on cortical cholinergic input.
Asunto(s)
Fibras Colinérgicas/química , Neuropéptido Y/análisis , Prosencéfalo/química , Prosencéfalo/citología , Somatostatina/análisis , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos , Recuento de Células , Muerte Celular/efectos de los fármacos , Fibras Colinérgicas/enzimología , Lóbulo Frontal/química , Lóbulo Frontal/citología , Inmunohistoquímica , Inmunotoxinas , Masculino , Neuropéptido Y/inmunología , Lóbulo Occipital/química , Lóbulo Occipital/citología , Lóbulo Parietal/química , Lóbulo Parietal/citología , Ratas , Ratas Sprague-Dawley , Somatostatina/inmunologíaRESUMEN
The role of basal forebrain-derived cholinergic afferents in the development of neocortex was studied in postnatal rats. Newborn rat pups received intraventricular injections of 192 IgG-saporin. Following survival periods ranging from 2 days to 6 months, the brains were processed to document the cholinergic lesion and to examine morphological consequences. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrate a loss of approximately 75% of the cholinergic neurons in the medial septum and nucleus of the diagonal band of Broca in the basal forebrain. In situ hybridization for glutamic acid decarboxylase mRNA reveals no loss of basal forebrain GABAergic neurons. Acetylcholinesterase histochemistry demonstrates a marked reduction of the cholinergic axons in neocortex. Cholinergic axons are reduced throughout the cortical layers; this reduction is more marked in medial than in lateral cortical areas. The thickness of neocortex is reduced by approximately 10%. Retrograde labeling of layer V cortico-collicular pyramidal cells reveals a reduction in cell body size and also a reduction in numbers of branches of apical dendrites. Spine densities on apical dendrites are reduced by approximately 20-25% in 192 IgG-saporin-treated cases; no change was detected in number of spines on basal dendrites. These results indicate a developmental or maintenance role for cholinergic afferents to cerebral cortical neurons.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Colinérgicos/farmacología , Dendritas/efectos de los fármacos , Inmunotoxinas/farmacología , Células Piramidales/ultraestructura , Corteza Visual/citología , Acetilcolinesterasa/análisis , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Axones/enzimología , Recuento de Células , Tamaño de la Célula/efectos de los fármacos , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/enzimología , N-Glicosil Hidrolasas , Prosencéfalo/citología , Prosencéfalo/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Tálamo/citología , Tálamo/crecimiento & desarrollo , Corteza Visual/crecimiento & desarrolloRESUMEN
During development, the sympathetic neurons innervating sweat glands undergo a neurotransmitter switch from noradrenergic to cholinergic between postnatal day (P) 4, when the sympathetic neurons first contact the sweat glands, and P21. Several in vitro experiments suggest that norepinephrine (NE), produced by sympathetic neurons, stimulates sweat glands to produce a factor that then induces the phenotypic switch. We tested this hypothesis in vivo using dopamine beta-hydroxylase-deficient mice (DBH -/-), which are unable to synthesize NE and epinephrine, and tyrosine hydroxylase-deficient mice (TH -/-), which are unable to synthesize any catecholamines. The cholinergic agonist pilocarpine and electrostimulation of the sciatic nerve both elicited a sweat response in adult DBH -/- mice that was indistinguishable from the response of controls, and the cholinergic antagonist atropine effectively blocked these responses. We did note, however, a 1- to 2-week delay in the acquisition of the sweat response in DBH -/- mice. Although diminished in magnitude, a sweat response to pilocarpine was also noted in TH -/- mice at P21. Immunohistochemistry demonstrated that TH and vasoactive intestinal peptide were detectable at P14 and increased to adult levels by P21 in DBH +/- and DBH -/- mice. These observations indicate that NE is not essential for the acquisition of the cholinergic phenotype, but it may facilitate its postnatal development.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Norepinefrina/farmacología , Glándulas Sudoríparas/embriología , Glándulas Sudoríparas/inervación , Acetilcolina/fisiología , Agonistas alfa-Adrenérgicos/metabolismo , Animales , Fibras Colinérgicas/química , Fibras Colinérgicas/enzimología , Dopamina beta-Hidroxilasa/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Norepinefrina/genética , Parasimpaticomiméticos/farmacología , Pilocarpina/farmacología , Glándulas Sudoríparas/efectos de los fármacos , Sudoración/efectos de los fármacos , Sudoración/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Péptido Intestinal Vasoactivo/análisisRESUMEN
In addition to cortical pathology, Alzheimer's disease is characterized by a loss of cholinergic neurons in the basal forebrain and the ventral striatum. Since cholinergic neurons which degenerate in Alzheimer's disease are sensitive to nerve growth factor, a link between nerve growth factor sensitivity and the vulnerability of cholinergic neurons has been suspected. The purpose of this study was to determine, in cholinergic neurons, the level of expression of TrkA, the high affinity receptor for nerve growth factor, in control subjects and Alzheimer patients. The study was performed by in situ hybridization using a 35S-labeled RNA probe complementary to human TrkA mRNA on immunohistochemically identified cholinergic neurons of the nucleus basalis of Meynert, the ventral striatum, and the putamen in postmortem brains of patients with clinically and neuropathologically confirmed Alzheimer's disease and control subjects. In patients with Alzheimer's disease, a decrease in TrkA mRNA expression was observed in the nucleus basalis of Meynert (-75%, P < 0.001) and the ventral striatum (-41%, P < 0.01), where the cholinergic neurons degenerate, and also in the anterior (-43%, P < 0.01) and posterior (-51%, P < 0.01) parts of the putamen, where they are spared but display precocious signs of cell alterations. These results, taken in conjunction with the reduced choline acetyltransferase activity and our previously published data showing a loss of high affinity nerve growth factor binding in both the dorsal and the ventral striatum of patients with Alzheimer's disease, indicate that receptor loss and the consequent decrease in trophic support may be associated with the degeneration of cholinergic neurons during Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Fibras Colinérgicas/química , Neostriado/citología , Neuronas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factor de Crecimiento Nervioso/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Autorradiografía , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/enzimología , Femenino , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Neostriado/química , Neuronas/enzimología , Neuronas/ultraestructura , Prosencéfalo/química , Prosencéfalo/citología , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor trkA , Receptores de Factor de Crecimiento Nervioso/metabolismo , Tinción con Nitrato de PlataRESUMEN
This study characterized the influence of full-term gestational ethanol exposure on choline acetyltransferase (ChAT)-immunoreactive neurons that project to the hippocampus, within the medial septal (MS) nucleus and the vertical limb of the diagonal band of Broca (DBv). On gestation days 1-22, pregnant dams were fed either a vitamin fortified ethanol-containing liquid diet, pair fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In a previous study, we found that chronic prenatal exposure to ethanol, in this manner, resulted in a significant decline in the ontogenetic upregulation of ChAT activity in the septal area during the second postnatal week, but was followed by recovery to control levels by adulthood. On postnatal days 14 and 60 (P14 and P60) the brains were prepared for ChAT immunocytochemistry. Ethanol exposure had little influence on the number of ChAT-positive neurons in the MS nucleus of animals at either age. Ethanol exposure had no effect on neuronal size or ChAT staining intensity of MS or DBv neurons when compared to chow-fed offspring. Although age-related increases in cholinergic neuronal numbers and decreases in neuronal size were observed between juvenile and adult animals, prenatal ethanol exposure did not appear to influence these postnatal changes in the population as a whole. Overall, these findings suggest that the anatomical maturation of septal cholinergic neurons may be relatively insensitive to prenatal ethanol exposure under conditions of a vitamin-rich dietary supplementation, while biochemical development within this region may be more susceptible to early ethanol influences.
Asunto(s)
Fibras Colinérgicas/efectos de los fármacos , Etanol/farmacología , Hipocampo/citología , Neuronas/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Núcleos Septales/citología , Animales , Recuento de Células , Tamaño de la Célula/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Femenino , Hipocampo/efectos de los fármacos , Inmunohistoquímica , Neuronas/enzimología , Embarazo , Ratas , Ratas Endogámicas , Núcleos Septales/efectos de los fármacos , Factores de TiempoRESUMEN
The subnormal choline acetyltransferase (ChoAcTase) activity in the cerebral cortex of patients with Alzheimer disease (AD) is thought to originate from the loss of cholinergic neurons in the nucleus basalis of Meynert (nbM). To examine possible changes in the functional activity of the remaining cholinergic neurons in the nbM of patients with AD, the level of expression of ChoAcTase mRNA was evaluated. A procedure for double-labeling cholinergic neurons to detect ChoAcTase mRNA and the corresponding protein in the same cell was developed, taking advantage of an anti-ChoAcTase antibody and the recently isolated cDNA complementary to a sequence of the human ChoAcTase mRNA. In the study of three controls and four patients with AD, the presence of both ChoAcTase mRNA and protein was observed in the same large neurons in both nbM and putamen. Specificity of in situ hybridization was further supported by the absence of neuronal staining with a sense probe. In AD patients a subnormal level of expression of ChoAcTase mRNA per cholinergic cell was detected in the nbM but not in the putamen. Our data support the hypothesis that expression of ChoAcTase mRNA might be down-regulated in the surviving cholinergic neurons in the nbM of patients with AD, raising the possibility of functional restoration by stimulating ChoAcTase synthesis.
Asunto(s)
Enfermedad de Alzheimer/genética , Colina O-Acetiltransferasa/genética , Sustancia Innominada/fisiología , Fibras Colinérgicas/enzimología , Expresión Génica , Humanos , Hibridación in Situ , ARN Mensajero/genéticaRESUMEN
The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.
Asunto(s)
Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/fisiología , Hipocampo/citología , Núcleo Accumbens/citología , Núcleos Septales/citología , Tálamo/citología , Animales , Fibras Colinérgicas/enzimología , Cuerpo Estriado/citología , Femenino , Microscopía Electrónica , Terminaciones Nerviosas/ultraestructura , Vías Nerviosas , Núcleo Accumbens/enzimología , Ratas , Ratas EndogámicasRESUMEN
Coronal vibratome sections of the rostral part of the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) nuclei were studied by an immunocytochemical technique using a monoclonal antibody against choline acetyltransferase (ChAT) and a double histochemical method for detection of acid phosphatase (AcPase) and nucleoside diphosphatase (NDPase) activity. The electron microscopic morphology of ChAT-immunoreactive and non-immunoreactive neurons was compared with similar neurons showing both AcPase and NDPase activity. ChAT-labeled and non-labeled neurons were well differentiated by the organization of the endomembrane system and especially by the structure of the rough endoplasmic reticulum (RER) and associated lamellar bodies. These results support the theory that the peculiar ultrastructure of the lamellar bodies in each neuron is related to the pattern of organization of the endomembrane system and its function. The significance of the lamellar bodies is discussed, and the data of the present work, together with findings described by other investigators. These data suggest that these bodies are predominant in efferent projection neurons in the basal forebrain nuclei.
Asunto(s)
Ácido Anhídrido Hidrolasas , Fibras Colinérgicas/ultraestructura , Lóbulo Frontal/ultraestructura , Membranas Intracelulares/ultraestructura , Neuronas/ultraestructura , Núcleos Septales/ultraestructura , Fosfatasa Ácida/metabolismo , Animales , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Lóbulo Frontal/citología , Lóbulo Frontal/enzimología , Histocitoquímica , Inmunohistoquímica , Membranas Intracelulares/enzimología , Masculino , Microscopía Electrónica , Neuronas/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Núcleos Septales/citología , Núcleos Septales/enzimologíaRESUMEN
We examined the electrophysiological properties of cholinergic and non-cholinergic neurons in the medial septum-diagonal band complex (MSDB) of the rat in the in vitro slice preparation. Cells were identified electrophysiologically, filled with Lucifer yellow, fixed and processed for immunohistochemistry with fluorescent labeled anti-choline acetyltransferase (ChAT) antibody. Cholinergic and non-cholinergic neurons differed in action potential parameters, spike afterpotentials and in current-voltage relationships. In addition, cholinergic neurons expressed a potent transient outward rectification in response to a depolarizing current pulse.
Asunto(s)
Fibras Colinérgicas/fisiología , Lóbulo Frontal/fisiología , Núcleos Septales/fisiología , Potenciales de Acción , Animales , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Inmunohistoquímica , Técnicas In Vitro , RatasRESUMEN
Mouse 3T3 fibroblasts were genetically modified by transfection with a mammalian expression vector containing the rat beta-nerve growth factor (NGF) gene. The transfected cell line, designated 3E, contains several hundred copies of the rat NGF gene and secretes high levels of biologically active NGF. Pieces of collagen gel containing the NGF-secreting 3E cells were grafted to the brains of unilaterally fimbria-fornix-lesioned rats. Grafts of the genetically modified NGF-producing cells rescued axotomized basal forebrain cholinergic neurons and significantly reduced cholinergic cell death in the medial septum as compared with rats treated with grafts of the parental 3T3 cells. Grafted fibroblast cells were detected, and rescue effects were noted up to 6 weeks after grafting. Local effects of NGF secreted by grafted cells were also seen at the gel-brain border in the form of sprouting acetylcholinesterase immunoreactive host cortical fibers. We suggest that implantation of genetically modified cells producing NGF may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease.
Asunto(s)
Fibras Colinérgicas/fisiología , Fibroblastos/trasplante , Lóbulo Frontal/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas Recombinantes/genética , Acetilcolinesterasa/metabolismo , Animales , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/enzimología , Femenino , Fibroblastos/metabolismo , Lóbulo Frontal/citología , Lóbulo Frontal/efectos de los fármacos , Inmunohistoquímica , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Ratas , Ratas Endogámicas , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Following fimbria/fornix transection, cells in the medial septum appear to undergo retrograde degeneration as shown by Nissl and acetylcholine esterase (AChE) staining. Recent studies using immunocytochemical techniques have also demonstrated loss of choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr) labeling of neurons in this region. Whether the apparent loss of ChAT- and NGFr-positive neurons is the result of the actual death of these neurons, or is instead a loss of ChAT enzyme or NGFr expression below levels detectable by immunocytochemical methods, remains an unresolved issue. In order to address this question, rhodamine-labeled fluorescent latex microspheres were injected into the hippocampus where they retrogradely transported to the cell bodies of the medial septum. Five days later these animals received either unilateral or bilateral fimbria/fornix lesions and were allowed to survive an additional 4 weeks. Compared to unlesioned control animals, unilaterally lesioned animals showed a 91% loss of fluorescently labeled cells and bilaterally lesioned animals showed a 93% loss. The inability to detect the fluorescent microspheres in the medial septum suggests that the majority of medial septal cells die after fimbria/fornix transection. ChAT and NGFr immunohistochemical staining were also performed. Cells stained for ChAT were reduced in number by 92% in animals with unilateral lesions and by 75% in animals with bilateral lesions, while NGFr-stained cells were reduced in number by 75% in animals with unilateral lesions and by 68% in animals with bilateral lesions.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Fibras Colinérgicas/fisiología , Lóbulo Frontal/citología , Hipocampo/fisiología , Degeneración Nerviosa , Acetilcolinesterasa/metabolismo , Animales , Fibras Colinérgicas/enzimología , Femenino , Lóbulo Frontal/fisiología , Inmunohistoquímica , Ratas , Ratas EndogámicasRESUMEN
The study evaluates whether any degenerative changes affect forebrain cholinergic systems during natural aging. The medial septal nucleus, the nuclei of the diagonal band, the neostriatum, and the basal nucleus were studied in adult and aged Wistar rats. Butcher's technique for acetylcholinesterase allowed us to identify neurons located in these forebrain nuclei, which stained intensely or moderately for the enzyme and were putatively cholinergic. The size of forebrain regions containing stained neurons, and the number and size of stained perikarya located therein, were measured. In aged rats, the size of forebrain cholinergic nuclei was reduced by an average of 26%. The density of neurons located in these regions was also significantly lower in aged rats than in controls; intensely stained neurons displayed a mean reduction of 27.81%, while intensely and moderately stained perikarya together were reduced by 25.43%. Cross-sectional area of the stained perikarya was also reduced in aged rats by 32.87%. These data show that the number of forebrain acetylcholinesterase-containing neurons is reduced in aged rats. They are consistent with the hypothesis that natural aging brings about a diffuse and homogeneous depletion of forebrain cholinergic perikarya. Neurons which are viable, and can be selectively stained, show morphological alterations, which are likely to be related to a degenerative process.
Asunto(s)
Envejecimiento/patología , Fibras Colinérgicas/patología , Lóbulo Frontal/patología , Acetilcolinesterasa/metabolismo , Animales , Recuento de Células , Fibras Colinérgicas/enzimología , Masculino , Ratas , Ratas EndogámicasRESUMEN
Application of acetylcholinesterase (AChE) method to the hypothalamus of the cobra Naja naja revealed a clearly discernible pattern of activity in the nuclear groups. The magnocellular neurons distributed in various subdivisions showed strong reaction. Among the parvocellular groups, intense enzyme activity was observed in the cells of nucleus subfornicalis, nucleus hypothalamicus medialis, dorsal and ventral subdivisions of the nucleus hypothalamicus lateralis and the nucleus mamillaris. The paraventricular organ displayed strong amorphous activity accompanied by the intense reaction in the nucleus of the paraventricular organ. The neurons of the lateral preoptic area, nucleus periventricularis posterior, nucleus hypothalamicus dorsomedialis, nucleus lateralis recessus infundibuli and nucleus medialis recessus infundibuli showed heterogenous population revealing varying degrees of staining intensity. The perikarya of the nucleus suprachiasmaticus, nucleus hypothalamicus ventromedialis and nucleus arcuatus showed light staining. The rest of the nuclear groups were unstained. Whereas, lateral forebrain bundles and supraoptic decussation showed some AChE activity in the fibers, anterior commissure and optic chiasma were distinctly negative. The AChE-positive neuropil was confined to the lateral preoptic, and circumscribed periventricular tuberal areas. In the median eminence, a layer of fine AChE-reactive granules restricted to the subependymal zone was found; the ependymal layer was equipped with large, intensely positive somata.