Oligomers of ß-amyloid protein (Aß1-42) induce the activation of cyclooxygenase-2 in astrocytes via an interaction with interleukin-1ß, tumour necrosis factor-α, and a nuclear factor κ-B mechanism in the rat brain.

Despite growing evidence indicating the effects of cytokines, including interleukin-1beta (IL-1ß) and tumour necrosis factor-α (TNFα), and the enzyme cyclooxygenase-2 (COX-2) in Alzheimer's diseases, little is known about the signalling mechanisms that mediate its activation in response to beta-amyloid protein (Aß). The aim of this study was first to investigate whether Aß1-42 peptide induced the up-regulation of COX-2. We then examined the expression of COX-2 and cytokines, such as IL-1ß and TNFα, in reactive astrocytes. Finally, we analyzed the role of nuclear factor kappa-B (NF-κB) as a signalling pathway in early stages of Aß-toxicity. In Wistar rats anaesthetised with equitesine, a single microinjection of Aß1-42 oligomers was made in the left retrosplenial cortex. Control animals were injected with Aß42-1 peptide into the corresponding region of the cerebral cortex. By COX-2 immunoblotting, we detected two immunopositive protein bands, at 70 and 50 kDa molecular mass. In the Aß1-42-injected animals the 50 kDa fragment showed a significant increase at 3 and 14 days, as compared with that seen in control animals. The 70 kDa fragment showed a maximal increase at 14 days. In the Aß1-42-injected animals immunoblot staining of NF-κB detected an active protein band at 50 kDa molecular mass, showing a maximal increase at the 72 h time point. Confocal analysis revealed that COX-2 protein co-localized with Aß-IR material at the injection site and in endothelial blood vessels, increasing at 72 h. In the Aß oligomer-treated animals, COX-2, IL-1ß, and TNFα proteins were expressed in reactive astrocytes surrounding the injection site and blood vessels at early stages of Aß toxicity. Double-labelling immunofluorescence studies also revealed that GFAP and COX-2 proteins co-localized with NF-κB-positive material at early time-points. In conclusion, our results suggest that in reactive astrocytes and in COX-2 positive cells NF-κB may mediate pro-, and/or inflammatory gene expression and that, develop strategies that target the GFAP/NF-κB and COX-2/NF-κB pathways might contribute to reducing Aß-induced toxicity.

Soluble oligomeric forms of beta-amyloid (Abeta) peptide stimulate Abeta production via astrogliosis in the rat brain.

The aim of this study was to investigate the interaction between beta-amyloid (Abeta) peptide and astrogliosis in early stages of Abeta toxicity. In Wistar rats, anaesthetised with equitesine, a single microinjection of Abeta1-42 oligomers was placed into the retrosplenial cortex. Control animals were injected with Abeta42-1 peptide into the corresponding regions of cerebral cortex. Immunocytochemical analysis revealed an intense Abeta immunoreactivity (IR) at the level of Abeta1-42 injection site, increasing from the first 24 h to later (72 h) time point. Control injection showed a light staining surrounding the injection site. In Abeta oligomers-treated animals, Abeta-immunopositive product also accumulates in cortical cells, particularly in frontal and temporal cortices at an early (24 h) time point. Abeta-IR structures-like diffuse aggregates forms were also observed in hippocampus and in several cortical areas, increasing from the first 24 h to later (72 h) time point. In control animals no specific staining was seen neither in cortical cells nor in structures-like diffuse aggregates forms. Injections of Abeta oligomers also induce activation of astrocytes surrounding and infiltrating the injection site. Astrocyte activation is evidenced by morphological changes and upregulation of glial fibrillary acidic protein (GFAP). By GFAP immunoblotting we detected two immunopositive protein bands, at 50 and 48 kDa molecular mass. Confocal analysis also showed that GFAP co-localized with Abeta-IR material in a time-dependent manner. In conclusion, our results indicate that astrocyte activation might have a critical role in the mechanisms of Abeta-induced neurodegeneration, and that should be further studied as possible targets for therapeutic intervention in AD.

Beta-amyloid peptide-induced modifications in alpha7 nicotinic acetylcholine receptor immunoreactivity in the hippocampus of the rat: relationship with GABAergic and calcium-binding proteins perikarya.

The effects of the injected beta-amyloid (Abeta) protein on the alpha7 subtype of nicotinic acetylcholine receptor protein (alpha7nAChR) in the hippocampus were studied in rats. Injections of Abeta into the retrosplenial cortex resulted in a decrease in alpha7nAChR-immunoreactivity in the hippocampus. Quantitative analysis revealed a significant reduction in alpha7nAChR-immunoreactivity in the dorsal part of the CA1 ipsilateral to the Abeta-injected side as compared to the corresponding hemisphere of non-treated control animals and with that seen in the contralateral hemisphere, which corresponds to the control (PBS)-injected side. A significant decrease in alpha7nAChR-immunoreactivity was also found in the dorsal part of the ipsilateral CA1 as compared with that in the ventral part of the CA1, in CA2, and in CA3 ipsilateral to the Abeta-injected side. The analysis also revealed a significant decrease in alpha7nAChR-immunoreactivity in the dentate gyrus ipsilateral to the Abeta-injected side as compared to the corresponding hemisphere of non-treated control animals and with that in the PBS-injected side co-localization studies showed that the alpha7nAChR protein is highly localized in GABA- and Parv-immunoreactive cells, while only few Calb-positive cells expressed immunoreactivity for alpha7nAChR. In addition, injections of Abeta protein resulted in a significant reduction in the number of GABA- and Parv-immunoreactive cells in the dorsal part of the ipsilateral CA1 as compared to the corresponding region of non-treated control animals and with that in the corresponding region of the PBS-injected side. Our findings suggest that Abeta induces a reduction in alpha7nAChR-containing cells, which may contribute to impairment of GABAergic synaptic transmission in the hippocampus.

Effects of beta-amyloid peptide on the density of M2 muscarinic acetylcholine receptor protein in the hippocampus of the rat: relationship with GABA-, calcium-binding protein and somatostatin-containing cells.

AIMS: The deposition of amyloid peptides (A beta) in the cortex and hippocampus is the primary trigger of Alzheimer's disease (AD). Recent studies also indicated that the M2 subtype of muscarinic acetylcholine receptors (M2mAChR) may be a key molecule involved in cognitive dysfunction. Thus, the purpose of this study was to determine the effects of extracellular deposition of A beta on the density of M2mAChR in the hippocampus of the rat by M2mAChR-immunohistochemistry. METHODS: Special attention was paid to discerning any interaction between A beta and M2mAChR in GABA-, and calcium-binding protein containing cells by double-labelling immunohistochemistry. Densitometric analysis of M2mAChR-immunoreactivity was performed using Scion Image Beta Software. Quantitative analysis of GABA-, and calcium-binding protein interneurones containing M2mAChR protein was performed using a NeuroLucida morphometric system. RESULTS: Injections of A beta into the retrosplenial cortex resulted in a significant reduction in M2mAChR-immunoreactivity in the CA1 ipsilateral to the A beta-injected side as compared with the corresponding hemisphere of non-treated control animals and with that in the corresponding region of the CA1 in the phosphate-buffered saline-injected side. Co-localization studies showed that the M2mAChR is localized in a subset of GABA-positive cells of the hippocampus, in cells that contain calcium-binding proteins, and in a subpopulation of cells that contain the neuropeptide somatostatin. CONCLUSIONS: Our findings suggest that A beta induces a significant reduction in M2mAChR-immunoreactivity in the CA1 of the hippocampus and a reduction in GABAergic interneurones containing M2mAChR, which may contribute to impairment of GABAergic synaptic transmission in area CA1 of hippocampus.

Interaction between â-amyloid protein andthe á7 nicotinic acetylcholine receptorin cholinergic, gabaergic and calcium-bindingproteins-containing neurons in theseptum-diagonal band complex of the rat

The effects of intracerebral injection of thebeta-amyloid protein (Aâ1-40) on the á7subtype of nicotinic acetylcoline receptor protein(nAChR) in neurons of the septum-diagonalband (MS-nDBB) complex were studiedin rats. Focal deposition of Aâ in the retrosplenialcortex resulted in a selective reductionin the number of á7nAChR-immunoreactivecells in different parts of the MS-nDBB complex,especially in the horizontal nucleus ofthe diagonal band of Broca (HDB). The analysisrevealed a significant decrease of 37.27%in the number of á7nAChR-immunoreactivecells in the HDB ipsilateral to the Aâ-injectedside as compared to the correspondinghemisphere of non-treated control animals,and a reduction of 31.55% was observed inthe HDB ipsilateral to the Aâ-injected side ascompared to the contralateral HDB, whichcorresponds to the control (PBS)-injected side.A significant reduction (up to 20%) ofá7nAChR-containing neurons was also foundin the medial septal nucleus when comparedwith the corresponding hemisphere in nontreatedcontrol animals. The results alsorevealed that á7nAChR-positive immunoreactivityis highly localized within cytoplasmicgranules in cholinergic neurons and in a smallsubset of putative GABAergic cells of the MSnDBBcomplex. In conclusion, these findingssuggest an interaction of Aâ1-40 with theá7nAChR, which may contribute to impairmentsin cholinergic and GABAergic transmissionin the MS-nDBB complex (AU)

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Effects of lipids and aging on the neurotoxicity and neuronal loss caused by intracerebral injections of the amyloid-beta peptide in the rat.

The influence of diet and age on the area of lesion and on the neuronal density in the cerebral cortex was studied in rats following local injections of the amyloid-beta peptide (Abeta1-40) in PBS vehicle into the left frontal and cingulate cortices and compared with effects of injections of PBS alone into the corresponding regions of the right hemisphere The experiments were carried out in two groups of animals: one group of young adult rats and a second group of aged rats. Each group of animals, depending on the diet received, was divided into high-cholesterol, high-fat, and a control group. In order to evaluate the interaction of Abeta/PBS-cholesterol and of Abeta/PBS-fat, animals without dietary manipulation receiving Abeta and PBS injection were used as controls. The results showed that the greatest area of lesion was at Abeta injection sites in the high-cholesterol fed group of aged animals. The results also revealed a significant variance in the neuronal density by group and by injection type. Thus, high-cholesterol fed animals showed a greater reduction in neuronal density at Abeta and PBS-injected sites than that seen in the high-fat or control groups. The results also indicate that the loss of neurons at the Abeta injection site exceeds that seen in the PBS-injected area. The greatest reduction in the neuronal density was found at Abeta-injected site in the high-cholesterol fed group of aged animals. In conclusion, our findings indicate an interaction between lipids, age, and Abeta neurotoxicity, and might provide insights into the basic mechanisms involved in a short-term (acute-to-subchronic) response to Abeta peptide.

Amyloid beta peptide-induced cholinergic fibres loss in the cerebral cortex of the rat is modified by diet high in lipids and by age.

The influence of diet and age on the effects of intracerebral injection of beta-amyloid peptide (Abeta1-40) in vehicle phosphate-buffered saline (PBS) and on the effects of vehicle alone on cholinergic fibres of the cerebral cortex was studied in rats. The experiments were carried in two groups of animals: one group of young adult rats and a second group of aged rats. Each group of animals, depending on the diet received, was divided into high-cholesterol, high-fat, and a control diet group. In order to evaluate the interaction of Abeta/PBS-cholesterol and of Abeta/PBS-fat, animals without dietary manipulation receiving Abeta and PBS injection were used as controls. High-cholesterol fed animals showed a statistically significant reduction of 49.62% in the number of cholinergic fibres at the Abeta injection site as compared with that at PBS injection site, while the high-fat and control animals showed a significant reduction of 28.13 and 26.81%, respectively. In all diet groups, the loss of cholinergic fibres caused by Abeta as compared to that caused by PBS injection was significantly greater in aged rats in comparison with that observed in the young animals. Furthermore, the results of a multivariate linear regression model revealed that the greatest reduction in cholinergic fibres was in the high-cholesterol fed animals (35 fibres/mm) as compared with that seen in the high-fat and control animals. A significantly greater reduction was also observed at Abeta injection site (28 fibres/mm) as compared with that caused by PBS injection, and a reduction of 16 cholinergic fibres per mm was found in aged animals as compared to that seen in young adult rats. These results show that high-cholesterol diet enhances the toxicity of Abeta peptide and that this is also age-dependent. Therefore, this study increases the evidences of the role of cholesterol in the pathology of Alzheimer's disease (AD).

Effects of beta-amyloid protein on serotoninergic, noradrenergic, and cholinergic markers in neurons of the pontomesencephalic tegmentum in the rat.

The effects on serotoninergic, noradrenergic and cholinergic markers on neurons of the pontomesencephalic tegmentum nuclei were studied in rats following local administration of fibrillar beta-amyloid peptide (Abeta1-40) into the left retrosplenial cortex. Focal deposition of Abeta in the retrosplenial cortex resulted in a loss of serotoninergic neurons in the dorsal and median raphe nuclei. The dorsal raphe nucleus showed a statistically significant reduction of 31.7% in the number of serotoninergic neurons and a decrease (up to 17.38%) in neuronal density in comparison with the same parameters in uninjected controls. A statistically significant reduction of 50.3%, together with a significant decrease of 53.94% in the density of serotoninergic neurons, was also observed in the median raphe nucleus as compared with control animals. Furthermore, a significant reduction of 35.07% in the number of noradrenergic neurons as well as a statistically significant decrease of 56.55% in the density of dopamine-beta-hydroxylase-immunoreactive neurons were also found in the locus coeruleus as compared with the corresponding hemisphere in uninjected controls. By contrast, a reduction of 24.37% in the number of choline acetyltransferase-positive neurons and a slight decrease (up to 22.28%) in the density of cholinergic neurons, which were not statistically significant, was observed in the laterodorsal tegmental nucleus in comparison with the same parameters in control animals. These results show that three different neurochemically defined populations of neurons in the pontomesencephalic tegmentum are affected by the neurotoxicity of Abeta in vivo and that Abeta might indirectly affect serotoninergic, noradrenergic and cholinergic innervation in the retrosplenial cortex.

Alteration of cholinergic, excitatory amino acid and neuropeptide markers in the septum-diagonal band complex following injections of fibrillar Beta-amyloid protein into the retrosplenial granular cortex of the rat / Alteraciones en los sistemas colinérgico, aminoacidérgicos y neuropeptidérgicos del prosencefalo basal tras inyectar la proteína beta-amiloide(1-40) en la corteza retroesplenial de la rata

The effects on cholinergic, excitatory amino acid and neuropeptide markers in the basal forebrain of an intracerebrally-injected synthetic peptide corresponding to the first 1-40 amino acids of beta-amyloid protein (A?1-40) was studied in rats. Focal deposition of A? in the retrosplenial granular cortex resulted in a significant loss of cholinergic and glutamate-immunoreactive neurons in the septum-diagonal band complex. The medial septal nucleus showed a reduction of 39% ± 1% in the number of cholinergic neurons and a significantly reduced number of cholinergic neurons (up to 22% ± 10%) was also found in the horizontal nucleus of the diagonal band of Broca in comparison with the same paramaters in uninjected controls. A markerd loss of glutamate-containing neurons was also found in the medial septal nucleus (up to 19% ± 6%) together with a decrease of approximately 22% ± 3% in the horizontal nucleus of the diagonal band of Broca when compared with uninjected control animals. Furthermore, a moderate reduction in fibres and structures such as terminals immunoreactive for glutamate, substance P, and neurotensin was observed in the septum-diagonal band complex. These results show that two different neurochemically defined populations of neurons in the basal forebrain are affected by the neurotoxicity of A? in vivo (AU)

En ratas (Wistar, 200-300 g), se realizaron microinyecciones (2 µg/ 1 µl) del péptido Beta-amiloide (Aß1-40) en diferentes regiones de la corteza retroesplenial (parte caudal de la corteza cingular) en el hemisferio izquierdo. En la misma sesión quirúrgica y como control, se realizaron microinyecciones de la solución tampón de fosfato salino (1 µl PBS) en diferentes regiones de la corteza retroesplenial en el hemisferio derecho. Tras periodos de entre una y dos semanas, los animales se perfundieron y el material se procesó mediante técnicas de inmunocitoquímica, utilizando diversos anticuerpos. Inyecciones de Aß en la corteza retroesplenial determinaron alteraciones significativas en los sistemas cholinérgico y glutamatérgico del prosencefalo basal, especialmente en el núcleo septal medial y en núcleo de la banda diagonal de Broca. En el colinérgico, se observó una pérdida de un 39 ñ 1 por ciento en el número de neuronas colinérgicas en el núcleo septal medial, mientras que en el núcleo de la banda diagonal de Broca se apreció una reducción de un 22 ñ 10 por ciento, respecto al control. En el sistema glutamatérgico, la inyección de Aß determinó una marcada pérdida de neuronas inmunorreactivas ante glutamato en la porción horizontal del núcleo de la banda diagonal de Broca (22 ñ 3 por ciento) y en el núcleo septal medial (19 ñ 6 por ciento). Además, en el complejo nuclear de la banda diagonal de Broca se apreciaron alteraciones en algunos sistemas neuropeptidérgicos, especialmente se observó una marcada disminución de fibras y terminales inmunorreactivos ante substancia P y neurotensina. En conclusión, el estudio demuestra la susceptibilidad de dos poblaciones de neuronas del prosencefalo basal, colinérgicas y posiblemente glutamatérgicas, ante el péptido Aß1-40 in vivo y apoya el papel de estos sistemas de neurotransmisión en la etiología de la enfermedad de Alzheimer (AU)

Localization of excitatory amino acid and neuropeptide markers in neurons of the subicular complex projecting to the retrosplenial granular cortex of the rat / Caracterización inmunocitoquímica de marcadores excitadores aminoacidérgicos y neuropeptidérgicos en neuronas del complejo subicular que proyectan a la corteza granular retroesplenial de la rata

Retrograde labelling was combined with immunohistochemistry to localize neurons containing glutamate and different neuropeptides such as neurotensin, leu-enkephalin, and substance P-like immunoreactivity in the projection pathways from the presubiculum and subiculum to the retrosplenial granular cortex of the rat. Injections of horseradish peroxidase conjugated to subunit B of cholera toxin (CT-HRP) into the retrosplenial granular cortex labelled large numbers of neurons in the presubiculum. A significant number of retrogradely-labelled neurons was seen in the dorsal subiculum, whereas small numbers of CT-HRP-labelled neurons were also found in the ventral subiculum. In the presubiculum, 90-95% of the CT-HRP-labelled neurons (30-32 per section) were also immunoreactive for glutamate, and small numbers of retrogradely-labelled neurons also displayed neurotensin-, leu-enkephalin- or substance P-immunoreactivity. In the subiculum, approximately 90-95% of the CT-HRP-labelled neurons (19-20 per section) were also immunoreactive for glutamate, and a significant number of retrogradely-labelled neurons (70-75%, 14-15 per section) also displayed neurotensin immunoreactivity. In addition, small numbers of CT-HRP-labelled neurons in the subiculum were immunoreactive for leu-enkephalin or substance P. These results suggest that the complexity of the neurotransmitter(s)/neuromodulator(s) of the subicular projections to the retrosplenial granular cortex of the rat should be taken into account when considering the mechanisms of the retrosplenial cortical neurons thought to play a role in memory (AU)

En ratas (Wistar, 250-300 g), se realizaron microinyecciones (0.2?l-0.6?l) de una solución acuosa al 2 por ciento de peroxidasa de rábano conjugada con la toxina del cólera (CT-HRP) en diferentes regiones de la corteza retroesplenial (parte caudal de la corteza cingular). Tras periodos de supervivencia entre 24 y 48 horas, los animales se perfundieron con una solución de paraformaldehido al 2 por ciento y glutaraldehido al 0.1 por ciento en solución tampón de fosfato (pH 7.2). Los cerebros se seccionaron en vibratomo y series adyacentes de 50 µm de grosor se procesaron para poner de manifiesto la presencia de CT-HRP mediante una reacción histoquímica utilizando tetrametilbenzidina (TMB) como crómogeno y la distribución de neuronas inmunorreactivas ante glutamato y ante diversos neuropéptidos, como neurotensina, encefalina y substancia P, utilizando DAB como cromogeno y técnicas de inmunocitoquímica con antisueros anti-glutamato, anti-neurotensina, anti-encefalina y anti-substancia P. El análisis del material muestra la presencia de diversas poblaciones neuronales marcadas retrógradamente por el complejo CT-HRP en la corteza hipocampal, especialmente en el presubiculum y en la región dorsal del subiculum, y por lo tanto son neuronas de proyección a la zona de inyección en la corteza retroesplenial. En el presubiculum el 90-95 por ciento de las neuronas marcadas retrógradamente eran inmunorreactivas ante glutamato y una pequeña proporción de neuronas de proyección al lugar diana expresaban inmunorreactividad ante los diferentes sistemas neuropeptidérgicos analizados, neurotensina, encefalina o substancia P. En el subiculum, aproximadamente el 90-95 por ciento de las neuronas marcadas retrógradamente expresaban inmunorreactividad ante glutamato y una población neuronal significativa, entre el 70-75 por ciento, expresaba inmunorreactividad ante neurotensina. Además, en el subiculum se apreció una pequeña población de neuronas marcadas retrógradamente por el complejo CT-HRP inmunopositivas ante encefalina o substancia P. En conclusión, el estudio indica que la proyección de la corteza hipocampal (subiculum y presubiculum) a la corteza cingular está mediada por diversos sistemas de neurotransmisión, esencialmente aminoácidos excitadores y neuropeptidos, y sugiere que el complejo sistema de neurotransmisión hipocampo-cortical (corteza cingular) puede tener un papel importante en los mecanismos cognitivos en los que está implicado este sistema, tales como memoria y aprendizaje (AU)

Glutamatergic components of the retrosplenial granular cortex in the rat.

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 microm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V-VI; some were also present in layers I-III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.

Localization of amino acids, neuropeptides and cholinergic markers in neurons of the septum-diagonal band complex projecting to the retrosplenial granular cortex of the rat.

Retrograde labelling was combined with immunohistochemistry to localize neurons containing choline acetyltransferase, gamma-aminobutyric acid (GABA), glutamate, leu-enkephalin, neurotensin, and substance P-like immunoreactivity in the projection pathways from the septum-diagonal band complex to the retrosplenial granular cortex in the rat. Injections of horseradish peroxidase conjugated to subunit B of cholera toxin (CT-HRP) into the retrosplenial granular cortex resulted in retrogradely labelled neurons in the ipsilateral nuclei of the diagonal band of Broca, especially in the horizonatal nucleus of the diagonal band, and small numbers of CT-HRP-labelled neurons were also found in the medial septal nucleus. In the horizontal and vertical nuclei of the diagonal band of Broca, 90-95% of CT-HRP-labelled neurons (35-45 per section) were immunoreactive for choline acetyltransferase and small numbers of retrogradely labelled neurons (2 to 4-5 per section) were also immunoreactive for GABA, glutamate, neurotensin, leu-enkephalin, or substance P. In the medial septal nucleus approximately 75-80% of the retrogradely labelled neurons (8-10 per section) were immunoreactive for choline acetyltransferase and up to 25% of the CT-HRP labelled neurons (1-3 per section) in the medial septal nucleus also displayed GABA-, glutamate-, neurotensin-, leu-enkephalin-, or substance P-immunoreactivity. These results suggest that the complexity of the neurotransmitter(s)/neuromodulator(s) of septum-diagonal band complex projections to the retrosplenial granular cortex should be taken into account when considering the mechanisms of cortical activation.

Immunoelectron microscopic study of glutamate inputs from the retrosplenial granular cortex to identified thalamocortical projection neurons in the anterior thalamus of the rat.

We have carried out an ultrastructural study to determine the characteristics and distribution of glutamate-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to glutamate and a postembedding immunogold detection method in animals in which the neurons of AD/AV projecting to the cortex had been retrogradely labelled and the terminals of corticothalamic afferents anterogradely labelled by injection of cholera toxin-horseradish peroxidase (HRP) into the retrosplenial granular cortex. The heaviest immunogold labelling was over axon terminals 0.42 to 2.2 microm in diameter containing round synaptic vesicles and establishing Gray type 1 (asymmetric) synaptic contact (type 1 terminals) on HRP-labelled or non-labelled dendrites. Mean gold particle densities over such terminals were 3-4 times higher than the densities over the dendrites to which they were presynaptic and 5-6 times higher than over terminals establishing Gray type 2 (symmetric) synaptic contacts (type 2 terminals). Gold particle densities over neuronal cell bodies and dendrites and over a subpopulation of myelinated axons were intermediate between the densities over type 1 and type 2 terminals. In adjacent serial sections immunoreacted for gamma aminobutyric acid, type 2 terminals were heavily immunolabelled whereas type 1 terminals and other profiles with moderate gold particle densities after glutamate immunoreaction displayed very low labelling. A subpopulation of small type 1 axon terminals (up to 1 microm diameter) contained HRP reaction product identifying them as cortical in origin; they contacted small dendritic profiles (most <1 microm diameter) many of which also contained HRP reaction product. We conclude that terminals of the corticothalamic projection from retrosplenial granular cortex to AD/AV are glutamatergic and innervate predominantly distal dendrites of thalamocortical projection neurons.

Neurotransmitter characteristics of neurons projecting to the supramammillary nucleus of the rat.

Retrograde labelling was combined with immunohistochemistry to localize neurons containing choline acetyltransferase, gamma-aminobutyric acid (GABA), glutamate, serotonin, somatostatin, Leu-enkephalin, neurotensin, and substance P-immunoreactivity in neurons projecting to the supramammillary nucleus in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the supramammillary nucleus resulted in retrogradely labelled neurons in the medial septal nucleus, the nuclei of the diagonal band of Broca, the infralimbic cortex, the medial and lateral preoptic nucleus, the subiculum, the laterodorsal tegmental nucleus, the compact subnucleus of the central superior nucleus and the dorsal raphe nucleus. In the medial septal nucleus and in the nuclei of the diagonal band of Broca, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for choline acetyltransferase and small numbers of WGA-HRP-labelled neurons were immunoreactive for GABA, glutamate, neurotensin or substance P. In the medial preoptic nucleus, 85-90% of retrogradely labelled neurons (25-30 per section) were immunoreactive for somatostatin and a few WGA-HRP-labelled neurons displayed neurotensin-immunoreactivity. In the rostroventral part of the subiculum, small numbers of retrogradely labelled neurons were also immunoreactive for neurotensin or for glutamate. In the laterodorsal tegmental nucleus, 90% of WGA-HRP-labelled neurons (20-25 per section) were immunoreactive for choline acetyltransferase and small numbers of retrogradely labelled neurons also displayed substance P immunoreactivity. In the compact subnucleus of the central superior nucleus, 50-60% of retrogradely labelled neurons (15-20 per section) were also immunolabelled for GABA and approximately 30-40% of WGA-HRP-labelled neurons (10-12 per section) were immunoreactive for Leu-enkephalin. The compact subnucleus of the central superior nucleus also contained small numbers of retrogradely labelled neurons that displayed neurotensin immunoreactivity. In the dorsal raphe nucleus, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for serotonin and small numbers of retrogradely labelled neurons displayed neurotensin or glutamate immunoreactivity. These results suggest that the multiple neurochemicals contained in ascending and descending projections to the SuM participate in complex interactions in the transmission process of SuM neurons.

[Changes in neurotransmission systems after the injection of beta-amyloid protein beta (12-28) in the hypothalamus and anterior thalamus of the rat]. / Alteraciones en los sistemas de neurotransmisión tras inyectar la proteína beta-amiloide beta (12-28) en el hipotálamo y en el tálamo anterior de la rata.

INTRODUCTION AND OBJECTIVE: Alzheimer's disease (AD) is a degenerative central nervous system disorder. The histopathologic features include senile plaques, which are composed primarily of insoluble aggregates of amyloid beta-protein (A beta). The purpose of this study was to analyse the effects of A beta on several neurotransmitter/neuromodulator systems as a possible model for the neuropathological changes that occur in AD. MATERIAL AND METHODS: Adult rats (Wistar, 200-300 g) received injections of A beta (12-28) (0.5 microliter-2 microliters, 1 microgram-4 micrograms) into the hypothalamus (mammillary complex) and anterior thalamic nuclei (ATN). After a postinjection survival period of 3 days-3 weeks the rats were perfused and the brains processed for the immunocytochemical localization of several neurochemicals markers. RESULTS AND CONCLUSIONS: Following injections of A beta into the mammillary nuclei and into ATN a significant decrease in fibres and terminal like-structures immunoreactive for ChAT were found in the cortex and hippocampus. Loss and degeneration of cholinergic neurons was also observed in the basal forebrain (medial septal nucleus, nuclei of the diagonal band of Broca and magnocellular nucleus of Meynert). A similar pattern of changes was also found in the glutamatergic system. Thus, injections of A beta into the mammillary nuclei caused a moderate loss of glutamate-immunoreactive neurons in the ATN and tegmental nuclei of Gudden, whereas injections of A beta into the ATN caused a moderate loss of glutamate-immunoreactive neurons in the retrosplenial cortex, hippocampus and mammillary nuclei. No significant changes were found in the GABAergic system. The alteration of certain neuropeptides, such us the somatostatin, is also a consistent finding. These results indicate the possibility that A beta in vivo caused alteration of different neurotransmitter/neuromodulator markers in the rat brain.

Immunoelectron microscopic study of gamma-aminobutyric acid inputs to identified thalamocortical projection neurons in the anterior thalamus of the rat.

We have carried out a semi-quantitative ultrastructural study to determine the characteristics and distribution of gamma-aminobutyric acid (GABA)-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to GABA and a postembedding immunogold detection method in animals in which the cortical projection neurons of these nuclei had been labelled by retrograde transport of cholera toxin/horseradish peroxidase (HRP) injected into the retrosplenial granular cortex. Two types of GABA-immunopositive structures were identified, with gold particle densities 4-40 times higher than the highest densities over blood-vessel lumens and areas of empty resin: (1) an apparently homogeneous population of axon terminals with Gray type-2 (symmetric) synaptic contacts corresponding to F-axon terminals; and (2) small-medium sized myelinated axons scattered individually or in small groups within the neuropil which may be their parent axons. These axons and terminals may originate from the ipsilateral thalamic reticular nucleus; others may arise from the basal forebrain or brainstem. The GABA-immunopositive terminals comprised approximately 16% of all axon terminal profiles in AD and 12% in AV, a significant difference. However, because the immunoreactive axon terminals in AD were significantly larger than those in AV (1.09+/-0.47 microm2 vs 0.90+/-0.43 microm2) and would therefore be encountered more frequently, it is not possible to conclude that the GABAergic innervation of AD is heavier than that of AV. The GABA-positive terminals established synaptic contacts with cell bodies and dendrites of all sizes (some of which were HRP-labelled) with the following frequency distribution (AD/AV, no significant difference): somata 5%/7%; large dendrites (> or = 1.5 microm) 14%/9%; medium dendrites (1.00-1.49 microm) 35%/45% and small dendrites (< 1 microm) 46%/40%. Despite evidence from previous studies, we found no evidence in this study for the presence of GABAergic interneurons or for GABA-containing projection neurons in AD or AV.

Localization of amino acids, neuropeptides and cholinergic neurotransmitter markers in identified projections from the mesencephalic tegmentum to the mammillary nuclei of the rat.

Retrograde labelling has been combined with immunohistochemistry to localize neurons containing GABA, glutamate, choline acetyltransferase, leu-enkephalin, neurotensin and substance P-like immunoreactivity in the projection pathways from the midbrain tegmental nuclei to the mammillary nuclei in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the medial mammillary nucleus resulted in retrogradely labelled neurons in the ventral tegmental nucleus of Gudden, whereas injections into the lateral mammillary nucleus resulted in large numbers of retrogradely labelled neurons in the ipsilateral dorsal tegmental nucleus of Gudden and in the laterodorsal tegmental nucleus. In the ventral tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also immunolabelled for GABA and approximately ten to 18 WGA-HRP-labelled neurons per section were immunoreactive for leu-enkephalin. In addition, small numbers of WGA-HRP-labelled neurons in the principal subnucleus of the ventral tegmental nucleus were immunoreactive for Glu whereas small numbers of retrogradely labelled neurons in the compact subnucleus of the central superior nucleus displayed neurotensin-like immunoreactivity. In the ventral subnucleus of the dorsal tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also GABA-immunoreactive and approximately ten to 14 WGA-HRP labelled neurons per section were immunoreactive for leu-enkephalin. The ventral subnucleus of the dorsal tegmental nucleus also contained small numbers of retrogradely labelled neurons that displayed either glutamate or substance P-like immunoreactivity. In addition, moderate to small numbers of WGA-HRP-labelled neurons (five to 20 per section) in the laterodorsal tegmental nucleus were immunoreactive for choline acetyltransferase. These results are compatible with the possibility that tegmentomammillary projection neurons use several different neurochemicals as neurotransmitter(s) and/or neuromodulator(s).

Glutamate/aspartate and leu-enkephalin immunoreactivity in mammillothalamic projection neurons of the rat.

We have used retrograde transport and immunohistochemistry to study glutamate, aspartate, and enkephalin-like immunoreactive pathways from the mammillary nuclei to the anterior nuclei of the thalamus. Injections of wheat germ agglutinin conjugated to horseradish peroxidase into the anterodorsal thalamic nucleus resulted in retrogradely labelled cell bodies in the lateral mammillary nucleus, bilaterally, whereas injections into the anteroventral thalamic nucleus resulted in retrogradely labelled neurons in the ipsilateral medial mammillary nucleus. In three parallel series of sections immunoreacted for glutamate, aspartate, and enkephalin, respectively, 50-60% of the retrogradely labelled cell bodies were also immunolabelled for glutamate, 50-60% for aspartate, and 40-50% for enkephalin. The enkephalin-immunoreactive neurons may coincide with or constitute a separate population from the glutamate/aspartate-containing neurons. These results are compatible with the possibility that mammillothalamic projection neurons may use glutamate and/or aspartate and enkephalin as neurotransmitters.

Evidence for collateral projections to the retrosplenial granular cortex and thalamic reticular nucleus from glutamate and/or aspartate-containing neurons of the anterior thalamic nuclei in the rat.

Small, stereotaxically guided injections of true blue (TB) were made into the retrosplenial granular cortex (RSg) and of diamidino yellow (DY) into the dorsal portion of the rostral pole of the thalamic reticular nucleus (TRN) in 16 adult rats to determine whether axons projecting from the anterior thalamic nuclear complex (ATN) to the TRN are branches of axons also projecting to the RSg. Following injections of the fluorescent dyes, serial coronal sections of the brain revealed single retrogradely labelled, and large numbers of double retrogradely labelled neuronal cell bodies in the ipsilateral anteroventral and anterodorsal nuclei and smaller numbers in the anteromedial nucleus of the ATN complex. In a second series of six adult rats with similar double injections of TB and DY, two sections in three were immunoreacted, one with antiserum against glutamate and one with antiserum against aspartate, using indirect immunofluorescence with rhodamine to detect reactive cells. The great majority of both single and double retrogradely labelled cell bodies were also immunoreactive for aspartate or glutamate. In addition, a moderate to small number of non-immunolabelled neurons projecting to the TRN and/or to the RSg were also found in all three nuclei of the ATN complex. These results are compatible with the possibility that large numbers of neurons in the ATN send axonal branches to both the RSg and the TRN, and that many such neurons use glutamate and/or aspartate as transmitters. The findings also suggest that the projections from the ATN might be heterogeneous with respect to transmitter phenotype.

Glutamate and aspartate immunoreactivity in the reciprocal projections between the anterior thalamic nuclei and the retrosplenial granular cortex in the rat.

We have used retrograde and anterograde labelling with wheat germ agglutinin-horseradish peroxidase and immunohistochemistry with antibodies against glutamate and aspartate to examine the reciprocal connections between the anterior thalamic nuclei and the retrosplenial granular cortex in the rat, and to characterize those projection neurones that contain glutamate and/or aspartate. Injections into superficial layers of the retrosplenial granular cortex resulted in retrogradely labelled cell bodies in the anterodorsal, anteroventral, and to a lesser extent the anteromedial subnuclei. Approximately 70% of these cell bodies were also immunolabelled for glutamate or aspartate. Injections confined to deep layers (V-VI) resulted in the presence, in anterior thalamic neuropil, of anterogradely labelled fibre and terminal-like structures, many of which appeared to be immunolabelled for glutamate or aspartate. Injections into the anterior thalamic nuclei resulted in retrogradely labelled pyramidal cells in layers V-VI of the retrosplenial granular cortex. Most (90-95%) of these cells were immunolabelled for glutamate or aspartate. Thus, approximately 70% of thalamocortical and 90-95% of corticothalamic projection neurones in these circuits may use glutamate and/or aspartate as neurotransmitters.