RESUMEN
The principal relay nuclei of the thalamus receive their cholinergic innervation from two midbrain cholinergic groups: the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus. The different thalamic nuclei exhibit populations of cholinergic axons which vary in density and morphology when examined at the light microscopic level. However, the ultrastructure of the cholinergic terminals in different thalamic nuclei has not been described. This study was undertaken to confirm that synaptic contacts are formed by cholinergic axons in several principal thalamic relay nuclei, to describe their ultrastructural morphology, and to identify the types of postsynaptic elements contacted by cholinergic synaptic terminals. The thalamic nuclei examined in this study are the dorsal lateral geniculate nucleus, ventroposteromedial nucleus, ventroposterolateral nucleus, and anteroventral nucleus. Our results confirm that cholinergic axons form synaptic terminals in these thalamic nuclei. Cholinergic synaptic terminals contact structures outside the characteristic synaptic glomeruli, are never postsynaptic, and have morphologies and postsynaptic targets which differ among the thalamic nuclei. In the ventroposterior nuclei, cholinergic terminals form asymmetric synaptic contacts onto larger dendrites in the extraglomerular neuropil. In the anteroventral nucleus, cholinergic terminals form both symmetric and asymmetric synaptic contacts onto dendrites and somata. Cholinergic terminals in the anteroventral nucleus are larger than those in other nuclei. In the dorsal lateral geniculate nucleus, cholinergic terminals contact both somata and dendrites in the extraglomerular neuropil, but the synaptic contacts in this nucleus are symmetric in morphology.
Asunto(s)
Colina O-Acetiltransferasa/química , Cuerpos Geniculados/ultraestructura , Sinapsis/ultraestructura , Núcleos Talámicos/ultraestructura , Animales , Fibras Colinérgicas/ultraestructura , Cuerpos Geniculados/enzimología , Masculino , Microscopía Electrónica , Ratas , Ratas Endogámicas , Sinapsis/enzimología , Núcleos Talámicos/enzimologíaRESUMEN
The thalamic reticular nucleus has been shown to receive cholinergic innervation from both the nucleus basalis of Meynert in the forebrain and the pedunculopontine and laterodorsal tegmental nuclei in the brainstem (Steriade et al.: Brain Res. 408:372-376, '87; Levey et al.: Neurosci. Lett. 74:7-13, '87). Relatively dense populations of choline acetyltransferase-(ChAT) immunoreactive axons and terminallike varicosities have been shown to be distributed throughout this nucleus (Levey et al.: J. Comp. Neurol. 257:317-332, '87). In this study, the ultrastructure of ChAT-immunoreactive axons and of their synaptic terminals in the reticular nucleus was examined in the electron microscope. All ChAT-immunoreactive axonal profiles in the reticular nucleus were presynaptic; the postsynaptic elements were exclusively dendritic profiles; and no axo-axonic or axosomatic contacts from labelled axons were observed. Most ChAT-immunoreactive synaptic contacts were made by profiles less than 0.25 micron in minor diameter. Single ChAT-immunoreactive axons made synaptic contact with several dendritic profiles as the axons were followed through serial sections. These results suggest that the cholinergic innervation of the reticular nucleus will modulate the function of reticular neurons by synapsing onto the dendrites of its neurons without direct effect on the corticothalamic and thalamocortical terminals which also innervate the reticular nucleus.
Asunto(s)
Axones/ultraestructura , Colina O-Acetiltransferasa/análisis , Dendritas/ultraestructura , Sinapsis/ultraestructura , Núcleos Talámicos/ultraestructura , Animales , Masculino , Microscopía Electrónica , Ratas , Ratas Endogámicas , Núcleos Talámicos/análisisRESUMEN
Previous studies have suggested that the pedunculopontine tegmental nucleus (PPTn) is reciprocally connected with extrapyramidal motor system nuclei (EPMS) whereas other studies have implicated the PPTn in behavioral state control phenomena such as sleep-wakefulness cycles. Many of these studies define the nonprimate PPTn as an area of mesopontine tegmentum which is labeled from injections of anterograde tracers into the basal ganglia. Recently, we have defined the rat PPTn as a large-celled, cholinergic nucleus. The rat PPTn is cytologically distinct from a group of smaller, noncholinergic neurons that are medially adjacent to the PPTn. This noncholinergic group is further distinguished from the PPTn by its afferent input from the globus pallidus, entopeduncular nucleus, and substantia nigra. We refer to the latter area as the midbrain extrapyramidal area (MEA). Using combined choline acetyltransferase immunohistochemistry of the PPTn and WGA-HRP retrograde tracing from the EPMS, we investigated the efferent connections of the MEA and PPTn to the EPMS in the rat. The noncholinergic MEA, rather than the PPTn, is the major source of tegmental innervation to the globus pallidus, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra, and motor cortex. In contrast, the cholinergic PPTn is the major source of tegmental innervation to the ventrolateral thalamic nucleus. This finding is in contradistinction to thalamic projections from the surrounding reticular formation, which are identified only after WGA-HRP injections into "nonspecific" thalamic nuclei. This body of evidence suggests that the noncholinergic MEA represents an additional component of the EPMS and may correspond to the "mesencephalic locomotor region." The cholinergic PPTn may play a role in more global thalamic functions such as the "reticular activating system" rather than a primary role in motor function.
Asunto(s)
Encéfalo/anatomía & histología , Fibras Colinérgicas/citología , Tegmento Mesencefálico/anatomía & histología , Animales , Encéfalo/citología , Mapeo Encefálico , Peroxidasa de Rábano Silvestre , Masculino , Vías Nerviosas/anatomía & histología , Vías Nerviosas/citología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Tegmento Mesencefálico/citología , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de TrigoRESUMEN
Ascending projections from the pedunculopontine tegmental nucleus (PPT) and the surrounding mesopontine tegmentum to the forebrain in the rat are here examined by using both retrograde and anterograde tracing techniques combined with choline acetyltransferase (ChAT) immunohistochemistry. The anterogradely transported lectin Phaseolus vulgaris-leukoagglutinin (PHA-L) was iontophoretically injected into the PPT in 12 rats. Anterogradely labelled fibers and varicosities were observed in the thalamic nuclei, confirming the findings of our previous retrograde studies (Hallanger et al: J. Comp. Neurol. 262:105-124, '87). In addition, PHA-L-labelled fibers and varicosities suggestive of terminal fields were observed in the anterior, tuberal, and posterior lateral hypothalamic regions, the ventral pallidum in the region of the nucleus basalis of Meynert, the dorsal and intermediate lateral septal nuclei, and in the central and medial nuclei of the amygdala. To determine whether these were cholinergic projections, the retrograde tracer WGA-HRP was injected into terminal fields in the hypothalamus, septum, ventral pallidum, and amygdala. Numerous ChAT-immunoreactive neurons in the PPT and laterodorsal tegmental nucleus (LDT) were retrogradely labelled from the lateral hypothalamus. These cholinergic neurons constituted over 20% of those retrogradely labelled in the dorsolateral mesopontine tegmentum; the balance consisted of noncholinergic neurons of the central tegmental field, retrorubral field, and cuneiform nucleus. Following placement of WGA-HRP into dorsal and intermediate lateral septal regions, the vast majority (greater than 90%) of retrogradely labelled neurons were cholinergic neurons of the PPT and LDT, with few noncholinergic retrogradely labelled neurons in the adjacent tegmentum. In contrast, fewer cholinergic neurons were retrogradely labelled following placement of tracer into the nucleus basalis of Meynert or into the central, medial, and basolateral nuclei of the amygdala, while numerous noncholinergic neurons of the central tegmental field rostral to the PPT and of the retrorubral field adjacent to the PPT were retrogradely labelled in these cases. These anterograde and retrograde studies demonstrate that cholinergic PPT and LDT neurons provide a substantial proportion of mesopontine tegmental afferents to the hypothalamus and lateral septum, while projections to the nucleus basalis and the amygdala are minimal.
Asunto(s)
Encéfalo/anatomía & histología , Mesencéfalo/anatomía & histología , Puente/anatomía & histología , Ratas Endogámicas/anatomía & histología , Vías Aferentes/anatomía & histología , Amígdala del Cerebelo/anatomía & histología , Animales , Transporte Axonal , Encéfalo/enzimología , Colina O-Acetiltransferasa/metabolismo , Globo Pálido/anatomía & histología , Peroxidasa de Rábano Silvestre , Hipotálamo/anatomía & histología , Técnicas para Inmunoenzimas , Masculino , Mesencéfalo/enzimología , Puente/enzimología , Ratas , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de TrigoRESUMEN
This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.
Asunto(s)
Puente/citología , Tegmento Mesencefálico/citología , Animales , Gatos , Colina O-Acetiltransferasa/análisis , Masculino , Vías Nerviosas/citología , Puente/análisis , Tegmento Mesencefálico/análisisRESUMEN
The origins of the cholinergic and other afferents of several thalamic nuclei were investigated in the rat by using the retrograde transport of wheat germ agglutinin conjugated-horseradish peroxidase in combination with the immunohistochemical localization of choline acetyltransferase immunoreactivity. Small injections placed into the reticular, ventral, laterodorsal, lateroposterior, posterior, mediodorsal, geniculate, and intralaminar nuclei resulted in several distinct patterns of retrograde labelling. As expected, the appropriate specific sensory and motor-related subcortical structures were retrogradely labelled after injections into the principal thalamic nuclei. In addition, other basal forebrain and brainstem structures were also labelled, with their distribution dependent on the site of injection. A large percentage of these latter projections was cholinergic. In the brainstem, the cholinergic pedunculopontine tegmental nucleus was retrogradely labelled after all thalamic injections, suggesting that it provides a widespread innervation to the thalamus. Neurons of the cholinergic laterodorsal tegmental nucleus were retrogradely labelled after injections into the anterior, laterodorsal, central medial, and mediodorsal nuclei, suggesting that it provides a projection to limbic components of the thalamus. Significant basal forebrain labelling occurred only with injections into the reticular and mediodorsal nuclei. Only injections into the reticular nucleus resulted in retrograde labelling of the cholinergic neurons in the nucleus basalis of Meynert. The results provide evidence for an organized system of thalamic afferents arising from cholinergic and noncholinergic structures in the brainstem and basal forebrain. The brainstem structures, especially the cholinergic pedunculopontine tegmental nucleus, appear to project directly to principal thalamic nuclei, thereby providing a possible anatomical substrate for mediating the well-known facilitory effects of brainstem stimulation upon thalamocortical transmission.
Asunto(s)
Acetilcolina/análisis , Tronco Encefálico/anatomía & histología , Fibras Colinérgicas/análisis , Lóbulo Frontal/anatomía & histología , Núcleos Talámicos/anatomía & histología , Animales , Colina O-Acetiltransferasa/análisis , Peroxidasa de Rábano Silvestre , Masculino , Vías Nerviosas/anatomía & histología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Sustancia Innominada/anatomía & histología , Tegmento Mesencefálico/anatomía & histología , Núcleos Talámicos/análisis , Núcleos Talámicos/citología , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de TrigoRESUMEN
The distribution of choline acetyltransferase immunoreactivity in the rat thalamus was investigated by using a specific monoclonal antibody and was compared with the pattern of acetylcholinesterase staining. The only choline acetyltransferase-immunoreactive cell bodies in the thalamus were in the medial habenula. A wide range of densities of immunoreactive fibers and varicosities was found. The highest densities of stained varicosities were in the anteroventral, reticular, lateral mediodorsal, and intralaminar nuclei. At the other extreme, the anterodorsal, ventroposteromedial, and paraventricular nuclei were almost devoid of immunoreactive varicosities. A light density of fibers was observed in several medial nuclei, including parataenial, reuniens, and gelatinosus. Most other nuclei contained moderately dense regions of varicose fibers that were often heterogeneous or patchy. The pattern of choline acetyltransferase immunoreactivity in the thalamus was in general similar to that of acetylcholinesterase. A marked discrepancy, however, was found in the anterodorsal nucleus, which was intensely stained for acetylcholinesterase but contained no apparent choline acetyltransferase immunoreactivity. Numerous physiologic studies have demonstrated striking effects of acetylcholine on thalamic activity. The present study provides a description of choline acetyltransferase-immunoreactive structures in the thalamic nuclei, providing a first step toward elucidating the anatomical basis for the physiologic and functional importance of cholinergic transmission in the thalamus.
Asunto(s)
Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Tálamo/enzimología , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Monoclonales , Histocitoquímica , Técnicas Inmunológicas , Masculino , Ratas , Ratas Endogámicas , Núcleos Talámicos/citología , Núcleos Talámicos/enzimología , Tálamo/citologíaRESUMEN
The cholinergic neurons of the nucleus basalis of Meynert have been shown to provide the major cholinergic innervation of the cerebral cortex through which cholinergic transmission may modulate cortical activity. This study describes a projection from the cholinergic and non-cholinergic neurons of the nucleus basalis to the reticular nucleus of the thalamus, and a projection from the brainstem cholinergic neurons to the reticular nucleus as well as to other thalamic nuclei. The projection from the nucleus basalis to the reticular nucleus, which itself is synaptically interconnected with other thalamic nuclei, may provide an additional pathway for the modulation of cortical activity by the cholinergic basal forebrain and brainstem groups.
Asunto(s)
Ganglios Basales/anatomía & histología , Tronco Encefálico/anatomía & histología , Fibras Colinérgicas/anatomía & histología , Sustancia Innominada/anatomía & histología , Telencéfalo/anatomía & histología , Núcleos Talámicos/anatomía & histología , Animales , Histocitoquímica , Peroxidasa de Rábano Silvestre , Vías Nerviosas/anatomía & histología , Ratas , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de TrigoRESUMEN
We have previously demonstrated that neurons of the rat cerebral cortex which stain positively for acetylcholinesterase are not likely to be cholinergic since they do not colocalize with choline acetyltransferase immunoreactivity [Levey, Rye, Wainer, Mufson and Mesulam (1984) Neuroscience 9, 9-22]. These noncholinergic acetylcholinesterase-positive cells were similar in morphology to cortical neurons which localize gamma-aminobutyric acid or glutamate decarboxylase immunoreactivity. In order to investigate the possibility that the two substances may be colocalized to the same cortical neurons, gamma-aminobutyric acid immunohistochemistry and acetylcholinesterase histochemistry were combined in single sections of rat cerebral cortex. We found that 18% of gamma-aminobutyric acid-immunoreactive cortical neurons are also acetylcholinesterase-positive, and about 36% of acetylcholinesterase-positive cells are gamma-aminobutyric acid-immunoreactive. Neurons which colocalized both substances were multipolar and bipolar neurons in cortical laminae II-VI and were observed in every cortical area examined. The possibility that gamma-aminobutyric acid-immunoreactive/acetylcholinesterase-positive cortical neurons may be postsynaptic targets of cholinergic afferents to the cerebral cortex is discussed.
Asunto(s)
Acetilcolinesterasa/análisis , Corteza Cerebral/análisis , Ácido gamma-Aminobutírico/análisis , Animales , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Histocitoquímica , Técnicas Inmunológicas , Masculino , Ratas , Ratas EndogámicasRESUMEN
Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.