Locations of pontomesencephalic cholinergic projection neurons from the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei to midbrain dopaminergic nuclei were mapped. Stereotaxic microinjections of Fluoro-Gold- or rhodamine-labeled microspheres were made either to substantia nigra (SN) or ventral tegmental area (VTA) in rat. Choline acetyltransferase was visualized immunohistochemically. Labeled cells were digitally mapped at multiple levels of the nuclei using an interactive computer/microscope system. SN-projecting neurons were distributed predominantly ipsilaterally in distinct regions of the PPTg: either at its rostral pole or caudally in an area ventromedial to the superior cerebellar peduncle. Few SN-projecting neurons were found in LDTg. VTA-projecting neurons were distributed bilaterally throughout the cholinergic group, primarily in the densest regions of the LDTg and caudal PPTg. Neurons were not strictly segregated into these patterns. Scattered cells belonging to either projection could be found throughout the cholinergic group on either side. Hierarchical log-linear analysis showed these differences in topographic distribution to be statistically significant. Subtraction of cell density images demonstrated well delineated regions of the cholinergic group where the projections were predominately either to SN or VTA. These data indicate a high degree of internal organization within the pontomesencephalic cholinergic group based on the location of efferent projections to SN or VTA. These findings support the concept that this cholinergic group is functionally organized in a manner which selectively innervates motor (SN) and limbic (VTA) dopaminergic nuclei.

This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 micron in diameter, 1.6 microns 2 in surface, and 0.12 micron 3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II-III, 11% in IV, 21% in V, 14% in VI), for a I-VI average of 14%. These synaptic junctions were usually single, symmetrical (> 99%), and occupied a small portion of the surface of varicosities (< 3%). A majority were found on dendritic branches (76%), some on spines (24%), and none on cell bodies. On the whole, the ACh junctional varicosities were significantly larger than their nonjunctional counterparts, and both synaptic and nonsynaptic varicosities could be observed on the same fiber. A subsample of randomized single thin sections from these whole varicosities yielded similar values for size and synaptic frequency as the result of a stereological extrapolation. Also analyzed in single sections, the microenvironment of the ChAT-immunostained varicosities appeared markedly different from that of unlabeled varicosity profiles randomly selected from their vicinity, mainly due to a lower incidence of synaptically targeted dendritic spines. Thus, the normal ACh innervation of adult rat parietal cortex is predominantly nonjunctional (> 85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity.

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A method for determining the length of acetylcholine (ACh) axons and number of ACh axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase (ChAT) was used to estimate the areal and laminar densities of this innervation in the frontal (motor), parietal (somatosensory), and occipital (visual) cortex of adult rat. The number of ACh varicosities per length of axon (4 per 10 microm) appeared constant in the different layers and areas. The mean density of ACh axons was the highest in the frontal cortex (13.0 m/mm(3) vs. 9.9 and 11.0 m/mm(3) in the somatosensory and visual cortex, respectively), as was the mean density of ACh varicosities (5.4 x 10(6)/mm(3) vs. 3.8 and 4.6 x 10(6)/mm(3)). In all three areas, layer I displayed the highest laminar densities of ACh axons and varicosities (e.g., 13.5 m/mm(3) and 5.4 x 10(6)/mm(3) in frontal cortex). The lowest were those of layer IV in the parietal cortex (7.3 m/mm(3) and 2.9 x 10(6)/mm(3)). The lengths of ACh axons under a 1 mm(2) surface of cortex were 26.7, 19.7, and 15.3 m in the frontal, parietal, and occipital areas, respectively, for corresponding numbers of 11.1, 7.7, and 6.4 x 10(6) ACh varicosities. In the parietal cortex, this meant a total of 1.2 x 10(6) synaptic ACh varicosities under a 1 mm(2) surface, 48% of which in layer V alone, according to previous electron microscopic estimates of synaptic incidence. In keeping with the notion that the synaptic component of ACh transmission in cerebral cortex is preponderant in layer V, these quantitative data suggest a role for this innervation in the processing of cortical output as well as input. Extrapolation of particular features of this system in terms of total axon length and number of varicosities in whole cortex, length of axons and number of varicosities per cortically projecting neuron, and concentration of ACh per axon varicosity, should also help in arriving at a better definition of its roles and functional properties in cerebral cortex.

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