At the light microscopic level, enkephalin labeling was seen in cell bodies and fibers throughout the striatum (). Cellular and synaptic localization of enkephalin immunoreactivity was quantified in the caudate nucleus and the putamen of normal human tissue using stereology. Simple profile counts were used in addition to stereology for the localization of enkephalin in axon terminals. Montages covering a large field of neuropil and taken from serial sections were used for stereology (). They were viewed at a higher magnification to quantify profiles within the neuropil (). Results of the quantitative analysis of labeled profiles are summarized as proportions in . To find proportions for the two regions, n values were summed across individuals. Due to the nature of human tissue however, there was some interindividual variability. The variability of profile densities of only the salient points are shown in .
Light micrograph taken at 40X of enkephalin-labeled neurons in human striatum. Medium-sized neurons are labeled (large arrows) as well as processes (small arrows). Scale bar, 50 μm.
Figure 2 Representative subsections of neuropil montages used for stereology, constructed from 8 individual micrographs. (A) and (B) are adjacent serial sections in a ribbon of 16 sections from the caudate nucleus of case #M3. Distinguishable structures (asterisks) (more ...)
Figure 3 Higher magnification of nearby serial sections of area in rectangles in . (A) Two labeled spines (sp) emerging from the same dendrite (den) receive asymmetric synapses (closed black arrows) from unlabeled axon terminals (at). Synapses are also present (more ...)
Quantitative data and proportions.
The majority of the enkephalin-positive striatal neurons observed in this study were medium-sized and characterized by a large nonindented nucleus and a moderate amount of cytoplasm (). Some of the neurons (3/85) showed an indentation or notch in the nucleus, but were otherwise similar. Organelles present in labeled cell bodies include mitochondria, rough endoplasmic reticulum, polyribosomes, lysosomes, and occasionally (11% of cells) the Golgi apparatus. An average of 8 lysosomal bodies was present in each soma. Additionally, a perisomatic glial cell had adjacent membranes with 39% of the labeled neurons. Enkephalin-like immunoreactivity was diffuse throughout the cytoplasm of cell bodies and dendrites, localized primarily on polyribosomes and was also present on rough endoplasmic reticulum. Cell nuclei of neurons were also positive for enkephalin, with markedly lighter staining than that of the surrounding perikarya, with immunoreactivity localized in small clusters on euchromatin.
Figure 4 Medium spiny neuron and spine morphology. (A) A medium spiny neuron contains small clusters of enkephalin immunoreactivity throughout the perikarya, dendrite, and nucleus. (B) A labeled spine (sp) with a large head and thin neck emerges from a labeled (more ...)
Labeled postsynaptic profiles were abundant throughout the caudate nucleus and putamen and received asymmetric and symmetric synapses. Labeled dendrite shafts and dendritic spines of various shapes were postsynaptic to terminals. The morphology of enkephalin-labeled spines included long necks with elongated heads (), thin necks with large heads (), wide necks with large heads (), thin necks with small heads (), mushroom shaped (), and spines with spinules (). Labeled spines receiving synapses had similar densities in both regions () however, a smaller density of total spines (labeled and unlabeled) receiving synapses in the putamen results in a significant difference in the proportions of labeled spines between the two regions; 32% of spines in the caudate nucleus and 44% of spines in putamen contained enkephalin immunoreactivity (p<0.001, n=1680).
Figure 5 Density (per 100 μm3) of synapses formed on spines (total spines; labeled and unlabeled), synapses formed on labeled spines (+Sp), perforated (perf) synapses, and perforated synapses on labeled spines in the caudate and putamen. Densities were (more ...)
Synapses with perforated postsynaptic densities were formed with spines (91%) and occasionally with dendrites (9%; ). Using stereology, the caudate nucleus and putamen had similar densities of perforated synapses (). Perforated synapses on labeled spines had similar densities in the two regions (), however the proportions were somewhat uneven. In the putamen, perforated synapses targeted a slightly higher proportion of labeled spines (59%) than unlabeled spines (41%), while in the caudate nucleus, spines receiving perforated synapses were labeled 47% of the time. The difference in the proportions of perforated synapses targeting labeled versus unlabeled spines was significantly different (p=0.02, n=405) between the putamen and the caudate nucleus. All dendrites receiving perforated synapses in the putamen were immuno-positive, but perforated synapses showed no preference for immuno-positive versus immuno-negative dendrites in the caudate nucleus.
Both myelinated and unmyelinated axons revealed enkephalin immunoreactivity (). Axon terminals containing immunoreactivity varied in size, but were usually small, and typically had round, clear synaptic vesicles with reaction product deposited around the vesicles (). Large dense core vesicles not associated with the synapse were also present in some terminals. Using stereology, synapses formed by labeled axon terminals were very infrequent (1.1% and 4.3% of all synapses in the putamen and the caudate nucleus, respectively). The densities of synapses formed by labeled terminals () were not significantly different between the caudate and the putamen (p=0.08, n=5). Many labeled axon terminals observed in multiple planes of section never made contact with a postsynaptic target (), or made contact but failed to fulfill all three synaptic criteria (not shown). Labeled axon terminals forming synapses that met all three synaptic criteria terminated on cell bodies (), dendrites (), and spines (). They typically had symmetric postsynaptic membrane specializations (82% in the caudate and putamen combined), and primarily formed synapses with unlabeled profiles (86% in the caudate and putamen combined). The density of labeled terminals forming symmetric synapses was significantly (p=0.043, n=5) higher in the caudate than in the putamen (). Labeled terminals forming asymmetric synapses were occasionally identified in both regions. They had similar densities in the caudate nucleus and putamen () but asymmetric synapses comprised a significantly (p=0.029, n=49) larger proportion of the synapses formed by labeled terminals in the putamen (42%) than in the caudate nucleus (11%). Using stereology, labeled axon terminals tended to form more synapses with spines rather than dendrites (58% vs. 42%) in putamen, while no difference was observed in the caudate nucleus (49% vs. 51%); these differences were not statistically significant (p=0.74, n=49). Enkephalin-labeled terminals forming asymmetric synapses with spines tended to synapse on the spine head, whereas those forming symmetric synapses with spines tended to synapse on either the spine neck or head. The labeled terminals forming symmetric synapses onto spine heads often synapsed on spines receiving convergent input from an unlabeled terminal forming an asymmetric synapse. Labeled axon terminals forming synapses on cell bodies were often long with multiple contact points (). Similar results were found with simple profile counts ().
Figure 7 Labeled axon terminals making synapses on labeled and unlabeled profiles. (A) Myelinated axons containing enkephalin immunoreactivity (+ma). Note nearby unlabeled myelinated axons (asterisks). (B) A large, lightly labeled axon terminal (+at) makes an (more ...)
Figure 8 Density (per 100 μm3) of all labeled terminals making synapses, labeled terminals forming symmetric synapses (Sym) and labeled terminals forming asymmetric synapses (Asym) in the caudate and putamen. Densities were averaged over cases. Error bars, (more ...)
Figure 6 (A) A long, labeled axon terminal (+at, arrowheads) makes a symmetric synapse (open black arrow) with a labeled cell body. Serial sections of the labeled terminal, shown in C-E, show the terminal forming multiple symmetric synapses with the soma. The (more ...)