Two types of degenerating axon terminals in the basilar pontine nuclei of the opossum following cerebral cortical lesions

Subsequent to large cerebral cortical lesions in adult opossums, small boutons (less than 2 μm) contacting small dendritic profiles were observed to undergo the dark, electron dense type of degeneration in most cases. When lesions were restricted to post-orbital (sensorimotor) regions, the majority of degenerating boutons were again small and dark. However, if the lesion was restricted to occipital (visual) regions, most degenerating boutons were somewhat larger (1–3 μm), contacted some intermediate and proximal dendrites and underwent an initial filamentous reaction before becoming electron dense. Thus, it was postulated that at least two different systems of corticofugal axons reach the pontine nuclei, one (small dark boutons) perhaps arising as collaterals or corticospinal axons, the other (filamentous boutons) representing a more direct corticobulbar or corticopontine system.     

Vesicular glutamate transporter-1 colocalizes with endogenous opioid peptides in axon terminals of the rat locus coeruleus

We have previously shown that a subset of axon terminals in the locus coeruleus (LC) containing methionine(5)-enkephalin (ENK) forms type I (asymmetric-type) synaptic specializations that are characteristic of excitatory-type transmitters. In addition, we previously provided ultrastructural evidence showing that ENK is colocalized with glutamate using a combination of pre- and postembedding immunohistochemistry. To examine cellular substrates for interactions between glutamate and other endogenous opioid peptides in the LC, we examined the localization of the vesicular glutamate transporter 1 (VGLUT1), a transporter protein involved in the accumulation of the transmitter glutamate into synaptic vesicles, with either ENK or preprodynorphin (ppDYN). Dual-immunofluorescence and electron microscopy showed prominent coexistence of VGLUT1 and ENK in varicose processes of the LC, confirming our previous report using postembedding immunolabeling for glutamate. Likewise, VGLUT1 and ppDYN were identified in common varicose processes in the LC using confocal fluorescence microscopy. Immunoelectron microscopy using gold-silver labeling for VGLUT1 and peroxidase labeling for ppDYN established that this endogenous opioid peptide also colocalizes with glutamate transporters. The majority of these formed asymmetric-type synapses. Taken together, these results demonstrate that excitatory LC afferents are enriched with endogenous opioid peptides and are positioned to modulate LC neuronal activity dually.     

Morphology of Purkinje cell axon terminals in intracerebellar nuclei following inferior olive lesion

We have examined the ultrastructural changes of axons and synaptic boutons in the intracerebellar nuclei of the rat at 3 days to one year after inferior olive lesion performed by means of electrocoagulation or 3-acetylpyridine injection. A large number of preterminal segments and axons terminals undergoes remarkable ultrastructural changes after total or subtotal olivary lesion. Large membrane bound vacuoles and clusters of small synaptic vesicles characterize a good number of these terminals at 3 days up to one month after the lesion. Tightly packed tubules and cisternae of smooth endoplasmic reticulum appear during the first week in an increasing number of axon terminals. Boutons with large whorled bodies formed by smooth membranes increase in number during the second half of the first month and further increase in density until the sixth month. They are still present in large amounts at one year. Immunoreactivity for 3',5'-guanosine-phosphate-dependent protein kinase, which is specific for Purkinje neurons, can be detected in the axons and synaptic terminals displaying the ultrastructural changes described above. These results are discussed in relation to a possible trophic action of the climbing fibers on the Purkinje cells. We suggest that, at least in part, these alterations may be the consequence of the intense Purkinje cell hyperactivity which is present for up to one month from inferior olive lesion.     

Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract

The nucleus of the solitary tract is the site of termination of primary afferent fibers running in the facial, glossopharyngeal and vagus nerves. The present study was performed to map the distribution of glutamatergic axons terminals in the rat nucleus of the solitary tract using immunodetection of vesicular glutamate transporter 1 and vesicular glutamate transporter 2. The two vesicular glutamate transporters were differentially distributed among nucleus of the solitary tract subdivisions. Vesicular glutamate transporter 1 immunoreactivity was mostly found in the lateral part of the nucleus (ventrolateral, interstitial and intermediate subdivisions) whereas vesicular glutamate transporter 2 labeling was distributed throughout the nucleus of the solitary tract. Electron microscope examination indicated that vesicular glutamate transporter immunoreactivity was localized in axon terminals filled with round synaptic vesicles. After injection of cholera toxin B subunit in sensory ganglia, anterograde labeling was found in vesicular glutamate transporter 1, as well as vesicular glutamate transporter 2-immunoreactive boutons. Double immunolabeling experiments allowed distinctions between terminals expressing either vesicular glutamate transporter 1 or vesicular glutamate transporter 2 or both vesicular glutamate transporter 1 and vesicular glutamate transporter 2 immunoreactivities. The latter population, expressing both transporters immunolabeling, completely disappeared after deafferentation induced by removal of sensory ganglia. This study indicates that vesicular glutamate transporter content identifies three different subpopulations of glutamatergic boutons in the nucleus of the solitary tract and provides definitive evidence that primary afferent neurons contribute glutamatergic terminals to the nucleus of the solitary tract.     

Pericapillary and distant axon terminals in the nuclei of the cat amygdala: a morphometric study

According to some ultrastructural studies, the pericapillary axon terminals in the central nervous system (CNS) are functionally connected with the capillary vessel wall. Thus, it may be expected that the population of pericapillary axon terminals will be morphologically distinct from the terminals at a further distance from the capillary walls. To test this hypothesis, morphometrical analysis of 3,048 axon terminals was performed, comparing terminals situated in the close vicinity of the capillary vessel with those at a distance from the vessels in the lateral, basal, medial, central and cortical nuclei of the amygdaloid body of eight cats. The cross-sectional area and circumference of each identified axon terminal profile were measured, and the shape of synaptic vesicles and the presence of synaptic contacts and granular vesicles were recorded. The statistical evaluation of results was performed by means of the Newman-Keuls' test, Wilcoxon's test, Fisher's contingency table test and the test for two coefficients of structure. The morphometric examination revealed two ultrastructurally distinct groups of axon terminals, pericapillary and distant terminals, in all the nuclei of the amygdaloid body. The differentiating features were the shape of the synaptic vesicles, the number of synaptic contacts, and the size of the axon terminals. These results further support the hypothesis of a functional connection between axon terminals and the capillary vessel wall in the CNS.     

Mu-opioid receptor is present in dendritic targets of Endomorphin-2 axon terminals in the nuclei of the solitary tract

Endomorphins represent a group of endogenous opioid peptides with high affinity for the mu-opioid receptor. In the brainstem, Endomorphin-2 is found in trigeminal dorsal horn and the nuclei of the solitary tract, suggesting its presence in both nociceptive and visceral primary afferents. If Endomorphin-2 were an endogenous ligand for the mu-opioid receptor, we would expect to find the receptor at cellular sites in close association with the peptide. We used dual-labeling immunocytochemistry combined with electron microscopy to examine interactions between Endomorphin-2-immunoreactive and mu-opioid receptor-immunoreactive profiles within the nuclei of the solitary tract in the rat. Endomorphin-2-immunoreactivity was found primarily in unmyelinated axons and axon terminals in nuclei of the solitary tract and the majority of these terminals contained dense core vesicles. Endomorphin-2-immunoreactive axon terminals often formed asymmetric synapses with dendritic spines lacking mu-opioid receptor-immunoreactivity, but mu-opioid receptor-immunoreactivity was found in many of the larger dendritic targets of Endomorphin-2-immunoreactive terminals. Thus, mu-opioid receptor-immunoreactivity was found in the postsynaptic targets of Endomorphin-2-immunoreactive axon terminals, consistent with the hypothesis that Endomorphin-2 is an endogenous ligand for this receptor within the nuclei of the solitary tract. A small number of Endomorphin-2-immunoreactive somata, dendrites, and axon terminals also contained mu-opioid receptor-immunoreactivity. Cells that contain both the opioid peptide and its receptor may be a substrate for potential autoregulation of nuclei of the solitary tract neurons by opioid ligands. Finally, using tract tracing and confocal microscopy, we found Endomorphin-2-immunoreactivity in a subset of vagal afferents. Together these findings support the hypothesis that Endomorphin-2 is a ligand for the mu-opioid receptor within nuclei of the solitary tract and that the peptide is at least partially derived from primary visceral afferents.     

Profiles enriched in GABA-like immunoreactivity participate in serial synapses in the pontine nuclei of baboon (Papio anubis)

Summary Using a postembedding immunogold procedure with an antiserum against glutaraldehyde-fixed GABA, we demonstrate GABA-like immunoreactivity in two classes of synaptic profiles in the pontine nuclei of baboon. One is an axon terminal in symmetrical synaptic contact with small or medium-sized GABA-immunonegative dendrites, the other is a pale, vesicle-containing profile resembling a dendrite or dendritic process which participates in serial synaptic arrangements. These synaptic arrangements, or triads, consist of a GABA-like immunoreactive, pale vesicle-containing profile being postsynaptic to a GABA-immunonegative axon terminal, and presynaptic to a small or medium-sized GABA-immunonegative dendrite. In at least some of these triads, the GABA-immunonegative axon terminals also contact the GABA-immunonegative dendrite directly.     

Effects of taipoxin on the ultrastructure of cholinergic axon terminals in the mouse adrenal medulla.

Intravenous injection of the neurotoxin, taipoxin, to adult mice caused conspicuous alterations in the ultrastructure of axon terminals upon chromaffin cells in the adrenal gland. The alterations were in several respects similar to those previously described at the neuromuscular junction. The electron density of the axoplasm was greatly increased and the mitochondria swollen. In many instances the number of synaptic vesicles was reduced. The alterations were more marked the longer the period of toxin action.It is suggested that the toxin molecule, which has phospholipase A₂ activity, enters the axoplasm before exerting a hydrolytic and thereby neurotoxic action on cholinergic nerve terminals.     

Organization of the pontine nuclei.

The pontine nuclei provide the cerebellar hemispheres with the majority of their mossy fiber afferents, and receive their main input from the cerebral cortex. Even though the vast majority of pontine neurons send their axons to the cerebellar cortex, and are contacted monosynaptically by (glutamatergic) corticopontine fibers, the information-processing taking place is not well understood. In addition to typical projection neurons, the pontine nuclei contain putative GABA-ergic interneurons and complex synaptic arrangements. The corticopontine projection is characterized by a precise but highly divergent terminal pattern. Large and functionally diverse parts of the cerebral cortex contribute; in the monkey the most notable exception is the almost total lack of projections from large parts of the prefrontal and temporal cortices. Within corticopontine projections from visual and somatosensory areas there is a de-emphasis of central vision and distal parts of the extremities as compared with other connections of these sensory areas. Subcorticopontine projections provide only a few percent of the total input to the pontine nuclei. Certain cell groups, such as the reticular formation, project in a diffuse manner whereas other nuclei, such as the mammillary nucleus, project to restricted pontine regions only, partially converging with functionally related corticopontine connections. The pontocerebellar projection is characterized by a highly convergent pattern, even though there is also marked divergence. Neurons projecting to a single cerebellar folium appear to be confined to a lamella-shaped volume in the pontine nuclei. The organization of the pontine nuclei suggests that they ensure that information from various, functionally diverse, parts of the cerebral cortex and subcortical nuclei are brought together and integrated in the cerebellar cortex.     

Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic “hot spots.” Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. Electron microscopy (EM) analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles ∼30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway.     

An autoradiographic identification of Purkinje axon terminals in the cat

Summary The Purkinje axon boutons terminating in nuclei fastigii and interpositus in the cat have been identified after injection of³H leucine into the cerebellar cortex overlying the nuclei. The animals survived from 4–48 h after injection of the isotope. Semithin and ultrathin sections were coated and exposed for 3 and 14 weeks, respectively. The electron micrographs showed labelling over myelinated axons down to a diameter of 0.8 m, and over boutons.Fifty labelled boutons were used for identification of the shape of their synaptic vesicles. The statistical analysis including the test for skewness showed that 39 boutons (78%) fall in one group. Most of the synaptic vesicles in this group are elliptical (ration from 11.1 – 11.7). Slightly ovoid vesicles (ration up to 11.3) are frequent, but flattened vesicles (ration above 11.7) are relatively few in this group of boutons. 8% of the boutons have a rather homogeneous vesicle population (prevalence of round vesicles, ratio 11).Synaptic specializations of Gray's type II or of an intermediate type were found in the boutons belonging to the first group (78% of the boutons). Specializations of Gray's type I were found in the other bouton groups (8% of the boutons).     

Electron microscopic identification of reticulo-subthalamic axon terminals in the cat

Synaptic boutons emanating from axons of nucleus tegmenti pedunculopontinus origin were identified by electron microscopy in the neuropil of the subthalamic nucleus. Such boutons measure 1.5-3 microns, contain round synaptic vesicles and make asymmetrical axodendritic and axosomatic synaptic contacts with large subthalamic neurons. Very few contacts with vesicle-containing dendrites, and no contacts with the perikarya of the small neurons were observed. The present findings, in keeping with the relevant light microscopic and electrophysiologic data, furnish evidence for a substantial bilateral tegmenti pedunculopontinofugal projection that excites monosynaptically the relay subthalamic neurons.     

Visual cells in the pontine nuclei of the cat.

Two hundred and thirty-two visually activated neurones were recorded in a small area of the rostral pontine nuclei of cats. The location of visually activated neurones was coextensive with the input from visual areas of cat's cortex as determined by degeneration studies. 2. Pontine visual cells could only be driven by visual stimuli. Cells responsive to somatosensory or auditory stimuli were also found in different regions in rostral pontine nuclei. They too responded to only one modality. 3. 96% of the cells were directionally selective. 4. Pontine visual cells were responsive to a wide range of stimulus speeds. Some cells responded to targets moving as fast as 1000 degrees/sec without losing directional selectivity. No pontine visual cells gave a clearly sustained response to a stationary stimulus. 5. Exact stimulus configurations were not critical. Large fields containing many spots were the most effective stimuli for 50% of the cells. Inhibition of responses depending upon stimulus dimensions, direction of movement, or location in the visual field was found for many cells. 6. Receptive field dimensions were large, ranging in size from 3 degrees X 4 degrees to more than an entire hemifield. 7. 94% of the cells had receptive fields which were centred in the contralateral hemifield. 8. 98% of the cells could be driven from both eyes. 9. The properties of the pontine visual cells suggest a corticopontocerebellar pathway sensitive to a wide range of speeds and directions of movement, but not sensitive to precise form.     

Urotensin II receptor and acetylcholine release from mouse cervical spinal cord nerve terminals

Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are expressed in subsets of mammal spinal motoneurons. In fact, a role for the peptide in the regulation of motoneuron function at neuromuscular junction has been suggested, while roles for urotensin II at central synapses in spinal cord have never been addressed. We found that urotensin II receptors were closely associated with cholinergic terminals apposed to a subset of motoneuron and non-motoneuron cell bodies in the ventral horn of the adult mouse cervical spinal cord; urotensin II receptor was also expressed on non-cholinergic nerve terminals. In particular, urotensin II receptor appeared associated with both large cholinergic C-boutons and standard cholinergic terminals contacting some motoneuron perikarya. Cholinergic nerve terminals from mouse cervical spinal cord were equipped with functional presynaptic urotensin II receptors linked to excitation of acetylcholine release. In fact, functional experiments conducted on cervical spinal synaptosomes demonstrated a urotensin II evoked calcium-dependent increase in [³H]acetylcholine release pharmacologically verified as consistent with activation of urotensin II receptors. In spinal cord these actions would facilitate cholinergic transmission. These data indicate that, in addition to its role at the neuromuscular junction, urotensin II may control motor function through the modulation of motoneuron activity within the spinal cord.     

A regenerating release of acetylcholine from mouse motor nerve terminals treated with anticholinesterase agents

It was found by intracellular recording with glass microelectrodes that train stimulation (50-200 Hz) of the phrenic nerve of intact or cut mouse diaphragm induced an accumulative depolarization of the endplate and triggered after a few pulses an 'all-or-none' regenerative depolarization lasting for 300-900 ms when acetylcholinesterase was inhibited by neostigmine or diisopropylfluorophosphate. This depolarization was associated with a noise of the membrane potential and a failure of the end plate potential. Low Ca2+ prolonged whereas high Ca2+ shortened the duration of regenerative depolarization which needed no further stimulation once triggered. d-Tubocurarine abolished the depolarization while restoring the end plate potential. A regenerative release of acetylcholine due to an activation of presynaptic cholinoceptors is speculated.     

Intramembrane structure of the sensory axon terminals in bullfrog muscle spindles

Much physiologic and morphologic research has been done into the sensory mechanism of the frog muscle spindle. However, no freeze-fracture study has described in detail the shape and intramembrane structure of the nonmyelinated sensory axon terminals of the frog muscle spindle. In this study, muscle spindles were isolated from the red part of bullfrog semitendinous muscles. Chemically fixed spindles were subjected to freeze fracturing. The sensory axon endings were reconstructed, and the size and density of intramembrane particles (IMPs) were measured along the sensory nerve endings. The axon terminals had four distinctive parts: parent trunks (>0.5 μm in diameter), primary branches (0.15–0.5 μm), terminal branches (<0.1 μm), and varicosities (0.02–0.5 μm). IMPs ranged from 5 nm to 21 nm in diameter and were present in the intramembrane space of the plasma membrane all throughout the nonmyelinated sensory nerve endings. Mean IMP sizes in the protoplasmic face (PF) and the external face (EF), respectively, were 8.1 nm and 8.4 nm in the parent trunks, 8.8 nm and 8.8 nm in the primary branches, 9.4 nm and 9.0 nm in the varicosities, and 8.7 nm and 8.7 nm in the terminal branches. Mean IMP size in the PF was smallest in the parent trunk and largest in the varicosity. Mean IMP densities (numbers of IMPs per μm²) in the PF and the EF, respectively, were 2,500 and 700 in the parent trunks, 2,200 and 500 in the primary branches, 1,700 and 400 in the varicosities, and 1,000 and 300 in the terminal branches. Density decreased with the tapering of the axon terminal, with IMPs distributed evenly in the PF and the EF. The characteristic intramembrane structure of sensory nerve endings is discussed. Anat. Rec. 252:340–354, 1998. © 1998 Wiley-Liss, Inc.     

Electron microscopic identification of superior colliculo-pontine axon terminals

Synaptic boutons emanating from axons of superior colliculus origin were identified by electron microscopy in the neuropil of the basilar pontine nuclei. Such boutons were relatively small (0.6-2.0 microns) and exhibited electron-dense degeneration within a 1-2 day period following electrolytic lesions which involved much of the superior colliculus. Degenerating boutons were observed in synaptic contact with dendritic shafts and spines as well as neuronal somata. The reactive boutons were rapidly engulfed by phagocytic elements and were no longer visible in the neuropil after 6 days of survival.     

Carbachol in the pontine reticular formation of C57BL/6J mouse decreases acetylcholine release in prefrontal cortex

The prefrontal cortex and brainstem modulate autonomic and arousal state control but the neurotransmitter mechanisms underlying communication between prefrontal cortex and brainstem remain poorly understood. This study examined the hypothesis that microdialysis delivery of carbachol to the pontine reticular formation (PRF) of anesthetized C57BL/6J (B6) mouse modulates acetylcholine (ACh) release in the frontal association cortex. Microdialysis delivery of carbachol (8.8 mM) to the PRF caused a significant (P<0.01) decrease (-28%) in ACh release in the frontal association cortex, a significant (P<0.01) decrease (-23%) in respiratory rate, and a significant (P<0.01) increase (223%) in time to righting after anesthesia. Additional in vitro studies used the [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) assay to test the hypothesis that muscarinic cholinergic receptors activate guanine nucleotide binding proteins (G proteins) in the frontal association cortex and basal forebrain. In vitro treatment with carbachol (1 mM) caused a significant (P<0.01) increase in [(35)S]GTPgammaS binding in the frontal association cortex (62%) and basal forebrain nuclei including medial septum (227%), vertical (210%) and horizontal (165%) limbs of the diagonal band of Broca, and substantia innominata (127%). G protein activation by carbachol was concentration-dependent and blocked by atropine, indicating that the carbachol-stimulated [(35)S]GTPgammaS binding was mediated by muscarinic cholinergic receptors. Together, the in vitro and in vivo data show for the first time in B6 mouse that cholinergic neurotransmission in the PRF can significantly alter ACh release in frontal association cortex, arousal from anesthesia, and respiratory rate.     

Multiple axon terminals in reinnervated Pacinian corpuscles of adult rat

Summary The ultrastructure of Pacinian corpuscles localized beneath the crural interosseous membrane was examined two weeks to 18 months after crushing the sciatic nerve in adult rats. The Pacinian inner core and capsule remained preserved during the transient period of denervation. Regenerating axons reached Pacinian corpuscles approximately three weeks after nerve crush. Up to 15 axonal sprouts entered a single corpuscle at the initial stage of reinnervation, but only 1–3 axons increased in size, myelinated and formed axon terminals in the inner core, the excess sprouts being eliminated. Most corpuscles of the crural group were reinnervated by the end of the first month.Three to 19 months after nerve crush, 10% of corpuscles examined were found to be monoaxonal and monoterminal as before the operation; 74% contained multiple terminals; 16% remained denervated. Over half the multiterminal corpuscles were supplied with a single myelinated axon that branched inside the corpuscles; the rest received two or three myelinated axons which formed several terminals. The terminals were distributed at random, usually in the axial region between the lamellae of the inner core. They were cylindrical, with an oval profile; the larger terminals were filled with mitochondria and microtubules at their circumference and contained a core of neurofilaments. Lateral processes of the terminals were filled with vesicles and had membrane specializations as in normal corpuscles. The mean number of terminals in reinnervated corpuscles was 4.07 ± 0.37 (S.E.M.) at three months, and 3.26 ± 0.49 (S.E.M.) 6–18 months after nerve crush. This small decrease was apparently the result of degeneration occasionally observed in some axon terminals at later stages of reinnervation.These experiments thus demonstrate that most rat Pacinian corpuscles become reinnervated with multiple terminals after nerve injury and maintain multiterminal innervation permanently.     

A cerebellar projection onto the pontine nuclei in the albino rat

Summary Following removal of a significant part of the dentate nucleus and most of the interpositus nucleus in the rat cerebellum degenerated cerebellopontine fibres are shown to end in three fairly restricted regions in the contralateral pontine gray: in the paramedian position, in the middle and in the lateral third. The three regions are arranged in rostro-caudal longitudinal columns in the caudal three-quarters of the pons and these columns are continuous with one another by regions of scattered degeneration. The fibres appear to end in relation to distal dendrites of the pontine cells.This article has not been published in any journal before. The guiding principles in the care and use of animals approved by the American Physiological Society have been followed