Neurofibrils and the Nauta method.

When the pretreatment stages are omitted, the Nauta method for degenerating axons stains neurofibrils. Under the electron microscope the stain is closely related to neurofilaments. When one uses the comnplete Nauta technique, the stain is specific for mnembrane structures.

Plasticity in the rat olfactory cortex.

The relationship of age to deafferentation plasticity was studied in the rat olfactory cortex (OC). Ablation of a single olfactory bulb (OB) was performed in each of several rats of selected postnatal (PN) ages: PN2.5, 6, 9, 13, and 21 days and in adults of PN100 days. Following survival times sufficient to remove the resultant degeneration, a cortical lesion was placed in the ipsilateral OC. The patterns of degeneration from the OC lesion were studied and mapped in the adjacent deafferented OC. The results show a spread or sprouting of the usually deep-lying afferents (interrupted by the OC lesion), onto the deafferented superficial dendrites (normally occupied by the OB afferents) in all of the ages. The spread is most striking at PN2.5 to PN9, gradually reduced by PN13 to PN21, and least in the adult (PN100). There is also an apparent increase of afferents to the deeper dendrites nearer the cell bodies in all cases except in the PN 100 group. Shrinkage of layer I is not seen in PN2.5 subjects, is minimal by PN9, but is most marked in the adult PN100 with total OB lesions. Incomplete OB lesions sparing some lateral olfactory tract (LOT) fibers greatly reduce the shrinkage of layer I and the spread of afferents in all ages. Thus, a capacity for reorganization of afferents occurs at least through PN9, with PN13-21 a possible "critical period" after which plasticity is limited and transneuronal effects are more permanent. The association, centrifugal, and olfactory-entorhinal pathways are possible origins for this plasticity. Factors contributing to limitations in this reorganization are discussed.

Stereological analysis of synaptogenesis in the molecular layer of piriform cortex in the prenatal rat.

The development of synapses in the molecular layer of the rat piriform cortex was studied at embryonic days 15, 17, 19, and 21. The present study has sought to extend past studies of synaptogenesis by identifying not only changes in numbers of synapses, but also changes in numbers of potential precursors of synapses. A stereological method (Cruz-Orive, '80) was used to make volumetric estimations of the numbers of synapses, axonal puncta, vesicle-associated puncta, and unapposed postsynaptic specializations. This stereological method was preferred to other morphometric methods because it is not influenced by changes in the size, shape, or orientation of the structures of interest. This was considered important since such changes might be expected during development. Large numbers of unapposed axonal specializations (axonal puncta and vesicle-associated puncta) were found in all three sublaminae (lateral olfactory tract, Ia, and Ib) at all ages. The numerical density (number per unit volume of neuropil) and relative frequency of these structures changed significantly with time. In all three sublaminae, these changes were associated with changes in the number of synapses, although the numerical density and relative proportions varied between the sublaminae. These results suggested that axonal puncta could accumulate vesicles, thus becoming vesicle-associated puncta, and that vesicle-associated puncta could contact dendrites, thus forming synapses. In contrast, the numerical density of lone postsynaptic specializations remained low and no significant changes in their relative proportion in the population were found. This suggested that although lone postsynaptic sites were observed, they did not appear to play a major role in synaptogenesis in this region of the cortex. In addition to documenting developmental differences between the three sublaminae in the molecular layer, the results support a synaptogenic hypothesis in which the axon can form surface specializations that appear to be involved in synaptogenesis, independent of direct dendritic contact.

Ultrastructure of transganglionic degeneration in brain stem trigeminal nuclei during normal primary tooth exfoliation and permanent tooth eruption in the cat.

Electron microscopy is used to study changes in the axons and terminals in the cat brain stem trigeminal nuclei, main sensory, and partes interpolaris and caudalis, during the process of natural tooth shedding. Areas previously showing light optical argyrophilic degeneration products and adjacent areas lacking this degeneration are included. Various types of alteration occur early during tooth loss, including increased presumed glycogen, increased cytoplasmic density, flocculence, lucency, and neurofilamentous hyperplasia. By the stage of maximum exfoliation, terminals and axons of marked density become prominent in areas showing argyrophilia, whereas nondense forms occur elsewhere. By late eruption ages, all forms of degenerated terminals and axons are rare, but phagocytes are heavily laden with similar forms of debris. The sequence of ultrastructural events is discussed in light of recent studies of transganglionic degeneration, their correlation with light microscopic findings, and the potential implications for central plasticity in this system.

Nerve growth factor receptor (p75)-immunoreactivity in the normal adult feline trigeminal system and following retrogasserian rhizotomy.

The 75 kDa protein nerve growth factor receptor [NGFr(p75)] is a neurotrophin receptor that is able to bind different members of the neurotrophin family of molecules implicated in affecting neuronal survival. Here we describe the light microscopic distribution of NGFr(p75)-immunoreactivity (IR) within the feline trigeminal brainstem sensory nuclear complex and trigeminal ganglion of normal adult subjects and in subjects 10 and 30 days following retrogasserian rhizotomy. Within the trigeminal ganglion of normal subjects, numerous fibers and most of the neuronal cell bodies showed NGFr(p75)-IR that varied in intensity, while cells and fibers with NGFr(p75)-IR were less numerous within the mesencephalic trigeminal nucleus. Within the main sensory and spinal trigeminal nuclei, NGFr(p75)-IR formed a reproducible pattern that varied between the different subnuclei. The NGFr(p75)-IR consisted both of dense pockets and a low level NGFr(p75)-IR that was selective to the trigeminal neuropil. Following rhizotomy, most of the NGFr(p75)-IR was lost from the main sensory and spinal trigeminal nuclei, except in regions where the upper cervical roots and cranial nerves VII, IX, and X project. In contrast, examination of the central root that was still attached to the trigeminal ganglion showed increased NGFr(p75)-IR in fibers and supporting cells, as did the motor root within the peripheral mandibular division. These results indicate that the majority of the NGFr(p75)-IR within the main sensory and spinal trigeminal nuclei originates from primary trigeminal afferents and that retrogasserian rhizotomy leads to an up-regulation of NGFr(p75)-IR in the part of the central root that is contiguous with the ganglion.

Electron microscopy of immunoreactivity patterns for glutamate and gamma-aminobutyric acid in synaptic glomeruli of the feline spinal trigeminal nucleus (Subnucleus Caudalis).

We studied the ultrastructure of the synaptic organization in the feline spinal trigeminal nucleus, emphasizing specific neurotransmitter patterns within lamina II of the pars caudalis/medullary dorsal horn. Normal adults were perfused, and Vibratome sections from pars caudalis were processed for electron microscopy. Ultrathin sections were reacted with antibodies for the excitatory neurotransmitter glutamate (Glu) and for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) by using postembedding immunogold techniques. Both single- and double-labeled preparations were examined. Results with single labeling show that Glu-immunoreactive terminals have round synaptic vesicles and form asymmetric synaptic contacts onto dendrites. GABA-immunoreactive axon terminals and vesicle-containing dendrites have pleomorphic vesicles, and the axon terminals form symmetric contacts onto dendrites and other axons. Double labeling on a single section shows glomeruli with central Glu-immunoreactive terminals that are presynaptic to dendrites, including GABA+ vesicle-containing dendrites. These Glu+ terminals are also postsynaptic to GABA+ axon terminals, and these GABA-immunoreactive terminals may also be presynaptic to the GABA+ vesicle-containing dendrites. Quantitative analyses confirm the specificity of the Glu and GABA immunoreactivities seen in the various glomerular profiles. The results suggest that a subpopulation of Glu-immunoreactive primary afferents (excitatory) may be under the direct synaptic influence of a GABA-immunoreactive intrinsic pathway (inhibitory) by both presynaptic and postsynaptic mechanisms.

Ultrastructural localization of immunoreactivity in the developing piriform cortex.

The purpose of this study was to determine the ultrastructural basis for the immunoreactivity patterns in synaptic structures during development in layers I and II of the piriform cortex (PC) of rats. Antisera to cholecystokinin (CCK) and glutamic acid decarboxylase (GAD) were used at several different postnatal days (PN) and in adults to describe the distribution, characteristics, and relative frequency of labeled profiles--especially axons and terminals--with emphasis on details of the synaptic contacts. GAD-positive terminals occur from PN 2 to adulthood but only form contacts in deeper sublayers (Ib and II) initially. Contacts increase in layer I after PN 6 and are reduced in layer II after PN 21 when the GAD-labeled terminals and synapses take on adult features with flattened vesicles and symmetric contacts. CCK-labeled terminals are present in deeper sublayers at PN 2 but are few and rarely form contacts. Both terminals and contacts increase between PN 2 and 9, taking on distinctive shapes and vesicle morphology by PN 13. At PN 21 and older, CCK terminals have mainly flattened vesicles and mostly form symmetric contacts onto dendrites and somata in deeper layers (Ib and II). Superficial sublayer Ia has very few CCK-labeled synapses and axons. Thus immunoreactivity occurs in terminals prior to synapse formation; labeling of the presynaptic specializations precedes subsequent maturation; synaptic vesicle morphology and membrane specializations are similar for the vast majority of both CCK and GAD terminals; inhibitory (GABA) synapses are established sooner than the possibly excitatory CCK synapses; a deep to superficial gradient of synaptogenesis is associated with GAD-positive terminals in the PC; and the labeling patterns may be related to critical developmental or synaptogenic periods.

Ultrastructure of synaptic remodeling in piriform cortex of adult rats after neonatal olfactory bulb removal: an immunocytochemical study.

The purpose of this investigation was to study possible remodeling in synaptic structures of the piriform cortex (PC) of adult rats following neonatal deafferentation by removal of the olfactory bulb (OB) at birth. Emphasis was placed on possible qualitative changes in the ultrastructure and immunocytochemical localization of cholecystokinin (CCK, a possible excitatory neurotransmitter or modulator) and glutamic acid decarboxylase (GAD, precursor enzyme to the inhibitory transmitter GABA) in axons, terminals, and synaptic complexes. Light microscopic results in normal adult material show that GAD-positive terminals form a dense band subjacent to the lateral olfactory tract (LOT), become less dense in deeper Ib, and are rare in layer II. Following deafferentation, GAD-positive terminals appear denser and more homogeneously distributed throughout layer I and are also more prevalent in layer II. Ultrastructural results of normals and controls indicate GAD-positive terminals normally contain pleomorphic or flattened vesicles and form symmetric contacts onto dendritic shafts and branches throughout layer I. In deafferented layer I not only do there appear to be greater numbers of symmetric GAD-positive contacts, but in contrast to normals, asymmetric contacts mainly onto spines are now present. Light microscopic results from deafferented material also show an apparent proliferation with spread or sprouting of CCK-positive fibers or axonlike structures mainly into layer Ia, whereas these fibers are normally observed only in the LOT and are generally few in number. Also in normals the few CCK-positive terminals in the area subjacent to the LOT contain flattened or pleomorphic vesicles and form symmetric contacts. Deafferentation results in CCK-positive terminals throughout layer I with a greater frequency of synaptic contacts which now also include a few asymmetric contacts onto spines. The findings clearly show modifications in synaptic patterns of immunocytochemical-labeled terminals that might be compatible with the process of atypical reinnervation of deafferented postsynaptic sites and possible ingrowth of new axons.

Host primary olfactory axons make synaptic contacts in a transplanted olfactory bulb.

Previous light microscopic studies have shown that host olfactory neurons are able to grow into a transplanted fetal olfactory bulb, and behavioral studies have shown that animals with transplanted olfactory bulbs recover functional olfactory abilities. We examined the olfactory bulb transplant at the ultrastructural level to determine whether synaptic contacts are reestablished between host olfactory neurons and donor olfactory bulb. Mature rats that, as neonates, had received embryonic olfactory bulb transplants following olfactory bulb removal were studied. An antibody specific for olfactory marker protein was used to identify the primary olfactory neurons; it was bound by a gold-conjugated secondary antibody for visualization. To preserve the antigenicity of the olfactory marker protein for immunolabeling, Lowicryl K4M hydrophilic resin was used. Synaptic contacts were unmistakable between labeled axons of host olfactory neurons and unlabeled processes within glomerulus-like areas of the transplanted olfactory bulb. The surrounding neuropil contained other elements similar to those found in normal tissue, including synaptic contacts between unlabeled profiles. We clearly show that the transplanted olfactory bulb exhibits sufficient plasticity to form an array of normal synaptic contacts, including the contacts from host primary olfactory neurons.

Light microscopic localization of calcitonin gene-related peptide in the normal feline trigeminal system and following retrogasserian rhizotomy.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been implicated in the transmission and modulation of primary afferent nociceptive stimuli. In this study, we describe the light microscopic distribution of CGRP immunoreactivity (IR) within the feline trigeminal ganglion and trigeminal nucleus of normal adult subjects and in subjects 10 and 30 days following complete retrogasserian rhizotomy. Within the trigeminal ganglion of normal subjects, cell bodies and fibers showed CGRP-IR, whereas immunoreactive fibers were rare in the central root region. Within the normal spinal trigeminal and main sensory nuclei, CGRP-IR was seen to form a reproducible pattern that varied between the different nuclei. Following rhizotomy, most, but not all, of the CGRP-IR was lost from the spinal trigeminal and main sensory nuclei, except in regions where the upper cervical roots and cranial nerves VII, IX and X project into the trigeminal nucleus. The pattern seen at 10 days contained more CGRP-IR than that seen at 30 days and suggests that degenerating fibers still show CGRP-IR. In contrast to the decrease seen in the nuclei after rhizotomy, examination of the central root that was still attached to the trigeminal ganglion showed an increase in CGRP-IR within fibers, some of which ended in growth conelike enlargements. Rhizotomy induced a dramatic increase in CGRP-IR within trigeminal motoneurons and their fibers, which was strongest 10 days after rhizotomy and weaker at 30 days, which was still stronger than normal. These results indicate that the majority of CGRP-IR found in the trigeminal nucleus originates from trigeminal primary afferents and that an upregulation of CGRP-IR occurs in trigeminal motoneurons and in regenerating fibers in the part of the central root that was still attached to the ganglion. In addition, the persistence of CGRP-IR fibers in the trigeminal nucleus provides one possible explanation for the preservation of pain in humans following trigeminal rhizotomy.

Enhanced ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine reaction product.

Ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine (HRP-TMB) reaction product within trigeminal ganglion cells and brain stem axons and terminals following HRP injections into the pulpal chambers of cat teeth is enhanced by utilization of a modified osmication procedure that converts the reaction product to a markedly stable and electron-dense form. The results following the use of the modified osmication procedure (pH 5.0 phosphate buffer at 20 degrees C for 12 hours) are compared to results obtained by following Carson's osmication protocol (Carson KA, Mesulam M-M: J Histochem Cytochem 30:425, 1982; Carson KA, Mesulam M-M: In Tracing Neural Connections with Horseradish Peroxidase. Edited by M-M Mesulam. J Wiley, Chichester, England, 1982, p 153-184) (pH 6.0 phosphate buffer at 45 degrees C for 45 min). The results suggest that the conversion of the HRP-TMB reaction product to an electron-dense form during osmication is intimately associated with the pH of the phosphate buffer and the total time of osmication.

Dynamic studies of ototoxicity in mature avian auditory epithelium.

Hearing loss induced by ototoxicity is a worldwide problem despite the development of newer antibiotics and chemotherapy agents. The cellular mechanisms responsible for aminoglycoside-induced hearing loss are still poorly understood. We have developed two different methods of studying the dynamic cellular and subcellular changes in the chick auditory sensory epithelium that occur during hair cell death. The first study was performed in mature chicks after a single, high dose injection of gentamicin, which results in the rapid loss of all hair cells in the basal third of the cochlea. Chicks were sacrificed at discrete time points after drug treatment, and transmission electron microscopy was performed to study the ultrastructural changes in basal hair cells during the course of cell death. We noted various changes in the cell morphology including accumulation of cytoplasmic inclusion bodies, dispersion of the cytoplasmic polyribosomes, mitochondrial swelling, and cellular extrusion by 24 h after injection. The next two studies were performed using tissue cultures from mature avian auditory sensory epithelium. Cultured cells were labeled using vital fluorescent markers, and levels of intracellular calcium and reactive oxygen species within hair cells were studied following aminoglycoside exposure. We identified a dose-dependent increase in the levels of intracellular calcium, which was blocked by an inhibitor of voltage-gated calcium channels. We also found that levels of reactive oxygen species in hair cells greatly increased after exposure to gentamicin, and this response was blocked by two different antioxidants. These studies serve to identify key cellular and molecular changes in hair cells in response to ototoxic drugs. Further study of these processes may lead to a better understanding of how ototoxicity is induced and to potential preventative interventions.

Electron microscopy of plasticity in rat olfactory cortex.

Electron microscopy (EM) is being used to study the ultrastructural basis for the age-dependent reorganization of afferents in the olfactory cortex (OC) of rat after deafferentation of the area by removal of the ipsilateral olfactory bulb (OB). The double-lesion technique was used with a primary lesion of the OB at various postnatal (PN) ages between PN 0 and 30 and in the adult (PN 100). After appropriate survival times to remove initial lesion-degenerated terminals from the OB lesion, a second lesion was placed in the ipsilateral OC. One to 3 days later the tissue is prepared for EM with emphasis on a study of changes in the superficial and deep dendritic layer (Ia and Ib respectively) rostral to the lesion. In control litter mates with both OBs intact, but with a single OC lesion only, degenerating synaptic terminals occur onto dendritic spines and branches only in deeper Ib. However, in adults with OB lesions at PN 0-9, OC lesions produce degenerating terminals throughout Ia and Ib including immediately subjacent to the pia. In Ia degenerating terminals are greatly reduced in the PN 13 group and rare to absent in experiments with OB lesions at older ages (PN 30-100). Electron-dense debris within glia occurs throughout layer I in each double-lesion group but is greatest in experiments with OB lesions at older ages. Some transsynaptic alterations are seen throughout, especially in the PN 30-100 group even at a distance from the OC lesion. The results support earlier light microscopic (LM) findings, suggesting PN 9-13 as critical ages for developmental plasticity and prove that at least in the younger ages, synapses are involved in the phenomenon. This may be explained by either reinnervation of deafferented sites or persistence of synapses that would otherwise have been eliminated by afferents from the OB. In addition, some of the LM degeneration particles probably are engulfed masses of debris and not synaptic structures, especially in cases which were operated at older ages and survived for 3 days. The various afferent pathways involved in the events as well as factors that limit the phenomenon in older ages are discussed.

Synapse development within the spinal trigeminal nucleus.

Pars interpolaris of the spinal trigeminal nucleus of kittens has been studied with the electron microscope at birth and at several subsequent ages during the first month of life. Attention has been given to ultrastructural maturational changes that occur in this neuropil, especially events in synaptogenesis. The results of this investigation include the following observations: (1) the neuropil, even at the earliest ages studied (three-hour-old kittens), is strikingly mature, necessitating a quantitative assessment in order to determine subtle developmental changes in synaptic patterns; (2) the number of axoaxonic contacts at birth are few, and their emergence is essentially a postnatal phenomenon; (3) it appears that the immature Gray type II or symmetrical synapse possesses distinct cleft material and dense, parallel membrane specializations. Synaptic vesicle accumulation at this contact appears to occur after the membrane specializations have formed. A previous study by Kerr26 has shown a reduced potential for primary afferent reorganization with the spinal trigeminal nucleus when kittens are subjected to trigeminal rhizotomy after three days of age. Our observations on the development of axoaxonic synaptic arrangements in the neonatal period may provide an explanation for these earlier results.

Brain stem degeneration patterns following tooth extractions: visualization of dental and periodontal afferents.

The terminal central nervous system distribution of those primary afferent neurons from the teeth and periodontium in cats was studied by degeneration methods. Lesions were created by combinations of tooth extractions and the brain stem was examined at 14, 30 and 60 days survival for degeneration patterns, with 30 days being the optimal survival time. Degenerating axons and terminals are seen bilaterally, but somewhat less on the side contralateral to the lesions and are concentrated in the ventral half of pars interpolaris and pars caudalis near obex. The results are discussed with regard to the current controversies concerning the primary central termination of these trigeminal neurons.

Ultrastructure of transganglionic HRP transport in cat trigeminal system.

The ultrastructure of transganglionic transport of horseradish peroxidase (HRP) from the inferior alveolar (IA) nerve to the brainstem is being studied in the cat. The IA nerve was soaked in an HRP solution and following a two-day survival the animal was perfused transcardially with a paraformaldehyde-glutaraldehyde solution. The tissue was immediately dissected and postfixed for 1-3 h in perfusate. Sections of 75 micron thickness were cut with a Vibratome and reacted utilizing tetramethyl benzidine (TMB) as the chromagen. Optimum results for electron microscopy were obtained by osmication in a pH 6.0, 1% osmium tetroxide solution for 45 min at 45 degrees C, followed by rapid dehydration and embedment in Epon. The resulting HRP-TMB reaction product was characterized and identified ultrastructurally in ganglion cells, peripheral and central axons and in brainstem terminals. The HRP-TMB reaction product varied in density but had consistent crystalline-like laminations of a repeating unit and characterized by a membrane 4-5 nm in diameter. Some of the HRP-TMB reaction product found in terminals and axons was below the limit of resolution of the light microscope.

Unmyelinated axons in the pyramidal tract of the cat.

Ultrastructural preparations revealed the presence of unmyelinated axons in the pyramidal tract (PT) of the adult cat. At the level of the medulla oblongata, unmyelinated axons constituted 8-15% of the total PT population. Axon diameters ranged from 0.05 to 0.06 micron with a mean of 0.18 micron. Although axons were distributed throughout the PT, their density was highest in the medial part.

A morphometric study of the effects of maturation and aging on synaptic patterns in the spinal trigeminal nucleus of the cat.

Morphometric methods have been used to study the synaptic and terminal patterns in cat trigeminal nucleus, pars interpolaris, during development and aging. Ages 1, 3, 6, 11, 16, 21, 27, 110, 600 days and 8 and 11 years were studied. Both proportions and densities (number per unit area) of certain terminals and synapses showed significant changes with age. Axoaxonic synapses especially showed two major periods of increase (3-6 days and 21-27 days). The values of most parameters increased in the 21-27 day period to peak levels and then decreased gradually with age. The results indicate two separate critical synaptogenic periods of development and a loss of synaptic elements in aging. Factors contributing to these changes are discussed as is the potential for plasticity in the different afferents at each period.

Electron microscopic localization of nerve growth factor receptor (p75)-immunoreactivity in pars caudalis/medullary dorsal horn of the cat.

Previous studies have demonstrated the presence of nerve growth factor receptor [NGFr(p75)]-immunoreactivity (IR) in the spinal trigeminal nucleus of both 8-10 week-old kittens and mature cats. Most of the NGFr(p75)-IR is lost following retrogasserian rhizotomy, indicating that the majority of the NGFr(p75)-IR within the spinal trigeminal nucleus is of trigeminal primary afferent origin. Here, we examined the ultrastructural localization of NGFr(p75)-IR within lamina II outer of pars caudalis/medullary dorsal horn in the mature cat. Lamina II outer represents a location where dense NGFr(p75)-IR is seen with the light microscope. The NGFr(p75)-IR identified with the electron microscope was located within small thinly myelinated and unmyelinated axons and within axon terminals. The terminals with NGFr(p75)-IR typically formed asymmetric synaptic specializations onto dendritic profiles and at times were postsynaptic to other axon terminals at symmetric synaptic specializations. The terminals with NGFr(p75)-IR were either simple (associated with a single profile) or more complex, such as those that typically formed the central element in synaptic glomeruli. The NGFr(p75)-IR in terminals was especially prominent on microtubules and the plasmalemma and these findings are consistent with proposed roles for NGFr(p75) in axoplasmic/neuronal transport and as a membrane protein, respectively. The profiles with NGFr(p75)-IR seen with the electron microscope indicate a primary afferent origin and show some similarities when compared to other markers of primary afferent fibers such as calcitonin gene-related peptide. In addition, a possible role for NGFr(p75) in the transmission of nociceptive stimuli is also discussed.

Toxic ricin demonstrates a dual dental projection.

Toxic ricin was used to study the central distribution of dental afferents in the cat. Following intrapulpal ricin injections ganglion cell degeneration is seen in the II and III ganglion divisions. Central argyrophilic degeneration occurs in the dorsal portion of all ipsilateral trigeminal nuclei. Ventral degeneration is seen in the pars interpolaris and pars caudalis. No contralateral degeneration was observed. The results are discussed with regard to previous studies of the central location of dental afferents.