Deafferentation-induced alterations in the rat dorsal horn: II. Effects of selective poisoning by pronase of the central processes of a peripheral nerve

Studies of deafferentation and regeneration, as well as studies requiring several tracing techniques, would benefit from availability of a substance that would selectively lesion the central components of a single peripheral nerve. Pronase, a combination of proteolytic enzymes, was tested for this purpose. Three weeks following microinjection of Pronase (5-25 mg) into the rat sciatic nerve, many ganglia cells in the L3-L5 ganglia were degenerated. Degeneration of primary afferents also was evident in the dorsal horn, as detected by silver Fink-Heimer methods. Patterns of terminal fields coincided with those mapped in normal rats for the sciatic nerve by using HRP transport. Ultrastructural changes were similar to those seen at 3 weeks following sciatic nerve section or rhizotomy, as described in our companion paper. However, degenerative changes following Pronase injection of the sciatic nerve were quantitatively greater than those following sciatic nerve section alone. Degenerating terminals were either electron lucent and swollen, electron dense, or filamentous with loss of vesicles. Postsynaptic dendrites, and occasionally somata, also showed signs of degeneration. Some became electron dense, others accumulated osmiophilic floccular material, but most became electron lucent and developed large membrane-bound cavities. Glial processes expanded around degenerating elements, wrapping around both terminals and dendrites. Glial sheets covered denervated dendritic and somatic spines, separating them from their terminals. Labyrinth formations of glial sheaths around debris were also found. Pronase appears to mimic the effects of mechanical destruction of primary afferents, but when compared to rhizotomy, is selective for the afferents of a single nerve, and, when compared to nerve section, produces a greater effect. Further, the substance is relatively safe for investigators compared to other toxins such as ricin.     

Glial cell responses,complement, and clusterin in the central nervous system following dorsal root transection

We have examined the glial cell response, the possible expression of compounds associated with the complement cascade, including the putative complement inhibitor clusterin, and their cellular association during Wallerian degeneration in the central nervous system. Examination of the proliferation pattern revealed an overall greater mitotic activity after rhizotomy, an exclusive involvement of microglia in this proliferation after peripheral nerve injury, but, in addition, a small fraction of proliferating astrocytes after rhizotomy. Immunostaining with the phagocytic cell marker ED1 gradually became very prominent after rhizotomy, possibly reflecting a response to the extensive nerve fiber disintegration. Lumbar dorsal rhizotomy did not induce endogenous immunoglobulin G (IgG) deposition or complement expression in the spinal cord dorsal horn, dorsal funiculus, or gracile nucleus. This is in marked contrast to the situation after peripheral nerve injury, which appears to activate the entire complement cascade in the vicinity of the central sensory processes. Clusterin, a multifunctional protein with complement inhibitory effects, was markedly upregulated in the dorsal funiculus in astrocytes. In addition, there was an intense induction of clusterin expression in the degenerating white matter in oligodendrocytes, possibly reflecting a degeneration process in these cells. The findings suggest that 1) complement expression by microglial cells is intimately associated with IgG deposition; 2) axotomized neuronal perikarya, but not degenerating central fibers, undergo changes which induce such deposition; and 3) clusterin is not related to complement expression following neuronal injury but participates in regulating the state of oligodendrocytes during Wallerian degeneration. GLIA 23:221–238, 1998. © 1998 Wiley-Liss, Inc.     

The distribution of dorsal root axons to laminae IV,V, and VI of the macaque spinal cord: A quantitative electron microscopic study

The projections of dorsal root axons to the deeper laminae (IV, V, and VI) of the Macaque spinal cord were examined by the use of experimentally induced degeneration following dorsal rhizotomy or by injection of dorsal root ganglia with tritiated amino acids followed by light and electron mi-croscopic autoradiography. Following dorsal rhizotomy, neurofilamentous degeneration of synaptic profiles occurs in each of the three deep laminae, more commonly in laminae IV and V than in lamina VI. The neurofilamentous degeneration is seen both in central glomerular (C) profiles and in many of the round vesicle (R) profiles. Neurofilamentous degeneration occurs as early as 18 hours following rhizotomy and the degenerating terminals are most numerous at 3–4 days postrhizotomy. None are seen after 7 days survival. The neurofilamentous profiles form axodendritic and, occasionally, axosomatic synapses with neurons of the dorsal horn. They are also seen to be postsynaptic to flat vesicle (F) profiles in axoaxonal synapses. A second type of degeneration, electron-lucent degeneration, is seen in laminae V and VI, and only occasionally in lamina IV. The lucent degeneration occurs somewhat later after rhizotomy than does the neurofilamentous degeneration and reaches its peak at 5 days postrhizotomy. No lucent terminals are seen after 7 days survival. Electron-dense degeneration, so common in lamina II, is not seen in the deeper dorsal horn. Autoradiographic techniques show that both C and R terminals are labelled in the deeper dorsal horn. Both of these terminals form axodendritic synapses and a significant number are found to be postsynaptic in axoaxonal synapses. Most of the C terminals degenerate following rhizotomy or are labelled following injection of the parent dorsal root ganglia with tritiated amino acids. Approximately one-fifth of the R profiles are derived from dorsal roots. F profiles do not appear to be of dorsal root origin in any case. It is concluded that neurofilamentous alterations represent the degeneration of larger-diameter (Aβ) axons which distribute to the deeper dorsal horn and that electron-lucent degeneration represents the termination of Aδ fibers. Electron-dense degeneration thought to represent the termination of nonmyelinated axons (C fibers) in the superficial dorsal horn is not seen in the deeper dorsal horn and it is concluded that C fibers do not project to the deeper laminae.     

An electron microscopic study of the afferent connections of the lateral reticular nucleus of the cat

The mode and pattern of termination of the afferents to the lateral reticular nucleus (LRN) of the cat were examined at the cellular level through the ultrastructural localization of induced degeneration. Examination of the LRN following hemicordotomy at the fifth and sixth cervical levels revealed that most of the degenerating terminals were in contact with intermediate and distal dendrites, and that most of these degenerating terminals were small and contained round vesicles. Fewer degenerating terminals were observed on the somata and proximal dendrites after spinal hemisection, and most of these terminals were large and contained round vesicles. Following lesions of the pericruciate cortex, small degenerating terminals were occasionally observed making contact onto intermediate and distal dendrites. Degenerating rubral terminals were observed synapsing on somata, somatic and dendritic spines, proximal dendrites and most commonly on intermediate and distal dendrites following lesioning of the red nucleus. The degenerating axosomatic rubro-LRN terminals belonged to the large, round-vesicle terminal population, while those degenerating terminals contacting intermediate and distal dendrites belonged to the small, round-vesicle population. Small, degenerating terminals were occasionally seen following lesions of the fastigial nucleus, and they made synaptic contact mainly onto intermediate and distal dendrites and dendritic spines. The present ultrastructural observations taken together with the convergence pattern of LRN afferents and the available electrophysiological data on inputs to the LRN suggest an extensive integration of converging impulses from two or more afferent sources to the rostral LRN neurons. The results of this study therefore support the view that the rostral LRN functions as a comparator of command signals from the motor cortex and red nucleus and feedback signals from the spinal cord and cerebellum during ongoing movement.     

Electron microscopic analysis of lesion-induced changes in synaptic structure and immunogold labeling of neurotransmitters within the feline trigeminal nucleus

This report uses lesion and postembedding immunogold protocols to examine the ultrastructural details of lesion-induced synaptic and neurotransmitter changes in the feline trigeminal nucleus. Electron microscopic (EM) analysis concentrated on lamina II (substantia gelatinosa) of the subnucleus pars caudalis (PC) which is one relay site of trigeminal fibers involved in nociception. Special attention was directed to analysis of reoccupation of synaptic sites vacated by primary afferent degeneration. Primary afferents were caused to degenerate by performing unilateral retrogasserian rhizotomy. After survival times of 1, 2, 6, and 7 days, sections of PC were processed for postembedding immunogold labeling with antibodies to the neurotransmitters gamma aminobutyric acid (GABA) and glutamate (Glu). The results show: (1) degenerating primary afferent terminals were easily identified in various stages of degeneration; (2) Glu immunoreactivity was observed in early forms of degenerated primary afferent terminals with clumped vesicles as well as in the highly distorted, electron dense terminals of later degeneration; and (3) some GABA immunoreactive terminals formed atypical synapses which exhibited both asymmetric (excitatory) and symmetric (inhibitory) synaptic densities. A possible model is presented of the progression of events following trigeminal nerve lesion which results in atypical synapse formation. Such altered synaptic relationships seen in PC following trigeminal rhizotomy may be related to hyperactivity that is seen in animals and to the atypical facial pain following nerve lesions in humans. © 1996 Wiley-Liss, Inc.     

Septal efferent axon terminals identified by anterograde degeneration show multiple sites for modulation of neuropeptide Y-containing neurons in the rat dentate gyrus

The ultrastructure and cellular associations of septal efferent terminals identified by anterograde degeneration with neurons containing neuropeptide Y (NPY) in the rat dentate gyrus were examined quantitatively. For this, the septal complex (i. e., medial septal and diagonal band nuclei) of adult male rats was injected with the neurotoxin ibotenic acid (1%; 150 nl) and following a 2–4-day survival period, the hippocampal formation was processed for the electron microscopic immunocytochemical demonstration of NPY using the avidin-biotin complex method. Terminals with the morphological characteristics of anterograde degeneration, in particular an increase in osmiophilia, and neurons containing NPY-like immunoreactivity (NPY-LI) were most abundant in the hilus of the dentate gyrus. In this region, degenerating terminals (n = 109) were usually small (0.2–0.4 μm in diameter) and formed both asymmetric and symmetric synapses with small (distal) dendrites. The degenerating terminals contacted either single NPY-containing (19%) perikarya or dendrites or unlabeled (48%) perikarya or dendrites. Some degenerating terminals contacted the same perikarya or dendrites as an NPY-containing terminal (11%); these neurons were either immunoreactive for NPY or unlabeled. The remaining degenerating terminals were either directly apposed without glial intervention to unlabeled and NPY-labeled terminals (11%) or lacked associations with any neuronal processes in the plane of section analyzed (11%). The findings demonstrate that ibotenic acid injections in the septal complex can identify septal efferent terminals by degeneration and provide cellular substrates for the direct synaptic regulation as well as presynaptic modulation of hippocampal NPY-containing neurons by septal efferent terminals. © 1993 Wiley-Liss, Inc.     

Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord

Sciatic nerve section has been shown to reduce substance P (SP) in the dorsal horn of the spinal cord, but the mechanism which underlies the reduction is not understood. Whether SP levels subsequently recover as they do after dorsal rhizotomy has also been unknown. To test the hypothesis that transganglionic degeneration of primary afferents contributes to the reduction of SP, we have studied the changes in SP which result from section of the cat sciatic nerve and determined the extent of dorsal root ganglion (DRG) cell death. Sciatic nerve section resulted in DRG cell death, but the amount was variable and not seen in all animals. Reduction in dorsal horn and DRG SP was seen in all animals, and in the spinal cord it was followed by recovery. These sequelae resemble the changes which follow dorsal rhizotomy. After sciatic nerve section, the reduction in dorsal horn SP is small than after rhizotomy, the recovery more complete, and both the reduction and the recovery proceed more slowly. Evidence is presented that similar mechanisms may contribute to depletion of intraspinal SP after sciatic nerve section and after dorsal rhizotomy. The mechanisms contributing to recovery of spinal cord SP after sciatic nerve section may resemble known mechanisms of recovery that occur when the lesion is central.     

Differential distribution of diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine-specific phospholipase d immunoreactivity in the superficial spinal dorsal horn of rats

It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGL-α) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGL-α and NAPE-PLD showed a remarkably different distribution. DGL-α immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGL-α and NAPE-PLD. Glial processes immunostained for DGL-α were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGL-α, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. The 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from nonsynaptic dendritic and glial compartments.     

Interlaminar connections of rat visual cortex: An ultrastructural study

Thermal lesions were made in layers I, II, and upper part of layer III of rat visual cortex. The distribution of degenerating axons and axon terminals in layers IV, V, and VI was studied using electron microscopic techniques. Following supragranular thermal lesions, the majority of degenerating axon terminals were found in layer V, with extension into the adjacent part of layer VI. Neural profiles postsynaptic to degenerating axon terminals were found in these layers in the following distribution: 81.7% on spines of small to medium size dendrites; 18.2% on dendrite shafts; and <1% on neuronal perikarya. Few degenerating terminals were found on or near apical dendrites. Degenerating terminals were identified on shafts of stellate-type dendrites found in the upper part of layer V. Degenerating axons oriented parallel to the cortical surface were found most often in deep layer IV and upper layer V. Degenerating axons were also seen in axon bundles coursing vertically through layer IV. Approximately 10% of the terminals within a grid square have undergone degeneration; no clustering of degenerating terminals was found in vertical or transverse sections through layers V and VI. We suggest that most axon terminals arising from pyramidal neurons in layers II and upper III synapse with spines and shafts of dendrite branches originating from pyramidal neurons in layer V and perhaps VI.     

Modification of astrocytes in the spinal cord following dorsal root or peripheral nerve lesions

Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to monitor the response of astrocytes in the rat spinal cord to either dorsal root or sciatic nerve lesions. Image analysis methods were used to provide a quantitative correlate of the reactive gliosis. Multiple dorsal root section elicited a rapid increase in GFAP immunoreactivity of astrocytes unilaterally within the spinal cord along the pathway of the degenerating dorsal root axons in the dorsal and ventral horns and this gliosis persisted in the dorsal horn beyond the time at which active phagocytosis of degenerative debris occurred. Labeling of proliferating cells using [3H]thymidine revealed that none of the dividing cells contained detectable GFAP, suggesting that the increased GFAP labeling represents primarily a hypertrophy rather than a proliferation of astrocytes. Comparison of animals that had been deafferented in the early neonatal period with those deafferented as adults indicated that the GFAP immunoreactive response persisted following neonatal lesions but that it was markedly less intense than after adult lesions. Sciatic nerve section in adults does not result in extensive frank degeneration but it does evoke a rapid and marked increase in staining of astrocytes both in the dorsal horn and in the ventral horn. Transganglionic changes in GFAP staining in the dorsal horn occur by 3 days post-operatively, which is much earlier than the time of dorsal root ganglion neuron death caused by the sciatic nerve lesion. These experiments indicate that astrocytes can respond to signals from a variety of changes in neurons, including not only Wallerian degeneration, but also retrograde and transganglionic changes.     

Quantitative autoradiographic localization of [125I]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy.

Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [125I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [125I]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P less than 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P less than 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [125I]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals.     

Fine structure of synapses and retinal innervation of substance P and adenosin deaminase containing neurons in the superior colliculus of the rat.

The fine structure of substance P (SP) and adenosine deaminase (ADA) immunoreactive structures in synaptic contacts localized to the superficial layers of the superior colliculus of the rat was investigated by means of immunoelectron microscopy. We also examined the possibility of retinal innervation of SP- and ADA- containing neurons by immunohistochemistry after degeneration of retinal terminals caused by enucleation. SP-like immunoreactive presynaptic terminals of the stratum griseum superficiale (SGS) formed both asymmetric and symmetric synaptic contacts. Presynaptic dendritelike structures were also observed. SP immunoreactive postsynaptic elements made contacts with terminals showing diverse features. ADA-like immunoreactive structures were seen only as postsynaptic elements to different kinds of nonimmunoreactive terminals and were mostly localized in the ventral third of the SGS and the dorsalmost stratum opticum (SO). After enucleation, degenerating retinal terminals were found to form synaptic contacts with SP and ADA immunoreactive structures. The highest number of such degenerating terminals on ADA immunoreactive structures was observed 2 days after retinal denervation, very few being seen after 5 days. These degenerating terminals were restricted to the ventral SGS and dorsal SO. SP immunoreactive structures postsynaptic to degenerating retinal terminals were most numerous 5 days after enucleation and mainly localized in the dorsal SGS. Occasionally, SP immunoreactive dendritelike processes forming synapses with degenerating retinal terminals were simultaneously presynaptic to other nonimmunoreactive profiles, defining, therefore, serial synapses. The present results suggest that SP-I and ADA-I collicular neurons may be part of distinct channels carrying visual information to the lateral posterior and lateral geniculate nuclei of the thalamus, respectively.     

The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study.

This study examines the projection of dorsal root fibers to the upper dorsal horn of the monkey lumbar spinal cord utilizing degeneration and autoradiographic methods. The animals survived dorsal rhizotomy for periods varying from 18 hours to 28 days. Electron microscopy reveals the earliest degeneration to be neurofilamentous alteration of large synaptic profiles in lamina III and the inner zone of the substantia gelatinosa (IIi). This degeneration begins 18 hours after rhizotomy, reaches a peak at three days postoperatively and disappears by the end of the first week. Degenerating myelinated axons in the spinal gray matter, dorsal column white matter and Lissauer's tract first appear three days postoperatively. The second tye of degeneration of synapses occurs in lamina I and outer gelatinosa (IIo) and consists of electron lucent alteration of moderate size synapses, especially those having large granular vesicles (LGVs) and some neurofilamentous and dense degeneration. This synaptic degeneration in lamina I begins two days following rhizotomy and reaches a peak between five to seven days, declines markedly by ten days and is absent at four weeks survival. The third type of degeneration occurs in the substantia gelatinosa (laminae IIo and IIi) initially as an enlargement of synaptic vesicles at two days and then progresses to large numbers of electron dense small synapses, the peak of degeneration occurring at seven days and persisting as long as four weeks postoperatively. Some of the dense synapses can be seen to arise from small, nonmyelinated axons. These axons are first seen to be degenerating in the gelatinosal and marginal layers at four days survival and the first definite degeneration of nonmyelinated axons in Lissauer's tract is at seven days postoperatively. It is concluded that the largest axons projecting to this region of the dorsal horn degenerate most rapidly and that these axons are distributed to laminae III and IIi. Axons of intermediate diameter degenerate next and are distributed principally to laminae I and IIo. Fine diameter axons, probably nonmyelinated, degenerate more slowly and terminate principally in the substantia gelatinosa (IIi and IIo). There is some overlap in these projection domains, in that the principal projection to lamina III extends into the lower part of the gelatinosa and the projection to the marginal layer overlaps the outer gelatinosa. The axon terminals in gelatinosa of C fibers are sometimes postsynaptic in axoaxonal synapses as are several of the axon terminals of larger A fibers in lamina III. Most of the synapses of primary afferent origin in lamina I are not involved in axoaxonal synapses. It is likely that the terminations of many primary afferent fibers in laminae II and III are subject to presynaptic inhibition and those in lamina I are not. Some of the primary afferents in all three laminae synapse upon presynaptic dendrites and thus may influence transmitter release from these profiles. The LGV profiles are distributed in a manner similar to the distribution of substance P and it is suggested that the degenerating LGV profiles may contain substance P. Most of the LGV profiles and many of the round vesicle profiles do not appear to be derived from dorsal root, but most of the central synaptic profiles are of primary afferent origin. In no case was there evidence that flat vesicle synapses were derived from primary afferents. Following dorsal root ganglia injections with H³ leucine, light microscopic autoradiography at short postoperative survival times demonstrated heavy grain distribution over marginal and gelatinosal layers with somewhat less numbers of grains over lamina III. There were also many grains over the dorsal column white matter and Lissauer's tract. Electron microscopic autoradiography revealed that the majority of labeled structures seen with fast axonal transport in the upper dorsal horn are not synapses but are myelinated and nonmyelinated axons. Labeled synapses were the same types as those undergoing degeneration following rhizotomy: round vesicle profiles, central synaptic profiles and LGV profiles. Each of the labeled types was distributed throughout the upper laminae, with the exception of LGV profiles which are uncommon in layers deep to the outer zone of the gelatinosa. It is concluded that fast axon transport autoradiography is not a selective label for synapses in the cord and light microscopic autoradiography does not provide direct estimates of synaptic densities in the dorsal horn.     

Phenylethanolamine N-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat

The ultrastructural morphology as well as neuronal and glial associations of phenylethanolamine N-methyltransferase (PNMT)-containing terminals in the intermediolateral cell column (IML) of the thoracic spinal cord were examined in the rat utilizing the peroxidase-antiperoxidase (PAP) method. The PNMT-immunoreactive terminals were 0.5-1.4 micron in diameter and contained a few mitochondria, a large population of small clear vesicles and from 1 to 6 large dense-core vesicles. The terminals formed synapses primarily with dendrites. The type of axodendritic association (i.e. symmetric or asymmetric) varied with the size of the dendrite, such that the majority of synapses on large dendrites were symmetric and those on smaller dendrites and dendritic spines were asymmetric. Moreover, most of the synaptic associations of PNMT-containing terminals were with the smaller dendritic processes. Many of the PNMT-labeled terminals, as well as their postsynaptic targets, were closely invested with, or apposed to fibrous astrocytic processes. In a subsequent set of experiments, we combined immunoautoradiographic labeling for PNMT with horseradish peroxidase (HRP) retrograde identification of sympathetic preganglionic neurons (SPNs) in the IML to determine whether or not SPNs receive direct synaptic input from the adrenergic terminals. In these sections, PNMT-containing terminals directly synapsed on the HRP-containing (i.e. retrogradely labeled SPNs) perikarya and dendrites. The axosomatic synapses observed between PNMT-labeled terminals and SPN perikarya were exclusively symmetric; whereas the type of axodendritic association varied depending upon the size of the dendrite such that the majority were asymmetric. The findings provide ultrastructural evidence that in the rat IML, adrenergic (i.e. PNMT-containing) terminals (1) may be either excitatory (asymmetric) or inhibitory (symmetric) depending on their site of termination and (2) can influence sympathetic nerve discharge through a direct effect on the SPN cell membrane.     

Electron microscopic study of the gerbil dentate gyrus after transient forebrain ischemia

Summary Silver impregnation performed 1–2 days after transient forebrain ischemia in the Mongolian gerbil demonstrated terminal-like granular deposits in the outer two-thirds of the hippocampal dentate molecular layer (perforant path terminal zone), even though neither the cell bodies of origin of the perforant path nor the dentate granule cells were destroyed. Electron microscopic studies of the dentate gyrus were performed in an effort to discover the identity of these degenerating structures. Electron microscopy revealed that the granular silver deposits corresponded to electron-dense profiles. Many of these were degenerating boutons and some were degenerating postsynaptic dendritic fragments, but most of them could not be identified with certainty. Electron-dense profiles were less numerous than expected from the density of granular silver deposits. These structures were probably the degenerating axons, axon terminals and dendrites of CA4 neurons. The granular silver deposits and electron-dense boutons observed in the inner third of the dentate molecular layer 5 days after transient ischemia can probably be explained by the ischemia-induced degeneration of CA4 mossy cells, which give rise to the dentate associational-commissural projection. Finally, most mossy fiber boutons in area CA4 and some boutons in the molecular layer appeared watery and enlarged on postischemia days 1 and 2. Mossy fiber boutons with this ultrastructural appearance have previously been observed in seizure-prone animals and in animals undergoing convulsant-induced seizures. Although no postischemic seizures occur under the conditions of this study, these findings support the idea that excitatory pathways become hyperactive after transient ischemia.Supported by NIH Stroke Center grant NS 06233     

An Ultrastructural Study of the Ciliary Ganglia of the Cat and Monkey (Macaca fascicularis) Following Preganglionic Axotomy

The present study describes ultrastructural changes in the ciliary ganglia of the cat and monkey following preganglionic axotomy. At 3, 5 and 7 days after operation, the nucleus of some neurons was irregular, with prominent indentations, and displaced to the periphery of the neuron. The surface of most neurons was irregular. Neurofilaments and glycogen-like granules were much increased in some neurons. At 21 and 28 days after operation, neurons again appeared normal. Dendritic profiles, packed with many mitochondria and glycogen-like granules, could often be observed from 3 days after operation. In longitudinal section such profiles represented expanded trunks of dendrites; dilated mitochondria and dense bodies were sometimes encountered within them. At later stages after operation, some of these profiles were synaptically contacted by, or closely associated with, axon terminals. In myelinated axons, mitochondria and glycogen-like granules were also increased in number and dilated profiles and dense bodies were found within the axoplasm. In unmyelinated axons, dilated profiles and myelin-like figures were present, as were vesiculo-tubular structures and dense bodies. Electron-dense and -lucent changes could both be observed in myelinated and unmyelinated axons. Almost all the axon terminals were affected 3 days after operation. Within such degenerating axon terminals, the synaptic vesicles had accumulated to form one or several clumps, sometimes the degenerating axon terminals had undergone filamentous hyperplasia. At 45 days after operation, hardly any axon terminals were encountered. Non-neuronal cells, including satellite cells, macrophages and Schwann cells, were actively involved in removing degenerating axons and other cell debris.     

Degenerative patterns in the ventral cochlear nucleus of the rat after primary deafferentation. An ultra-structural study

The anterior ventral cochlear nucleus (AVCN) has been studied with the electron microscope in normal rats and after cochlear nerve deafferentation. The degenerative changes are rapid and therefore it is possible to study their complete evolution at survival times ranging from the first to the ninth day following the primary lesion.At survival times between 1 and 3 days calyciform endings and some bouton-shaped terminals containing large rounded vesicles undergo degeneration. Two different degenerative patterns coexist for the same system of fibers. (1) The most frequent kind of degeneration corresponds to the clear and swollen type with lysis of synaptic vesicles and mitochondria. The distinct evolution and subsequent glial reaction are important factors in proving the existence of this type of degeneration. (2) The second kind of degenerative process corresponds to the dark and shrunken type. Intermediary forms between both degenerative patterns are a rare observation.At survival times between 5 and 9 days most degenerating axon terminals have disappeared, either by lytic breakdown or glial phagocytosis. Free postsynaptic sites, frequently attached to a presumed remnant of the presynaptic membrane, maintain their normal appearance.A new feature of the reactive astrocytic cytoplasm is described. It consists of the presence of numerous tubular and vesicular profiles of about 70–120 nm in diameter, containing a central core of variable density. Such astrocytic reaction is not specific to the deafferented AVCN. It probably represents a local increase of lytic enzymes, due to a hyperproduction of primary lysosomes.The fate of the denervated postsynaptic sites has been elucidated. According to the present results one of two different fates may occur in the evolution of these postsynaptic differentiations: (1) In some cases they are reoccupied by intact axon terminals. This reinnervation is here considered as due to a mechanical displacement, by a process of sliding, of nearby intact terminals rather than to collateral sprouting. (2) More often, the postsynaptic neuron is cleared of the free postsynaptic sites by engulfment into its cytoplasm. By this way of membrane sequestration the remnants of the synapses disappear thus preventing the induction of new but unspecific synaptic contacts.     

Changes in dorsal horn synaptic disc numbers following unilateral dorsal rhizotomy

The present study estimates the numbers of synaptic discs and numbers of degenerating synaptic terminals in laminae I-IV of the rat S2 dorsal horn ipsi- and contralateral to unilateral dorsal rhizotomy. These data allow us to estimate the loss of synapses of primary afferents and to correlate this loss with the rate of axon disappearance in the proximal stump of a transected S2 dorsal root. Our first findings are that 47% of the ipsilateral synapses and 27% of the contralateral synapses disappear within a day following unilateral rhizotomy. Conclusions are that the predominant synaptic population in this part of the rat spinal cord is of primary afferent origin and that there is an extensive bilateral projection of the dorsal root fibers. The contralateral projection is confirmed by the appearance of numerous degenerating terminals on the contralateral side. We also find that synaptic loss and appearance of degenerating terminals occur relatively synchronously in laminae I-IV. Finally we find that the time course of the synaptic loss correlates primarily with the disappearance of unmyelinated fibers in the proximal stump of the transected dorsal root.     

Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons

Following sciatic nerve axotomy, vasoactive intestinal polypeptide (VIP) immunoreactivity increases dramatically in the central terminal areas of the nerve whereas other primary afferent neuropeptides are depleted. The contribution of the peripheral nerve to VIP increases in the spinal cord was investigated by performing sciatic nerve section alone, dorsal rhizotomy of the lumbar roots, axotomy and rhizotomy in combination or section of other peripheral nerves terminating in the same segments as the sciatic nerve. VIP, and for comparison, substance P (SP), cholecystokinin (CCK), somatostatin (SOM), were localized in the lumbar spinal cord and corresponding sensory ganglia using unlabeled antibody immunohistochemistry. After sciatic nerve section, SP, CCK and SOM were depleted in the lumbar dorsal horn whereas VIP increased. After rhizotomy alone all neuropeptide staining including VIP was depleted; axotomy followed by rhizotomy prodiced the same result. Axotomy of other peripheral nerves terminating in the lumbar cord increased the area of neuropeptide depletion but correspondingly increased the area of VIP staining. A large proportion of small and medium diameter dorsal root ganglion cells were stained for VIP after nerve section or axotomy but not after rhizotomy alone. A radical change in neuropeptide metabolism of dorsal root ganglion cells occurs after peripheral axotomy, in the form of a maked increase in VIP synthesis. An intact dorsal root is necessary for increases in VIP in the spinal cord indicating the primary afferent origin of the response.     

Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area.

Physiological and pharmacological studies indicate that descending projections from the prefrontal cortex modulate dopaminergic transmission in the nucleus accumbens septi and ventral tegmental area. We investigated the ultrastructural bases for these interactions in rat by examining the synaptic associations between prefrontal cortical terminals labeled with anterograde markers (lesion-induced degeneration or transport of Phaseolus vulgaris leucoagglutinin; PHA-L) and neuronal processes containing immunoreactivity for the catecholamine synthesizing enzyme, tyrosine hydroxylase. Prefrontal cortical terminals in the nucleus accumbens and ventral tegmental area contained clear, round vesicles and formed primarily asymmetric synapses on spines or small dendrites. In the ventral tegmental area, these terminals also formed asymmetric synapses on large dendrites and a few symmetric axodendritic synapses. In the nucleus accumbens septi, degenerating prefrontal cortical terminals synapsed on spiny dendrites which received convergent input from terminals containing peroxidase immunoreactivity for tyrosine hydroxylase, or from unlabeled terminals. In single sections, some tyrosine hydroxylase-labeled terminals formed thin and punctate symmetric synapses with dendritic shafts, or the heads and necks of spines. Close appositions, but not axo-axonic synapses, were frequently observed between degenerating prefrontal cortical afferents and tyrosine hydroxylase-labeled or unlabeled terminals. In the ventral tegmental area, prefrontal cortical terminals labeled with immunoperoxidase for PHA-L were in synaptic contact with dendrites containing immunogold reaction product for tyrosine hydroxylase, or with unlabeled dendrites. These results suggest that: (1) catecholaminergic (mainly dopaminergic) and prefrontal cortical terminals in the nucleus accumbens septi dually synapse on common spiny neurons; and (2) dopaminergic neurons in the ventral tegmental area receive monosynaptic input from prefrontal cortical afferents. This study provides the first ultrastructural basis for multiple sites of cellular interaction between prefrontal cortical efferents and mesolimbic dopaminergic neurons.