Do subplate neurons comprise a transient population of cells in developing neocortex of rats?

Studies were undertaken to determine whether neurons of the subplate layer represent a transient or stable population of cells in developing neocortex of rat. The first set of studies sought to determine the fraction of subplate neurons that is lost during early postnatal development. The optical dissector method was used to analyze fluorescently stained material in animals the age of postnatal day 0 (P0) to P40. These results demonstrate a reduction of slightly less than half of the total number of subplate neurons from P0 to P40. Counts of labeled cells in littermates at varied ages after [(3)H]thymidine or BRDU treatment on gestational day 14 (G14 - birthdate of occipital subplate neurons) or G18 (birthdate of layers III-IV neurons) demonstrate loss of approximately 50% of neurons in the subplate layer between P0 and P40, somewhat greater than the loss of neurons from cortical layers III-IV. The second set of studies investigated whether subplate neurons display cellular atrophy during postnatal development. Analysis of subplate neurons injected intracellularly with Lucifer yellow in fixed slice preparations indicates no reduction in soma size, number of dendrites, or extent of dendritic fields of subplate neurons taken from animals age P0 to P60. The third set of studies investigated whether functional markers of subplate neurons are reduced during postnatal development. Analysis of tissue stained histochemically for cytochrome oxidase or acetylcholinesterase, or stained immunocytochemically for GABA, somatostatin, or neuropeptide Y, demonstrate a remarkable loss of expression of staining patterns from late gestational ages to P20. These data demonstrate that, although subplate neurons seem not to be a transient population of cells in the usual sense of being eliminated by cell death or structural atrophy, the loss of histochemical and immunocytochemical markers indicates that they may be a functionally transient population of cells.

Large-scale synaptic errors during map formation by regeneration optic axons in the goldfish.

During the formation of visual maps, growing axons initially form a map by using topographically distributed cues that direct their growth and branching to the appropriate target region. This initial map is typically roughly retinotopic and is subsequently refined through activity-dependent rearrangement or cell death. Although synaptic connections are thought to be rearranged during the later refinement phase, there is no clear evidence that synapses are being formed during the initial targeting phase of development. Also, because optic fiber growth can be accurately directed during normal development, it is unclear whether regenerative fibers that have more pathway disorder would behave similarly. This issue was addressed by using optic fibers of goldfish that have the capacity to regenerate a retinotopic projection and can reestablish a rough retinotopic order without impulse activity. The optic nerve of goldfish was crushed, and at various times later, a small number of optic fibers in ventronasal retina was labeled with wheatgerm agglutinin-horseradish peroxidase. The tectum was then processed for electron microscopy to look at the distribution of labeled synapses during regeneration. At 3 weeks, synapses were observed at the far anterior end of the tectum and none were yet seen at the correct posterior retinotopic position. At 4-5 weeks, synapses were seen in nearly equal numbers at the incorrect anterior end and at both correct (retinotopic) and incorrect posterior positions. At late stages of regeneration, synapses were restricted to their correct posterior retinotopic position in the tectum, as they were in normal fish. These findings show that the formation of global retinotopic order entails the formation and subsequent elimination of a large number of highly ectopic synapses. Synaptic rearrangement is a major feature of targeting in this system and may be required for the regeneration of a retinotopic projection.

Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain.

Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures. Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes. Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15-17, were seeded on to the slice cultures. Combined cultures were maintained for two to 14 days in vitro. Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate-putamen. Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers. Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used. Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate putamen and less apparent specificity of regional and cortical laminar attachment. Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon. By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex. These studies revealed several different types of cell-tissue interaction. The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue. This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.

Development of ventral cochlear nucleus projections to the superior olivary complex in gerbil.

The postnatal development of the projection from the ventral cochlear nucleus to the principal nuclei in the superior olivary complex in gerbil (Meriones unguiculatus) was studied in an age-graded series of pups ranging from 0 to 18 days old. Small crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were inserted into the ventral cochlear nucleus of aldehyde-fixed brains, and the labeled projections were examined with epifluorescence microscopy. Selected sections were photooxidized in a solution of diaminobenzidine and subsequently processed for electron microscopy to examine the development of labeled synapses in the target nuclei. Horseradish peroxidase was injected into the ventral cochlear nucleus of adult gerbils to assess the form and persistence of projections observed in the neonatal animals. In addition, electrophysiological responses to acoustic stimuli of single units in the adult auditory brainstem were analyzed to confirm the functionality of the novel projection from the ventral cochlear nucleus to the contralateral lateral superior olive. By the day of birth (P0), developing axons from the ventral cochlear nucleus have already established highly ordered pathways to the three primary nuclei of the superior olivary complex: the ipsilateral lateral superior olive, the contralateral medial nucleus of the trapezoid body, and at the lateral and medial dendrites of the ipsilateral and contralateral medial superior olive, respectively. Developing axons from the ventral cochlear nucleus that innervated the contralateral medial nucleus of the trapezoid body lacked the terminal morphology characteristic of the calyx of Held, but began to adopt a more characteristic form on P5. The mature calyx appeared around P14-16. Exuberant developmental projections to topographically inappropriate areas of the superior olivary complex were not observed at the postnatal ages studied. In addition to the projections of the ventral cochlear nucleus to the superior olivary complex described in other species, we observed the development and maintenance of a major direct projection from the ventral cochlear nucleus to the contralateral lateral superior olive. On P0, ventral cochlear nucleus axons decussate in the dorsal trapezoid body, form a plexus at the dorsal edge of the contralateral medial superior olive, and enter the ventrolateral limb of the contralateral lateral superior olive. Over the next 2 weeks, fascicles of fibers form on the hilar and ventral aspects of the ventrolateral limb. Fibers arising from these fascicles form converging, but nonoverlapping, arborizations within the ventrolateral limb at right angles to the curvature of the nucleus. The medial region was devoid of labeled axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of ectopic projections from the ventral cochlear nucleus to the superior olivary complex induced by neonatal ablation of the contralateral cochlea.

The ability of an animal to localize a sound in space requires the precise innervation of the superior olivary complex by the ventral cochlear nuclei on each side of the lower brainstem. This precise pattern of innervation could require an immutable recognition of appropriate targets by afferent processes arising from these nuclei. This possibility was investigated by destroying one cochlea of gerbil pups (Meriones unguiculatus) on the second postnatal day and assessing the projections from the ventral cochlear nucleus (VCN) on the unablated side to the superior olivary complex during the subsequent 2 weeks and after the animals had reached maturity. A crystal of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into VCN on the unablated side in animals ranging in age from 3 to 14 days. To assess the permanence of any altered pattern of innervation, horseradish peroxidase was injected into VCN on the unablated side in adult, neonatally ablated animals. Finally, electrophysiological responses to acoustic stimuli delivered to the ear on the unablated side were recorded in the superior olivary complex of adult animals to assess whether altered innervation patterns were functional. Normative data were derived from our accompanying study of the development of VCN projections to the superior olivary complex in normal gerbils (Kil et al., this issue). Whereas VCN normally projects to the lateral aspect of the ipsilateral medial superior olive and to the medial aspect of the contralateral medial superior olive in control animals, in experimental animals VCN on the unablated side projects to both sides of these nuclei. Whereas in the gerbil, VCN normally projects only to the hilar area and to the ventrolateral limb of the contralateral lateral superior olive, in experimental animals VCN on the unablated side projects throughout this nucleus. This induced projection is specific in that the efferents to each limb of the contralateral nucleus are linked to the normal projection to the homotopic region of the ipsilateral nucleus. Whereas VCN innervates the contralateral medial nucleus of the trapezoid body in control animals, in experimental animals VCN on the unablated side provides calyces of Held in the ipsilateral nucleus as well. The induced projections to these three major subnuclei of the superior olivary complex first appear within 24 hours of the cochlear ablation and continue to develop over at least the subsequent 11 days. Thus, prior to the day when the cochlea becomes functional, VCN has established specific ectopic projections to loci normally innervated by VCN on the ablated side.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal migration of neurons and formation of laminae in rat cerebral cortex.

Migration of neurons and formation of laminae in the developing neocortex were studied by means of thymidine autoradiography. Timed pregnant rats received a single pulse injection of [3H]thymidine in the morning of embryonic day (E)13, 14, 15, 16, 17, 18 or 19. Pups were killed on postnatal day (P)0, 1, 2, 3, 4, 6, 10, 30, or 60 and brains were processed for autoradiography. Neurons in posterior (visual) cortical areas labeled by [3H]thymidine administration on E13 or E14 were found predominantly in the cortical subplate; cells labeled on E15 in layer VI; cells labeled on E16 in layers VI and V, cells labeled on E17 in layers V and IV; E18 in layers IV and III; and E19 in layers III and II. By the day of birth (P0), neurons labeled from E13-16 injections were already in their mature laminae in cortex. Many of the cells labeled on E17 were still situated within the cell-dense cortical plate (CP) at P0, and within layer V by P1. Cells labeled on E18 were found in the most superficial part of the CP on P0, in the deep part of the CP on P1, and formed layer IV on P2 and P3. At P0, many E19 labeled cells appeared to be in migration to the cortex and were found in the CP on P1, in layer III by P4, and in layer II by P6. Cells in the auditory cortex labeled by [3H]thymidine injections on a particular day were situated more superficially than comparable labeled cells in the visual cortex, indicating a lateral to medial gradient in which the auditory cortex is formed earlier than the visual cortex. Distributions of labeled cells in the somatosensory cortex were similar to those in the visual cortex. These data provide a detailed and comprehensive description of the position of varied populations of cortical neurons during the early postnatal period, as well as a description of the formation of cortical laminae at times when major systems of afferents are growing into the cortex and making synaptic connections with their target cells.

Development of geniculocortical projections to visual cortex in rat: evidence early ingrowth and synaptogenesis.

Anterograde movement of DiI and transneuronal transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were used to study the temporal and laminar patterns of ingrowth of the geniculocortical projection to visual cortex in fetal and postnatal rats. The development of this projection was compared to patterns of migration and settling of [3H]-thymidine-labeled neurons destined for cortical layer IV, and to geniculocortical synapse formation. DiI-labeled geniculocortical axons were found in the intermediate zone beneath the lateral cerebral mantle at embryonic day (E)17 and in the subplate layer underlying visual cortex by E18. On E19 they appeared to accumulate and grow radially into an expanding subplate layer and into the deep part of developing cortical layer VI. By postnatal day (P)0, DiI or WGA-HRP-labeled geniculocortical axons were found in developing cortical layers VI and V. By P1, they invaded the deep portion of the cell-dense cortical plate, where they were in position to make initial contact with neurons that would later form layer IV. A few axons traversed the cortical plate to reach the marginal zone. Layer IV became an identifiable layer on P2, and a clear projection to layer IV was evident by P3. These results suggest that geniculocortical afferents grow continuously from the intermediate zone, initially into an expanding subplate layer and then sequentially into each of the developing cortical layers without evidence of "waiting." Electron microscopic data suggest that geniculocortical axons begin to form immature synapses with dendrites and neuronal perikarya as they first encounter cortical neurons, first in the subplate layer and then in developing layers VI, V and marginal zone, in addition to the primary target layer IV. The precise targeting and overall temporal and laminar patterns of ingrowth and synaptogenesis suggest that geniculocortical axons are directed to the visual cortex by guidance cues within the internal capsule and subplate. Further, they reach the occipital pole early enough to influence the specification and histogenesis of cortical area 17, perhaps by exerting an influence on the deep-to-superficial "wave" of neuronal differentiation in sequentially developing subplate and cortical layers VI, V and IV.

Transcellular retrograde labeling of radial glial cells with WGA-HRP and DiI in neonatal rat and hamster.

Topographically distinct populations of radial glial cells in the diencephalon and mesencephalon of neonatal rats and hamsters were transcellularly labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and with the lipophilic tracer DiI. A comparison of the histological distribution of the two tracers is suggestive of two different mechanisms of transcellular labeling. Intraocular injections of WGA-HRP resulted in the uptake of exogenously applied WGA-HRP by retinal ganglion cells, followed by anterograde axonal transport and exocytosis within the optic target nuclei. In addition to the transneuronal labeling, which is typical of such injections, we observed the transcellular labeling of the processes and somata of radial glial cells that were topographically associated with the terminal fields of the labeled axons. Similar transcellular labeling of radial glial cells associated with the axon terminal fields of the colliculogeniculate projection to the medial geniculate nucleus was observed following injections of WGA-HRP in the inferior colliculus. The transcellular labeling within the radial glial cells was discontinuous and somatopetally concentrated, indicating the existence of a retrograde active transport mechanism within the radial glial processes subsequent to its uptake following release of tracer from axons. This type of labeling can be referred to as transcellular retrograde glioplasmic transport. In contrast, DiI was used as a tracer through its capacity to diffuse within the plasmalemma. Topographically distinct populations of radial glial cells were transcellularly labeled following placements of DiI in the retina, inferior colliculus, or dorsal thalamus of fixed brains. The radial processes of labeled radial glial cells consistently extended into regions that also contained labeled axons. It is likely that the transcellular radial glial labeling with DiI occurred via transmembranous diffusion. These data indicate that a close structural and functional relation exists between axons and glial cells in the developing brain.

Development of cholinesterase histochemical staining in cerebellar cortex: transient expression of "nonspecific" cholinesterase in Purkinje cells of the nodulus and uvula.

Patterns of "nonspecific" cholinesterase (ChE) and acetylcholinesterase (AChE) activity were studied in developing rat cerebellar cortex by enzyme histochemistry and light and electron microscopy. Three types of ChE histochemical reaction product were observed in cerebellar cortex: (i) ChE is found in capillary endothelium throughout the cerebellum. Capillary ChE staining is present by the time of birth and continues into adulthood. (ii) ChE is found in radial glial fibers and their parent cell bodies, the Golgi epithelial cells. Radial glial fiber staining is mot intense during the first 3 weeks of postnatal life. (iii) ChE is found in Purkinje cells of the nodulus and ventral uvula. No ChE staining of Purkinje cells was seen in other parts of the cerebellum. ChE staining of Purkinje cells appears to be transient, first appearing at Postnatal Day 2 (P2), reaching peak intensity at P7-9, and decreasing to adult levels by P16. AChE activity displays a pattern markedly different from ChE, with staining in deep cerebellar nuclei, in putative mossy fiber terminals, and in Golgi neurons of cerebellar cortex. No evidence was found for transient AChE staining in Purkinje cells in any part of the cerebellum. The function of transiently expressed ChE activity in developing Purkinje neurons is unknown, but may be related to reorganization of cerebellar cortical circuitry associated with growth of mossy fiber afferents.

Primary auditory cortex in the rat: transient expression of acetylcholinesterase activity in developing geniculocortical projections.

A characteristic pattern of acetylcholinesterase (AChE) activity is expressed transiently in primary auditory cortex (cortical area 41) of developing laboratory rats during early postnatal life. This AChE activity occurs as a dense plexus in cortical layer IV and the deep part of layer III. This transient band of AChE activity is first detected by histochemical techniques on postnatal day (P) 3, reaches peak intensity at approximately P8-10, and declines to form the adult pattern by P23. The ventral nucleus of the medial geniculate body of the thalamus also displays prominent, and transient, staining for AChE. This intense staining for AChE, found within neuronal somata and neuropil, is detected at the time of birth, reaches peak intensity around P8, and declines to adult levels by P16. The areal and laminar patterns of the transient band of AChE activity in temporal cortex correspond to the patterns of anterograde transneuronal labeling of geniculocortical terminals following injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the inferior colliculus. Placement of lesions that include the medial geniculate nucleus or the geniculocortical axons results in a marked decrease in AChE staining in thalamorecipient layers of auditory cortex. Placement of lesions that include the medial globus pallidus reduce AChE staining of some axons in temporal cortex of developing rats, but the dense band of AChE in layers III and IV remains. Placement of lesions in the inferior colliculus in newborn animals results in marked decrease in AChE staining in cells of the ipsilateral ventral medial geniculate nucleus and in ipsilateral auditory cortex of developing pups. These data indicate that transiently expressed AChE activity is characteristic of geniculocortical neurons, including their somata in the medial geniculate body and their terminal axons in primary auditory cortex. This AChE activity is expressed early in postnatal development, probably during the time when thalamocortical axons are proliferating in cortical layer IV and forming synaptic contacts with cortical neurons.

Relationships between patterns of acetylcholinesterase activity and geniculocortical terminal fields in developing and mature rat visual cortex.

Intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) result in anterograde transneuronal labeling of geniculocortical axon terminals in cortical area 17. In area 17 of developing rat pups, transported WGA-HRP occurs primarily in layer I and in a band that includes layer IV and deep layer III; this pattern is virtually identical to the laminar pattern of endogenous acetylcholinesterase (AChE) activity. In adult animals, transported WGA-HRP again is localized in layer I and in deep layer III and layer IV, but the endogenous AChE activity is found most prominently in deep layer IV and layer V. These results indicate that geniculocortical terminal fields are co-extensive with transient patterns of AChE activity in the developing rat, but not with the mature pattern of AChE in the adult.

Glutamate-immunoreactivity in the retina and optic tectum of goldfish.

Glutamate was immunohistochemically localized in the goldfish retina and tectum at the light and electron microscopic (E.M.) levels using double affinity purified antisera against glutaraldehyde conjugated L-glutamate. In retina, glutamate-immunoreactivity (Glu+) was observed in cone inner segments, cone pedicles, bipolar cells, a small number of amacrine cells and the majority of cells in the ganglion cell layer. The latter were shown to be ganglion cells by simultaneous retrograde labeling. Centrally, Glu+ was observed in axons in the optic nerve and tract, and in stratum opticum and stratum fibrosum et griseum superficialis (SFGS) of the tectum. The Glu+ in the optic pathway disappeared four days after optic denervation and was restored by regeneration without affecting the Glu+ of intrinsic tectal neurons. In tectum, Glu+ was also observed in torus longitudinalis granule cells, toral terminals in stratum marginale, some pyramidal neurons in the SFGS, multipolar and fusiform neurons in stratum griseum centrale, large multipolar and pyriform projection neurons in stratum album centrale, and many periventricular neurons. Glu+ was also localized within unidentified puncta throughout the tectum and within radially oriented dendrites of periventricular neurons. At the E.M. level, a variety of Glu+ terminals were observed. Glu+ toral terminals formed axospinous synapses with dendritic spines of pyramidal neurons. Ultrastructurally identifiable Glu+ putative optic terminals formed synapses with either Glu+ or Glu- dendritic profiles, and with Glu- vesicle-containing profiles, presumed to be GABAergic. These findings are consistent with the hypothesis that a number of intrinsic and projection neurons in the goldfish retinotectal system, including most ganglion cells, may use glutamate as a neurotransmitter.

The relationship between CNS metabolism and cytoarchitecture: a review of 14C-deoxyglucose studies with correlation to cytochrome oxidase histochemistry.

Since the inception of the 14C-deoxyglucose method and its extension to in vivo imaging of regional cerebral glucose metabolism in humans by positron emission tomography, uncertainty has persisted concerning the type of work to which regional metabolism is coupled, as well as the distribution of this work within the neuron. 14C-deoxyglucose studies indicate that functionally-coupled neural metabolism is more apparent in axon terminals and perhaps dendrites than neuronal perikarya. Moreover, it appears that most of the metabolism in axon terminals is accounted for by Na+-K+-ATPase activity. Nevertheless, cytochrome oxidase histochemistry reveals the presence of intensely reactive mitochondria in soma-dendrite regions opposite presynaptic axon terminals, thereby indicating that continuous temporal and spatial summation of postsynaptic graded potentials is associated with increased metabolism. While the situation concerning the relative postsynaptic metabolic prices of EPSP's and IPSP's remains uncertain, the presence of elevated levels of cytochrome oxidase activity within certain classes of presynaptic terminals indicates that active excitation and inhibition is associated with increases in presynaptic metabolism. This observation has been confirmed in 14C-deoxyglucose studies. Nevertheless, studies of neonatal hippocampus indicate that, before metabolic activity shifts to dendritic and telodendritic regions of electrophysiological activity, metabolism is high in somal foci of biosynthesis.

Laminar and sublaminar ultracytochemical localization of cytochrome oxidase in the optic tectum of normal goldfish.

Distinct laminae and sublaminae in the goldfish optic tectum exhibit substantial differences in cytochrome oxidase (C.O.) reactivity. To determine whether these differences are associated with differential reactivity of different neuronal profiles, each tectal sublamina was examined at the ultrastructural level following C.O. treatment. The greatest abundance of darkly reactive mitochondria was found in the optically innervated layers within both pre- and postsynaptic profiles in correspondence with the most intense staining of these layers at the light microscopic level. Many reactive mitochondria were localized within terminals that were presumed to be optic on the basis of cytological criteria or were shown to be optic by filling optic fibers with HRP and processing so as to simultaneously demonstrate both mitochondrial C.O. reactivity and HRP labeling. These optic terminals tended to differ from each other in size and level of reactivity. The largest terminals were located within sublamina d of the stratum fibrosum et griseum superficials (SFGSd), and these were the most intensely reactive and contained the greatest number of darkly reactive mitochondria. Medium-sized terminals were found within sublaminae SFGSa, SFGSb, and a and c of the stratum album centrale (SACa,c). These were also darkly reactive but contained fewer mitochondria. Other medium-to-small optic terminals were found in stratum opticum a and b (60a,b), SFGSb, SFGSc, and stratum griseum centrale c (SGCc). These typically contained fewer mitochondria that also tended to be relatively less reactive, although darkly reactive mitochondria were also present. We suggest that the metalbolic differences within optic terminals of different size and sublaminar stratification arise from different ganglion cell classes and that the different optic layers of tectum are functionally substratified. As expected, darkly reactive mitochondria were most abundant in th intensely stained sublaminae, which included the optic lamina SFGS and nonoptic sublamina SGCa, and they were found not only within optic terminals but also within dendrites, presynaptic dendrites, and nonoptic terminals as well. Glial processes tended to contain less reactive mitochondria. The most prominent of the nonoptic terminals were the large-diameter P1 terminals, which contained pleomorphic vesicles and formed symmetric (presumed inhibitory) synapses. In stratum marginale most of the darkly reactive mitochondria were localized within dendrites. In the rest of the tectal layers most of the darkly reactive mitochondria were found in both presynaptic terminals and dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminar histochemical and cytochemical localization of cytochrome oxidase in the goldfish retina and optic tectum in response to deafferentation and during regeneration.

Cytochrome oxidase (C.O.) histochemistry and cytochemistry were used to examine the effects of optic denervation and subsequent optic fiber regeneration on oxidative metabolism in the retina and optic tectum of the goldfish. In the tectum, there was a dramatic and rapid decrease in C.O. activity within the optic layers 3-4 days after contralateral eye removal or optic nerve crush. At the E.M. level this was correlated with an initial decrease in mitochondrial reactivity within optic terminals followed by the subsequent degradation of mitochondria and phagocytosis of optic terminals. By 1 month after optic nerve crush, the entire tectum was reinnervated. However, the normal dark reactivity of the stratum fibrosum et griseum superficialis (SFGS), the main optic innervation layer, was not restored until after 3-4 months postcrush. The normal intense reactivity of the large-diameter optic axons and terminals at the bottom of the SFGS required an even longer period, about 7-8 months, for full recovery. The delayed restoration of C.O. reactivity was not due to a delay in synaptogenesis or in mitochondrial accumulation within optic terminals but to a delay in the maturation of mitochondrial reactivity. Following regeneration, the normal sublaminar stratification of C.O. bands was reestablished, suggesting that metabolically distinct classes of optic fibers may reinnervate at their original sublaminae. By using a distinct and persistent C.O. reactive sublamina, a of stratum griseum centrale (SGCa), just subjacent to the SFGS, it was possible to measure the thickness of the SFGS following optic denervation and subsequent reinnervation. At 1 week after optic nerve crush, the SFGS shrank by 35%. During regeneration, the thickness of the SFGS gradually increased to about 23% above normal at 2 months postcrush and this was maintained indefinitely. In the retina, ganglion cells were hypertrophic by 1 month postcrush and exhibited elevated levels of C.O. during the same period of time when optic terminals were unreactive. This indicates that oxidative metabolic activity within perikarya and axon terminals of the same neuron may be locally and independently regulated. It also suggests that in spite of the well-known elevation of axonal transport during the initial period of axon elongation and synaptogenesis, that oxidative metabolic energy production within the optic fibers is less than that of the mature projection.(ABSTRACT TRUNCATED AT 400 WORDS)

Histochemical localization of cytochrome oxidase in the retina and optic tectum of normal goldfish: a combined cytochrome oxidase-horseradish peroxidase study.

Cytochrome oxidase (C.O.) was histochemically localized in the normal retina and optic tectum of goldfish in order to examine the laminar and cellular oxidative metabolic organization of these structures. In the optic tectum, C.O. exhibited a distinct laminar, regional, and cellular distribution. The laminae with highest C.O. levels were those that receive optic input, suggesting a dominant role for visual activity in tectal function. This was demonstrated by colocalizing C.O. and HRP-filled optic fibers in the same section. However, the distribution of C.O. within the optic laminae was not uniform. Within the main optic layers, the SFGS, four metabolically distinct sublaminae were distinguished and designated from superficial to deep as sublaminae a, b, c, and d. The most intense reactivity was localized within SFGSa and SFGSd, followed by SFGSb, then SFGSc. In SFGSd, intense reactivity was found to occur specifically within a class of large diameter axons and terminals that were apparently optic since these were also labeled with HRP and cobaltous lysine applied to the optic nerve. Regional C.O. differences across the tectum were also noted. Low levels were found in neurons and optic terminals along the growing immature medial, lateral, and posterior edges of tectum, but were higher at the more mature anterior pole and central regions of tectum. This suggests that the oxidative metabolic activity is initially low in newly formed tectal neurons and optic axons, but gradually increases with neuronal growth and functional axon terminal maturation. Most C.O. staining was localized within neuropil, whereas the perikarya of most tectal neurons were only lightly reactive. Only a few neuron classes, mostly the relatively larger projection neurons, had darkly reactive perikarya. In the retina, intense C.O. reactivity was localized within the inner segments of photoreceptors, the inner and outer plexiform layers, and within certain classes of bipolar and ganglion cells. The large ganglion cells in particular were intensely reactive. Like the large diameter optic terminals in SFGSd, the large ganglion cells were preferentially filled with HRP, suggesting that they may project to tectum and are the source of the darkly reactive large diameter axons and terminals in sublamina SFGSd. We propose a new scheme to describe tectal lamination that integrates laminar differences in C.O. reactivity with classical histological work.(ABSTRACT TRUNCATED AT 400 WORDS)

Dense HRP filling in pre-fixed brain tissue for light and electron microscopy.

The use of neuroanatomical markers in tissues that have been pre-fixed has been virtually ignored, even though this approach could offer certain advantages over in vivo methods, in terms of convenience of application and choice of markers. We have found that HRP can be used on well-fixed brains of cats and goldfish to fill neurons, dendrites, axons, terminals, glial cells, and glial processes for high-resolution light microscopy and electron microscopy. Best results were obtained using brains that were perfusion-fixed with 2.5% depolymerized paraformaldehyde and 1.5% glutaraldehyde. Two methods of HRP application were used: optically guided injections of microliter quantities into various regions of cat brain, and optic nerve fills in goldfish by attaching an HRP-filled polyethylene tube for periods of 1 day to 2 weeks. HRP applied in these ways to pre-fixed tissue was found to fill neurons or glial cells with solid label in the anterograde and retrograde directions.

Differential effect of visual deprivation on cytochrome oxidase levels in major cell classes of the cat LGN.

Cytochrome oxidase histochemistry was used to examine the effects of visual deprivation on the development of neurons in the lateral geniculate nucleus of the kitten. Early postnatal monocular suture results in a decrease in reactivity within the neuropil of visually deprived binocular laminae A, A1, magnocellular C, and medial interlaminar nucleus. Within these regions, monocular suture has a greater effect on the relative numbers of, and the growth of darkly reactive (normally large), presumed Y-cells than on other less reactive geniculate neuronal classes. The decreases in the reactivity of the neuropil may be attributed to the decreases in the number of mitochondria, the number of darkly reactive mitochondria, and/or the number of darkly reactive mitochondria localized within dendrites. Although all classes of dendrites appear to be adversely affected, the decrease in C.O. reactivity was most dramatic within the presumed proximal dendrites of class 1 Y-cells. These dendrites were identified by the type of synaptic contacts they formed with retinal terminals (Rapisardi and Miles, '84, J. Comp. Neurol. 223:515-534; Wilson et al., '84, Proc. R. Soc. Lond. [Biol.] 221:411-436). As with Y-cells, the effects of monocular suture on the large darkly reactive cells were not as dramatic at sites where binocular interactions were either absent or where they had been experimentally eliminated. Based on the present and previously reported findings from several laboratories, it is likely that the selective physiological and morphological effects of monocular suture on Y-cells are accompanied by metabolic deficits involving both dendrites and perikarya. These effects appear to be due more to binocular interactions than to visual deprivation per se.

Localization of cytochrome oxidase in the mammalian spinal cord and dorsal root ganglia, with quantitative analysis of ventral horn cells in monkeys.

The spinal cord and dorsal root ganglia of mice, rats, cats, squirrel monkeys, and macaque monkeys were examined at both the light and electron microscopic levels for cytochrome oxidase activity. A similar histochemical pattern prevailed in all of the species examined. While the spinal gray exhibited a heterogeneous but consistent distribution of the enzyme, the white matter was only lightly stained. Highly reactive neurons were either singly scattered or aggregated into discrete clusters. The dorsal nucleus of Clarke, the lateral cervical nucleus (cat), the intermediolateral cell columns of the thoracic and upper lumbar levels, and selected groups of ventral horn neurons formed moderate to darkly reactive cell clusters, whereas fusiform and multipolar cells of Waldeyer in the marginal layer, small fusiform neurons in the ventral gray, funicular cells in the white matter, and ventral horn neurons of varying sizes tended to stand out against the neuropil as singly reactive neurons. At the electron microscopic level, reactive neurons were characterized by a greater packing density of darkly reactive mitochondria, while lightly reactive ones had fewer mitochondria, most of which showed very little reaction product. Reactive mitochondria were also found in the neuropil, mainly in dendritic profiles and some axon terminals. Glial cells, in general, were not very reactive. Ventral horn neurons from three macaque monkeys were measured for somatic areas and optical densities of cytochrome oxidase reaction product. A total of 1,770 neurons from representative sections of the cervical, thoracic, lumbar, and sacral cords of these animals were analyzed. The results indicated that the distribution of cell sizes as well as optical densities at every level of the cord fell on a continuum. Analysis of the regression coefficients revealed that the slopes were negative for all levels, indicating that there was a general inverse relationship between cell size and optical densities. However, there were representations of dark, moderate, and lightly reactive neurons in all three size categories (large, medium, and small). Thus, the level of oxidative metabolism of ventral horn neurons cannot be correlated strictly with size, but it is likely to reflect their total synaptic and spontaneous activities. Neurons of the dorsal root ganglia likewise exhibited heterogeneous distribution of cell sizes and levels of enzyme reactivity, while satellite cells, in general, were only lightly reactive. As in the case of the ventral horn, representatives of dark, moderate, and light levels of reactivity occurred in every size category of neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminar and cellular localization of cytochrome oxidase in the cat striate cortex.

Cytochrome oxidase (C.O.) was histochemically localized in the cat striate cortex at the light and electron microscopic levels. The results indicate that the oxidative metabolic activity within the cat striate cortex may vary between (1) different laminae, (2) neurons and glia, (3) different neuron types, (4) dendrite and soma of the same cell, (5) different types of dendrites, (6) different segments of the same dendrite, and (7) different classes of symmetric and asymmetric axon terminals. Maximal laminar C.O. staining was localized within geniculoreceptive layer IV. Darkly reactive neurons include the large (presumed corticotectal) pyramids of layer V, and various classes of large and medium-sized presumed GABAergic nonpyramidal cells sparsely distributed throughout layers II-VI. The small and medium-sized pyramids of layers II, III, V, and VI, as well as many of the smaller presumed GABAergic neurons, were only lightly or moderately reactive. The darkly reactive neurons tended to be those that received convergent or proximally localized asymmetric axosomatic synapses, implying that they are strongly driven by excitatory synaptic input. The darkly reactive nonpyramids resembled those that form GAD+, symmetric axosomatic synapses with pyramidal cells. The dark reactivity of the symmetric synaptic terminals indicates that they mediate strong inhibition of neuronal discharge. The dark reactivity of a class of large asymmetric terminals in layer IV is likely to represent highly active geniculocortical terminals. The predominant distribution of elevated C.O. reactivity in dendrites is correlated with reported sites of (1) convergent excitatory synaptic input, (2) maximal field potentials, (3) highly active ion transport, and (4) Na+, K+-ATPase.