Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.Key Words: motor cortex, neuronal transplantation, embryonic cells, GFP, GFAP, PLP     

Expression of the Axonal Membrane Glycoprotein M6a Is Regulated by Chronic Stress

It has been repeatedly shown that chronic stress changes dendrites, spines and modulates expression of synaptic molecules. These effects all may impair information transfer between neurons. The present study shows that chronic stress also regulates expression of M6a, a glycoprotein which is localised in axonal membranes. We have previously demonstrated that M6a is a component of glutamatergic axons. The present data reveal that it is the splice variant M6a-Ib, not M6a-Ia, which is strongly expressed in the brain. Chronic stress in male rats (3 weeks daily restraint) has regional effects: quantitative in situ hybridization demonstrated that M6a-Ib mRNA in dentate gyrus granule neurons and in CA3 pyramidal neurons is downregulated, whereas M6a-Ib mRNA in the medial prefrontal cortex is upregulated by chronic stress. This is the first study showing that expression of an axonal membrane molecule is differentially affected by stress in a region-dependent manner. Therefore, one may speculate that diminished expression of the glycoprotein in the hippocampus leads to altered output in the corresponding cortical projection areas. Enhanced M6a-Ib expression in the medial prefrontal cortex (in areas prelimbic and infralimbic cortex) might be interpreted as a compensatory mechanism in response to changes in axonal projections from the hippocampus. Our findings provide evidence that in addition to alterations in dendrites and spines chronic stress also changes the integrity of axons and may thus impair information transfer even between distant brain regions.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.     

Analysis of conduction of visual,somatic, and audiovibratory sensory information to the hippocamapal cortex in lizards

Acute electrophysiological experiments on lizards (Ophisaurus apodus) showed that electrical stimulation of the anterior dorsolateral thalamic nucleus and medial forebrain bundle evokes short-latency responses in the hippocampal (mediodorsal) cortex which coincides in distribution and configuration with responses in the same cortical area to sensory stimulation. Extensive destruction of these structures inhibits, or even completely blocks, the conduction of sensory (visual, somatic, audiovibratory) and tactile impulses to the hippocampal cortex. It is concluded that the anterior dorsolateral thalamic nucleus and medial forebrain bundle constitutes, if not the only, at least the principal pathway for transmission of these sensory impulses to the hippocampal cortex in lizards.     

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.     

lntracortical Connections in the Snakes Natrix sipedon and Thamnophis sirtalis

The association and commissural connections between the four cortical areas in water (Natrix sipedon) and garter (Thamnophis sirtalis) snakes were studied by placing lesions on the cortical surface and studying the resulting degeneration in Fink-Heimer preparations. Lateral cortex projects to the outer one third of layer 1 of ipsilateral medial cortex. Dorsal cortex projects to the middle third of layer 1 of ipsilateral medial cortex. Dorsomedial cortex projects bilaterally to the inner third of layer 1 and to layer 3 of me dial cortex. It also projects to layer 1 of contralateral dorsomedial cortex. Medial cortex projects ipsilaterally to each of the other cortical areas. With the apparent exception of the projection of medial cortex to lateral cortex, each projection is organized such that each rostrocaudal segment of a cortical area projects to all segments of the target area lying at the same or more caudal levels.     

Interhemispheric connections through the pallial commissures in the brain of Podarcis hispanica and Gallotia stehlinii (Reptilia,Lacertidae)

The cells-of-origin and the mode and site of termination of the interhemispheric connections passing through the anterior and posterior pallial commissures in the telencephalon of two lizards (Podarcis hispanica and Gallotia stehlinii) were investigated by studying the anterograde and retrograde transport of unilaterally injected horseradish peroxidase. The commissural projections arise mainly from pyramidal cells in the medial, dorsomedial, and dorsal cortices (medial subfield). Additionally some non-pyramidal neurons in the medial and dorsal cortices contribute to the commissural system. Medial cortex neurons project to the contralateral anterior septum through the anterior pallial commissure. The dorsomedial cortex projects contralaterally via the anterior pallial commissure to the dorsolateral septum and to the medial, dorsomedial, and dorsal cortices. The projection to the medial cortex terminates in two bands at the inner and outer border, respectively, of the cell layer; the projection to the dorsomedial and dorsal cortex ends in a zone in layer 1 which previously has been described to be Timm-negative, and in a diffuse band in the inner half of layer 3. The medial subfield of the dorsal cortex projects through the anterior pallial commissure to the dorsomedial and dorsal cortices with a similar pattern of termination to that found for the dorsomedial cortex. The posterior pallial commissure contains only the projections from the ventral cortex to its contralateral counterpart and to the ventral part of the caudal medial cortex. The similarities found between this commissural system and the mammalian hippocampal interhemispheric connections are discussed.     

Neuronal organization of the periamygdaloid cortex in the cat brain

Neuronal organization of the fields Pmm, Pml2, Pe and epm of the periamygdaloid cortex of the cat brain has been studied by means of Golgi and Nissl methods. The field Pmm essentially differs from other fields of this cortex by primitiveness of its cytoarchitectonic an neuronal organization (two layers uniform by the composition of their neurons are distinguished, the structure of the latter is relatively primitive). In the medial part of this field long axonal rarely branching short dendritic, and in the lateral part--poorly differentiating pyramidal and spindle-like cells predominate. The field Pmm can be considered as a transitional formation between the subcortex (the medial nucleus of the amygdaloid body) and other fields of the periamygdaloid cortex. The fields Pml2, Pe and epm are built more complexly: the cells are organized in 4 layers, more complexly differentiated by their form and size than in the field Pmm and correspondingly more various (long axonal densely branching cells are observed: pyramidal and spindle-like--of the cortical type and bushy--of the subcortical type, as well as long axonal rarely branching reticular cells). The short axonal cells in the fields Pml2, Pe and epm are rather variable in their form, size and direction of axons; in the field Pmm they are less numerous. The field Pmm and the complex of the fields Pml2, Pe and epm are perhaps different in their function, this is evident from different projection of their neurons. Axons of the cells in the field Pmm get into less differentiated and the most ancient medial nucleus of the amygdaloid body and into the ancient system of connections of the latter--terminal strip, and neurons of the fields Pml2, Pe and epm are projected into the basolateral part of the amygdaloid body and into the external capsule--phylogenetically younger structures. Besides, poverty of the axonal collateralies in the long axonal neurons and a small amount and uniformity of the forms of the short axonal cells in the field Pmm and contrary, rich collateralies and variety of short axonal cells in the composition of other fields demonstrate more complex internal integrative function, performing in that composition.     

Characterization of factors regulating lamina-specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.     

Regeneration of Identified Neurons and Their Synaptic Connections in the Central Nervous System of Aplysia

SYNOPSIS. In the CNS of Aplysia the number and sequence of lesionsinfluence the regeneration of identified neurons and their synapticconnections The left pleural ganglion giant neuron (LGC) failsto regenerate its severed axon following crushes of the cerebropleural (C-PL) connectses A conditioning lesion paradigm inwhich first a foot nerve and then 10 days later, the left C-PLconnective is crushed, results in the LGC regenerating its axonSynaptic inputs from identified neurons in the cerebral ganglionrarely regenerate when only the C-PL connectives are crushedConditioning lesions also result in the regeneration of synapticconnections from the cerebral A neurons Multiple and sequentiallesions appear to be more effective than single lesions in promotingboth axonal regeneration and synapse formation The possiblemechanisms by which axonal regeneration and synaptogenesis arecontrolled are discussed.     

The emergence of somatotopic maps of the body in S1 in rats: the correspondence between functional and anatomical organization

Most of what we know about cortical map development and plasticity comes from studies in mice and rats, and for the somatosensory cortex, almost exclusively from the whisker-dominated posteromedial barrel fields. Whiskers are the main effector organs of mice and rats, and their representation in cortex and subcortical pathways is a highly derived feature of murine rodents. This specialized anatomical organization may therefore not be representative of somatosensory cortex in general, especially for species that utilize other body parts as their main effector organs, like the hands of primates. For these reasons, we examined the emergence of whole body maps in developing rats using electrophysiological recording techniques. In P5, P10, P15, P20 and adult rats, multiple recordings were made in the medial portion of S1 in each animal. Subsequently, these functional maps were related to anatomical parcellations of S1 based on a variety of histological stains. We found that at early postnatal ages (P5) medial S1 was composed almost exclusively of the representation of the vibrissae. At P10, other body part representations including the hindlimb and forelimb were present, although these were not topographically organized. By P15, a clear topographic organization began to emerge coincident with a reduction in receptive field size. By P20, body maps were adult-like. This study is the first to describe how topography of the body develops in S1 in any mammal. It indicates that anatomical parcellations and functional maps are initially incongruent but become tightly coupled by P15. Finally, because anatomical and functional specificity of developing barrel cortex appears much earlier in postnatal life than the rest of the body, the entire primary somatosensory cortex should be considered when studying general topographic map formation in development.     

Cholecystokinin from the entorhinal cortex enables neural plasticity in the auditory cortex

Patients with damage to the medial temporal lobe show deficits in forming new declarative memories but can still recall older memories, suggesting that the medial temporal lobe is necessary for encoding memories in the neocortex. Here, we found that cortical projection neurons in the perirhinal and entorhinal cortices were mostly immunopositive for cholecystokinin (CCK). Local infusion of CCK in the auditory cortex of anesthetized rats induced plastic changes that enabled cortical neurons to potentiate their responses or to start responding to an auditory stimulus that was paired with a tone that robustly triggered action potentials. CCK infusion also enabled auditory neurons to start responding to a light stimulus that was paired with a noise burst. In vivo intracellular recordings in the auditory cortex showed that synaptic strength was potentiated after two pairings of presynaptic and postsynaptic activity in the presence of CCK. Infusion of a CCK_B antagonist in the auditory cortex prevented the formation of a visuo-auditory association in awake rats. Finally, activation of the entorhinal cortex potentiated neuronal responses in the auditory cortex, which was suppressed by infusion of a CCK_B antagonist. Together, these findings suggest that the medial temporal lobe influences neocortical plasticity via CCK-positive cortical projection neurons in the entorhinal cortex.     

Cytoarchitecture and connectivity of the amphibian medial pallium.

We investigated the cytoarchitecture and connectivity of the medial pallium of amphibians by intracellular recording and biocytin labeling. The experiments were carried out in a whole-brain in vitro preparation in the painted frog, Discoglossus pictus. Four types of neurons with specific axonal projection patterns and position in the medial pallium are distinguished, three types with extratelencephalic and one type with only intratelencephalic projections. Our findings corroborate the assumption that the anuran medial pallium is homologous to the subiculum and Ammon's horn of the mammalian hippocampus at a gross level, while the specific axonal projection patterns differ. Due to the absence of hippocampal neurons with only intrinsic projections, there seems to be no portion homologous to the dentate gyrus.     

Dopamine antagonist speeds up tail regeneration in lizards exposed to continuous darkness: evidence for prolactin involvement

In earlier studies, we demonstrated that continuous light (LL:LD, 24:0) stimulated tail regeneration whereas continuous darkness (DD:LD, 0:24) and pinealectomy depressed the same in the Gekkonid lizard, Hemidactylus flaviviridis, and, furthermore, exogenous prolactin significantly enhanced the regeneration process in lizards kept in 0:24 LD. However, the regeneration process in animals exposed to 24:0 LD was unaffected by the dopamine agonist, bromocriptine. This study with pimozide, an antipsychotic drug, and a potent dopamine receptor antagonist was conducted to ascertain whether the dopaminergic regulation of prolactin release is operative in lizards, as in mammals, and to provide further evidence for prolactin involvement in regenerative growth. Once daily intraperitoneal injection of 50 micrograms/kg pimozide to H. flaviviridis, 5 days prior to tail autotomy and 50 days thereafter, stimulated the regeneration process in lizards exposed to 0:24 LD. The initiation of regeneration, the total length of new growth (regenerate) produced by Day 50, and the total percentage replacement of the lost (autotomized) tails at the end of 50 days of experimentation were all significantly enhanced in pimozide-treated animals as compared with their counterparts injected with 0.6% sterile saline; in fact, better than saline-injected controls exposed to 24:0 LD of 638 lux intensity. The daily growth rate was also enhanced in pimozide-treated lizards. Interestingly, the pattern of regeneration as well as the final regenerate of pimozide-treated lizards were similar to those observed earlier in ovine prolactin-treated animals exposed to similar experimental photoperiodic schedules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tail regeneration in the lizards Anguis fragilis and Lacerta dugesii

Rates of tail regeneration in the Madeira wall lizard ( Lacerta dugesii ) and the slow-worm ( Anguis fragilis ) were studied.
L. dugesii regenerates very rapidly, the new tail sometimes attaining a maximum rate of growth of 2'6 mm a day during the fifth week after autotomy. By the twelfth week 90% of the original tail length has been replaced. Average regeneration rates of samples of lizards were reduced after repeated autotomies, but our investigation of this problem was probably complicated by another factor, the amount of tail lost, and is inconclusive.
The tip of the regenerate grows more rapidly than the rest; no elongation occurs at its cranial aspect.
Anguis , even when kept at 27°C, regenerates its tail very slowly, the best performance observed being a new tail of 5 mm after 14 weeks. The longest natural regenerate seen (16 mm) may have taken several years to produce in the wild.
The histological features of regeneration in Anguis are basically similar to those in other lizards. The new osteoderms are formed entirely in the subepidermal tissues but have a regular relationship with the scales. Some nerve fibres are regenerated with the ependymal tube.
The scales on the lizard's regenerating tail develop in a different manner from those in the lizard embryo and show suggestive resemblances to mammalian hairs.     

A Direct Projection from Mouse Primary Visual Cortex to Dorsomedial Striatum

The mammalian striatum receives inputs from many cortical areas, but the existence of a direct axonal projection from the primary visual cortex (V1) is controversial. In this study we use anterograde and retrograde tracing techniques to demonstrate that V1 directly innervates a topographically defined longitudinal strip of dorsomedial striatum in mice. We find that this projection forms functional excitatory synapses with direct and indirect pathway striatal projection neurons (SPNs) and engages feed-forward inhibition onto these cells. Importantly, stimulation of V1 afferents is sufficient to evoke phasic firing in SPNs. These findings therefore identify a striatal region that is functionally innervated by V1 and suggest that early visual processing may play an important role in striatal-based behaviors.     

Facts and reflections on thalamo cortical reverberation of impulses

Experiments on cats, either unanesthetized or anesthetized with various doses of pentobarbital, showed that the cortical rhythmic after-discharge ("slow after-activity"), which has been regarded as a manifestation of reverberation of impulses in thalamocortical circuits [17], consists of a burst of spontaneous "spindles" evoked by stimulation. This conclusion is supported by the following facts: Spontaneous "spindles" and the rhythmic after-discharge respond absolutely identically (disappear) to activation of the EEG and deepening of pentobarbital anesthesia. The absence of thalamocortical reverberation is also indicated by the preservation of a rhythmic after-discharge (to clicks), synchronous with the cortex, in the thalamic relay nucleus (the medial geniculate body) after cooling or after removal of its projection zone.     

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

Healing of "wire tunnels" in tibial diaphysis: data of local X-ray study and densitometry

Fifteen pelvic extremities from adult dogs were used to perform 60 cross tunnelization of tibial diaphysis with a wire having a pointed groove, 1.8-mm in diameter, at 1,500 rpm. Thirty five leg X-ray films in the lateral projection, which show changes on days 30, 60, 90, 120, 520 after the experiment, were studied. Bony diaphysis tunnelization was found to give rise to a regeneration focus wherein an area of tissue element degradation and an area of introduction of bony fragments into soft tissues were detectable. Changes in the regeneration focus were determined by a response to osseous fragments, which involved the formation of an bony regenerate during a month and to the resorption of the bony matter of an osseous regenerate and osseous fragments in future. Healing of diaphysis defects began with the resorption of a damaged cortical layer. Osteogenesis prevailed at month 2 and ensured healing of cortical layer defects following 4-6 months.     

Network structure implied by initial axon outgrowth in rodent cortex: empirical measurement and models

The developmental mechanisms by which the network organization of the adult cortex is established are incompletely understood. Here we report on empirical data on the development of connections in hamster isocortex and use these data to parameterize a network model of early cortical connectivity. Using anterograde tracers at a series of postnatal ages, we investigate the growth of connections in the early cortical sheet and systematically map initial axon extension from sites in anterior (motor), middle (somatosensory) and posterior (visual) cortex. As a general rule, developing axons extend from all sites to cover relatively large portions of the cortical field that include multiple cortical areas. From all sites, outgrowth is anisotropic, covering a greater distance along the medial/lateral axis than along the anterior/posterior axis. These observations are summarized as 2-dimensional probability distributions of axon terminal sites over the cortical sheet. Our network model consists of nodes, representing parcels of cortex, embedded in 2-dimensional space. Network nodes are connected via directed edges, representing axons, drawn according to the empirically derived anisotropic probability distribution. The networks generated are described by a number of graph theoretic measurements including graph efficiency, node betweenness centrality and average shortest path length. To determine if connectional anisotropy helps reduce the total volume occupied by axons, we define and measure a simple metric for the extra volume required by axons crossing. We investigate the impact of different levels of anisotropy on network structure and volume. The empirically observed level of anisotropy suggests a good trade-off between volume reduction and maintenance of both network efficiency and robustness. Future work will test the model's predictions for connectivity in larger cortices to gain insight into how the regulation of axonal outgrowth may have evolved to achieve efficient and economical connectivity in larger brains.