carbocyanines and biocytin

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Topics: Aged; Aged, 80 and over; Autopsy; Axons; Carbocyanines; Cell Body; Dendrites; gamma-Aminobutyric Acid; Glutamic Acid; Humans; Hypothalamus; Imaging, Three-Dimensional; Kisspeptins; Lysine; Male; Microscopy, Confocal; Microscopy, Immunoelectron; Middle Aged; Nerve Net; Neurons; Synapses; Vesicular Glutamate Transport Protein 2; Vesicular Inhibitory Amino Acid Transport Proteins

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole-cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction-permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin-Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47-deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47-double-deficient mice. Cx47-ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43-deficient animals, oligodendrocyte-astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30-double deficient mice, oligodendrocyte-to-astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Antigens; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Carbocyanines; Connexin 30; Connexins; Corpus Callosum; Gap Junction beta-1 Protein; Gap Junctions; Glial Fibrillary Acidic Protein; In Vitro Techniques; Lysine; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Patch-Clamp Techniques; Proteoglycans; Stem Cells; Streptavidin

The absence of a slice preparation with intact thalamocortical pathways has held back elucidation of the cellular and synaptic mechanisms by which thalamic signals are differentially transmitted to and processed in the anterior cingulate cortex (ACC). In this report we introduce an innovative mouse brain slice preparation in which it is possible to explore the electrophysiological properties of ACC neurons with intact long-distance inputs from medial thalamic (MT) nuclei by intracellular recordings; this MT-ACC neuronal pathway plays an integral role in information transmission. Biocytin-labeled fibers in a functional slice could be traced anterogradely or retrogradely from the MT via the reticular thalamic nuclei, striatum and corpus callosum to the cingulate cortical areas. Eighty-seven cells downstream of the thalamic projections in 49 slices were recorded intracellularly. Intracellular recordings in the ACC showed that thalamocingulate transmission involves both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors. Thalamus-evoked responses recorded extracellularly in the ACC were activated and progressed along a deep-superficial-deep trajectory loop across the ACC layers. We observed enhanced paired-pulse facilitation and tetanic potentiation of thalamocingulate synapses, suggestive of input-specific ACC plasticity and selective processing of information relayed by thalamocingulate pathways. Furthermore, we observed differential responses of ACC neurons to thalamic burst stimulation, which underscores the importance of MT afferents in relaying sensory information to the ACC. This new slice preparation enables the contribution of MT-evoked ACC synaptic transmission to short-term plasticity in the neuronal circuitry underlying sensory information processing to be examined in detail.

Topics: Action Potentials; Animals; Brain Mapping; Carbocyanines; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Gyrus Cinguli; Lysine; Male; Mice; Mice, Inbred C57BL; Neural Pathways; Neuronal Plasticity; Neurons; Organ Culture Techniques; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission; Thalamus

The superior colliculus (SC) transforms both visual and nonvisual sensory signals into motor commands that control orienting behavior. Although the afferent and efferent connections of this midbrain nucleus have been well characterized, little is know about the intrinsic circuitry involved in sensorimotor integration. Transmission of visual signals from the superficial (sSC) to the deeper layers (dSC) of the SC has been implicated in both the triggering of orienting movements and the activity-dependent processes that align maps of different sensory modalities during development. However, evidence for the synaptic connectivity appropriate for these functions is lacking. In this study, we used a variety of anatomical and physiological methods to examine the functional organization of the sSC-dSC pathway in juvenile and adult ferrets. Axonal tracing in adult ferrets showed that, as in other species, sSC neurons project topographically to the dSC, providing a route for the transmission of visual signals to the multisensory output layers of the SC. We found that sSC axons terminate on dSC neurons that stain prominently for the NR1 subunit of the NMDA receptor, a subpopulation of which were identified as tectoreticulospinal projection neurons. We also show that the sSC-dSC pathway is topographically organized and mediated by monosynaptic excitatory synapses even before eye opening in young ferrets, suggesting that visual signals routed via the sSC may influence the activity of dSC neurons before the emergence of their multisensory response properties. These findings indicate that superficial- to deep-layer projections provide spatially ordered visual signals, both during development and into adulthood, directly to SC neurons that are involved in coordinating sensory inputs with motor outputs.

Topics: Age Factors; Animals; Animals, Newborn; Biotin; Carbocyanines; Coloring Agents; Dextrans; Electric Stimulation; Excitatory Postsynaptic Potentials; Ferrets; In Vitro Techniques; Lysine; Neurons; Patch-Clamp Techniques; Psychomotor Performance; Reaction Time; Receptors, N-Methyl-D-Aspartate; Superior Colliculi; Synapses; Synaptic Transmission; Visual Pathways

Physiological studies have demonstrated a subcortical origin for orientation selectivity and the orientation columns of the primary visual cortex. However, there are no anatomical data showing how subcortical cells contribute to this important property. Optical imaging, combined with 1,1'-dioctadecyl-3,3,3,3'-tetramethylin-docarbocyanine perchlolate (DiI) and biocytin retrograde tracing, reveals that relay cells projecting to a single orientation column representing the horizontal meridian were clustered within 300 microm in the dorsal lateral geniculate nucleus (LGN). Interestingly, some labeled cells were located on a line parallel to an iso-elevation line in the LGN. Thus, according to the quantitative projection of the visual field to the LGN (J. Comp. Neurol. 143 (1971) 101), their receptive fields must distribute horizontally in alignment in the visual field providing the first anatomical evidence for Hubel and Wiesel's model of simple cell receptive fields (J. Physiol. 160 (1962) 106).

Topics: Action Potentials; Animals; Brain Mapping; Carbocyanines; Cats; Fluorescent Dyes; Geniculate Bodies; Lysine; Neurons; Orientation; Pattern Recognition, Visual; Photic Stimulation; Visual Cortex; Visual Fields; Visual Pathways

Synchronous oscillations in olfactory systems have been thought to play critical roles in encoding olfactory information. However, their role in determining behavior is unknown. As a first step toward understanding the decoding process of coherent oscillation, we looked for a neuron in the terrestrial slug Limax marginatus that receives output signals from the procerebrum (PC), which is the olfactory center of Limax. We identified a neuron in the metacerebrum that extends its neurites into both the PC and the metacerebrum, and named it the metacerebro-procerebral neuron (MPN). The MPN exhibited a membrane potential oscillation that was synchronous with the local field potential oscillation in the PC. When we cut the PC off, the membrane potential oscillation of the MPN disappeared. Numerous varicosities were found on the neurites in the metacerebrum, while no varicosities were found on the neurites inside the PC. From these morphological and physiological results, we conclude that the MPN is an output neuron from the PC. The MPN also receives monosynaptic inputs from the superior and inferior tentacle nerves. The MPN thus may receive olfactory information from two pathways, one directly from the sensory organ and the other by way of the PC, possibly functioning to integrate them.

Topics: Animals; Biological Clocks; Carbocyanines; Central Nervous System; Electric Stimulation; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Ganglia, Invertebrate; Glutamic Acid; Lysine; Membrane Potentials; Mollusca; Neurites; Neurons; Olfactory Pathways; Presynaptic Terminals; Reaction Time; Smell; Synaptic Transmission

The hypothalamus and perhaps its function appear to be similar among vertebrates. Thus, studying the teleostean hypothalamus could be a good model for understanding common neural circuits and mechanisms retained through the vertebrates. However, connections of the inferior lobe, which is considered the hypothalamus in teleosts, is poorly known. The corpus mamillare (CM) is a nucleus of the inferior lobe named after the mammalian mamillary body based on similarities in external morphology. Afferent connections of the CM have been reported only in cypriniform teleosts. These include projections from the nucleus pretectalis superficialis pars magnocellularis, a nucleus lacking in percomorph teleosts, and projections from the secondary gustatory nucleus. Efferent connections of the CM have not been reported in teleosts. In the present study, the CM and its subdivisions and the connections of these subnuclei were identified in isolated and maintained brains of tilapia Oreochromis niloticus by local DiI and biocytin injection. Afferent connections confirmed by reciprocal injections were from the nucleus diffusus lobi inferioris (NDLI) and the nucleus diffusus tori lateralis (NDTL). Efferent connections of each CM subnuclei were also reciprocally confirmed. These connections were to the area dorsalis pars medialis of the telencephalon, the nucleus ventromedialis (NVM) of the thalamus, the tectum opticum (TO), and the nucleus posterioris periventricularis. Because the NDLI is known to receive gustatory information in tilapia, the CM could relay gustatory inputs to multisensory areas, the TO and NVM, for which there are no current reports regarding gustatory inputs.

Topics: Afferent Pathways; Animals; Biotin; Carbocyanines; Dextrans; Female; Fluorescent Dyes; Histocytochemistry; Hypothalamus; Lysine; Male; Mammillary Bodies; Nerve Fibers; Tilapia

Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.

Topics: Amidines; Animals; Animals, Newborn; Axonal Transport; Calbindin 2; Carbocyanines; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Coculture Techniques; Entorhinal Cortex; Extracellular Matrix Proteins; Fluorescent Dyes; Hippocampus; Lysine; Microscopy, Video; Nerve Tissue Proteins; Neural Pathways; Neurons; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reelin Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Calcium Binding Protein G; Serine Endopeptidases

The corticopontine projection develops exclusively by collateral branches that form along the length of corticospinal axons days after they have passed their hindbrain target, the basilar pons. In vitro evidence suggests that the basilar pons releases a diffusible activity that initiates and directs the growth of collateral branches. This study investigates whether contact-dependent mechanisms may also influence the formation of collateral branches. By using immunocytochemistry, electron microscopy, and neuronal tracing techniques, we examined the region of the axon tract, the cerebral peduncle, overlying the basilar pons for cellular structures that correlate spatially and temporally with collateral branch formation. We found that radial glia are excluded from the tract. Oligodendrocyte precursors are found only at low density. Although mature astrocytes are absent, immature astrocytes are present throughout the tract. However, our evidence does not suggest a direct role for glial cell types in collateral branch formation. In contrast, dendrites of basilar pontine neurons are transiently present in the tract during the time of collateral branch formation. Although collateral branches are observed in regions of the tract devoid of dendrites, the orientation and location of most collateral branches correlates at the light microscopic level with dendrites. Electron microscopy reveals sites of increased collateral branch formation near neuronal cell bodies or dendrites. However, cell processes, whether dendritic or otherwise, are rarely found in direct contact with collateral branch points. A common and unexpected feature is the bundles of corticopontine collateral branches, oriented transversely to their parent corticospinal axons and directed across the tract to the basilar pons. Dendrites were often apposed to or embedded within the transverse bundles. These findings suggest that dendrites are not essential for collateral branch formation but that they may enhance this process and define discrete preferred locations for collateral branch initiation and elongation within the cerebral peduncle.

Topics: Animals; Astrocytes; Axons; Carbocyanines; Cerebral Cortex; Dendrites; Female; Lysine; Microscopy, Electron; Neural Pathways; Neuroglia; Pons; Pregnancy; Rats; Rats, Sprague-Dawley

Central fiber connections of the gustatory system were examined in a percomorph fish Oreochromis (Tilapia) niloticus by means of the horseradish peroxidase (HRP), biocytin, and carbocyanine dye tracing methods. The primary gustatory areas in tilapia are the facial, glossopharyngeal, and vagal lobes of the medulla. The secondary gustatory nucleus (SGN) is a dumb-bell-shaped structure located in the isthmic region. In the SGN, there are two or three layers of neurons lining the ventromedial periphery of the nucleus and a molecular layer constituting of the major part of the nucleus. The SGN receives bilateral projections from the facial lobes and ipsilateral projections from the glossopharyngeal and vagal lobes. Ascending fibers originating from the SGN form the ipsilateral tertiary gustatory tract. A major part of the tract courses rostrally and terminates ipsilaterally in several diencephalic nuclei: the preglomerular tertiary gustatory nucleus (pTGN), the posterior thalamic nucleus, the nucleus diffusus lobi inferioris, the nucleus centralis of inferior lobe, and the nucleus recessus lateralis. The remaining small fiber bundle enters the medial and lateral forebrain bundles and terminates directly in two telencephalic regions; the area ventralis pars intermedia (Vi) and the area dorsalis pars posterior (Dp). Ascending fibers from the pTGN pass through the lateral forebrain bundle and terminate ipsilaterally in the dorsal region of area dorsalis pars medialis (dDm) of the telencephalon. Following biocytin injections into the dDm, small, round cells were labeled in the pTGN. After biocytin injections into the Vi and Dp of the telencephalon, retrogradely labeled cells were found in the ipsilateral SGN. The results show that the ascending fiber connections of the central gustatory system in the percomorph teleost tilapia are essentially similar to those of mammals. That is, the pathway from the primary gustatory areas (facial, glossopharyngeal, and vagal lobes) through the SGN and pTGN to the dDm in tilapia corresponds with the mammalian gustatory pathway from the solitary nucleus through the pontine taste areas (nucleus parabrachialis) and the thalamic relay nucleus (ventral posteromedial nucleus) to gustatory neocortices. In addition, the pathway from the primary gustatory areas through the SGN to the Vi and Dp in tilapia corresponds with the pathway from the solitary nucleus through the pontine taste areas to the amygdala in mammals.

Topics: Afferent Pathways; Animals; Axonal Transport; Axons; Brain; Carbocyanines; Horseradish Peroxidase; Lysine; Models, Neurological; Nerve Endings; Nerve Fibers; Neurons; Taste; Telencephalon; Tilapia

The striatum receives excitatory input from virtually the entire cerebral cortex. In the adult, this input is segregated into two functionally distinct compartments of the striatum, the patch (striosome) and matrix regions. This study determined whether the patterning of corticostriatal afferents from the prelimbic cortex to the striatal patch compartment develops during the early period of collateral formation or instead at the time of peak synaptogenesis. Initial formation of corticostriatal axon collaterals was observed by embryonic day (E) 19. Quantification of corticostriatal collaterals revealed a significant increase in the number and complexity of collateral branches at postnatal day 6 as compared to E19. Concomitant with the increase in collateral branching, a heterogeneous pattern of collateralization consisting of parallel rows of corticostriatal collaterals was observed in the medial striatum. In addition to the rows, clusters of corticostriatal axons occurred more laterally. These clusters colocalized with patches of dense tyrosine hydroxylase-positive fibers, a marker for the striatal patch compartment in the neonatal mouse. Together, these data indicate that corticostriatal patterning occurs during the period of early axon collateralization resulting in a segregation of corticostriatal axon collaterals from the prelimbic cortex to the striatal patch compartment.

Topics: Animals; Animals, Newborn; Axons; Carbocyanines; Corpus Striatum; Embryonic and Fetal Development; Fluorescent Dyes; Limbic System; Lysine; Mice; Mice, Inbred Strains; Neural Pathways

A versatile technique for dye application in living tissue is described, which results in labeling of viable cells from which electrophysiological or optical recordings can be obtained. The dye-coated surface of a glass microelectrode tip is used to apply anatomical tracers or calcium sensitive probes with spatial precision. A total of three types of dyes have been applied in this way to find and record from olfactory interneurons in the terrestrial mollusc Limax maximus. Crystals of 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) formed on the tips of glass microelectrodes were placed in the procerebral lobe, the major olfactory processing center of Limax. Somata in buccal and pedal ganglia with processes extending several 100 microm to the procerebral lobe were stained within 4-6 h. Intracellular recordings from DiI stained buccal (B(PC)) and pedal (P(PC)) cells were obtained. Cross correlograms of the oscillatory field potential in the procerebral lobe and spontaneous action potentials in P(PC) or B(PC) show that P(PC) activity is weakly coupled to the oscillation in the procerebral lobe, while B(PC) activity is clearly coupled to the oscillation. Stimulation of the procerebral lobe with nitric oxide activated P(PC) cells but suppressed activity in B(PC) cells. Calcium green-10Kdextran coated electrodes were used to place calcium green in the cell body layer of the procerebral lobe. Bursting and nonbursting procerebral neurons incorporated and transported the calcium green-dextran. Optical recordings of changes in fluorescence signals from several bursting cells recorded simultaneously were used to test alternative mechanisms of bursting cell coupling. Application of biotin 3Kdextran to the midline of the cerebral ganglion revealed a group of cells in each procerebral lobe with processes crossing the midline of the cerebral ganglion. These cells may couple right and left procerebral lobe activity during odor processing.

Topics: Animals; Calcium; Carbocyanines; Discrimination Learning; Electric Stimulation; Electrophysiology; Fluorescent Dyes; Ganglia, Invertebrate; Interneurons; Lysine; Microelectrodes; Optics and Photonics; Organic Chemicals; Periodicity; Smell; Snails; Staining and Labeling

We investigated the anatomical connections of the pyramidal neurons located within the hilar region of the dentate gyrus of the human hippocampus, neurons which do not have a rodent equivalent. The myeloarchitectural patterns of the human hippocampus indicated the presence of a distinct fiber pathway, the endfolial fiber pathway, in the stratum oriens of the hilus and field CA3. By using the fluorescent lipophilic dye DiI in formalin-fixed human hippocampal tissue, we demonstrated that this is a continuous fiber pathway between the deep hilar region and CA2. This fiber pathway did not enter the fimbria or alveus along the entire distance of the traced pathway and ran exclusively in the stratum oriens of the hilus and CA3. Tracing studies with biocytin in in vitro human hippocampal slices indicated that the hilar and CA3 pyramidal neurons contributed to this pathway. Out distally in field CA3, the long transverse fibers became short and choppy, suggesting that they were beginning to move out of the plane of the tissue slice. Numerous fibers from this pathway were seen crossing the pyramidal layer. Based on comparative studies, we propose that the endfolial fiber system is a component of the hilar Schaffer collateral system in humans. The presence of a significant Schaffer collateral system from the pyramidal neurons in the hilar region would indicate that these neurons are anatomically related to the CA3 pyramidal neurons. Therefore, we suggest the inclusion of the human hilar pyramidal neurons within Lorente de No's field CA3 and, in particular, within subfield CA3c.

Topics: Adult; Aged; Aged, 80 and over; Brain Mapping; Carbocyanines; Epilepsy; Female; Fluorescent Dyes; Hippocampus; Humans; Lysine; Male; Middle Aged; Nerve Fibers, Myelinated; Neural Pathways

The development of trigeminal projections between the thalamus and cortex has been investigated in the marsupial mammal, the wallaby, by using a carbocyanine dye, horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), Neurobiotin, and biocytin as pathway tracers. The appearance of whisker-related patterns in the cortex in relation to their appearance in the brainstem and thalamus was examined, as was the presence or absence of a waiting period for thalamocortical afferents and the identity of the first cortical cells to project to the thalamus. Thalamic afferents first reached the cortex at postnatal day (P) 15 and were distributed up to the deep edge of the compact cell zone in the superficial cortical plate throughout development, in both dye and WGA-HRP labelled material, with no evidence of a waiting period. The initial corticothalamic projection, detected by retrograde transport of WGA-HRP from the thalamus, occurred at P60 from layer 5 cells. This was confirmed by labelling of corticothalamic axons after cortical injections of Neurobiotin and biocytin. Scattered, labelled cells seen before P60 after dye labelling from the thalamus presumably resulted from transcellular labelling via thalamic afferents. Clustering of afferents in layer 4 and cell bodies and their dendrites in layers 5 and 6 first occurred simultaneously at P76. There is a sequential onset of pattern formation, first in brainstem, then in thalamus, and finally in cortex, with a long delay between afferent arrival and pattern formation at each level. Independent regulation at each level, likely depending on target maturation, is suggested.

Topics: Animals; Axons; Biotin; Brain Mapping; Carbocyanines; Cerebral Cortex; Fluorescent Dyes; Lysine; Macropodidae; Neural Pathways; Thalamus; Time Factors; Trigeminal Nerve; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate

The molecular mechanisms that underlie dentate granule cell axon (i.e., mossy fiber) growth during development and following seizure-induced hippocampal injury remain unknown. Part of this process may involve specific factors that support dentate granule cells during differentiation, and molecular cues that allow the appropriate growth of mossy fiber axons toward their targets. To study this process, we developed an in vitro assay system to measure the activity of putative trophic, chemoattractant and chemorepulsive factors. Two-hundred-micrometer-thick transverse hippocampal sections were prepared from neonatal rats and microdissected to isolate the middle one-third of the superior blade of the dentate granule cell layer. These were embedded in a three-dimensional collagen matrix either alone or with microdissected regions of the CA2 pyramidal cell layer. Cultures were maintained in a defined medium and grown for two to three days in a standard culture environment. Results showed that numerous processes grew primarily from the hilar side of explants into the collagen matrix, often in excess of 500 microns in length. These were determined to be axons based on: (i) morphological criteria including size and presence of growth cones, (ii) synaptophysin and growth-associated protein-43 immunoreactivity, (iii) lack of glial fibrillary acidic protein immunoreactivity and (iv) contiguity of biocytin-filled processes with neuronal soma within the explant. Treatment of cultures with brain-derived neurotrophic factor caused a significant increase in axon number and length, and this effect was partially reversed by the addition of a trkB-immunoglobulin fusion protein that blocks the activity of brain-derived neurotrophic factor and neurotrophin-4/5. Basic fibroblast growth factor also caused a marked increase in axon number and length, and caused a migration of neuron-like cells out of the explant into the collagen. These results show that cultured dentate granule cell layer explants are capable of growing mossy fibers into a neutral collagen matrix, and the growth of axons can be modified by the addition of exogenous growth factors. Furthermore, since target tissue and point sources of purified factors can easily be co-cultured with the explants, this new system provides a direct means for testing the molecular cues that influence mossy fiber growth.

Topics: Animals; Axons; Brain-Derived Neurotrophic Factor; Carbocyanines; Collagen; Cytoplasmic Granules; Dentate Gyrus; Fibroblast Growth Factor 2; Immunohistochemistry; Lysine; Neural Pathways; Organ Culture Techniques; Pyramidal Cells; Rats; Receptor, Ciliary Neurotrophic Factor; Receptors, Nerve Growth Factor