Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex.

The developing neocortex influences the growth of thalamocortical projections. Layer 4 in particular receives the majority of input from the thalamus and is important in instructing thalamic afferents to terminate. Previous in vivo experiments demonstrated that disruption of layer 4 during corticogenesis in ferret somatosensory cortex by application of methylazoxy methanol acetate (MAM) prevents proper termination of thalamic afferents in appropriate cortical regions. To further explore the role of layer 4 in thalamocortical development, we prepared organotypic cocultures consisting of normal gestational day 0 (P0) ferret thalamus paired with normal, embryonic day 33 (E33), or E38 MAM-treated cortex obtained from ferrets at either P0 or P7. Injection of MAM on E33 disrupts layer 4 formation, whereas similar injections on E38 interfere with layer 2 formation. The cocultures grew together for a number of days, then discrete injections of either fluorescent dextrans or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were made into the thalamic piece. The labeled thalamic afferents that grew into the cortical slice were analysed and the sites of their terminations quantified after 3, 5, or 7-10 days in culture (DIC). Our results varied somewhat with the amount of time in culture, but the preponderance of thalamic fibers in normal cortex terminated in layer 4, whereas their counterparts in E33 MAM-treated cortex grew beyond the cortical plate and many fibers terminated inappropriately within lower cortical layers or white matter. Terminal distribution of thalamic fibers in E38 MAM-treated cortex looked similar to normal. These results demonstrate that the cells of layer 4 provide thalamic afferents with important positional and termination cues.

Disruption of layers 3 and 4 during development results in altered thalamocortical projections in ferret somatosensory cortex.

The precision of projections from dorsal thalamus to neocortex are key toward understanding overall cortical organization and function. To identify the significance of layer 4 cells in receiving the bulk of thalamic projections in somatosensory cortex, we disrupted layer 4 genesis and studied the effect on thalamic terminations in ferrets. Second, we ascertained the result of layer 4 disruption on functional responses and topographic organization. Methylazoxy methanol (MAM) was injected into pregnant ferrets on embryonic day 33 (E33), when most layer 4 neurons of somatosensory cortex are generated. This treatment resulted in dramatic reduction in the thickness of targeted layer 4. E38 MAM treatment was used as a control, when layer 2-3 neurons are generated. The projections of ventrobasal thalamus into somatosensory cortex were studied using DiI injections. We found only subtle differences between groups (normal, E33, or E38 MAM-treated) in the thalamic afferent pattern on postnatal day 1 (P1) and P7. On P14, thalamic terminations distribute almost equally throughout the remaining cortical layers in the E33 MAM-treated group compared with normal and E38 MAM-treated animals, in which the ventrobasal thalamus projects primarily to central layers. Electrophysiological recordings conducted on mature ferrets treated with MAM on E33 demonstrated that somatotopic organization and receptive field size are normal. These findings emphasize the importance of layer 4 in determining the normal laminar pattern of thalamic termination and suggest that, although its absence is likely to impact on complex neocortical functional responses, topographic organization does not arise from the influence of layer 4.

Neurons derived from radial glial cells establish radial units in neocortex.

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.

An excitatory GABAergic plexus in developing neocortical layer 1.

Layer 1 of the developing rodent somatosensory cortex contains a dense, transient GABAergic fiber plexus. Axons arising from the zona incerta (ZI) of the ventral thalamus contribute to this plexus, as do axons of intrinsic GABAergic cells of layer 1. The function of this early-appearing fiber plexus is not known, but these fibers are positioned to contact the apical dendrites of most postmigratory neurons. Here we show that electrical stimulation of layer 1 results in a GABA(A)-mediated postsynaptic current (PSC) in pyramidal neurons. Gramicidin perforated patch recording demonstrates that the GABAergic layer 1 synapse is excitatory and can trigger action potentials in cortical neurons. In contrast to electrical stimulation, activation of intrinsic layer 1 neurons with a glutamate agonist fails to produce PSCs in pyramidal cells. In addition, responses can be evoked by stimulation of layer 1 at relatively large distances from the recording site. These findings are consistent with a contribution of the widely projecting incertocortical pathway, the only described GABAergic projection to neonatal cortex. Recording of identified neonatal incertocortical neurons reveals a population of active cells that exhibit high frequencies of spontaneous action potentials and are capable of robustly activating neonatal cortical neurons. Because the fiber plexus is confined to layer 1, this pathway provides a spatially restricted excitatory GABAergic innervation of the distal apical dendrites of pyramidal neurons during the peak period of cortical synaptogenesis.

Extrinsic GABAergic innervation of developing neocortical layer 1 in organotypic slice co-cultures.

Afferents from the zona incerta (ZI) of the ventral thalamus contribute to the dense, transient gamma-aminobutyric acid (GABA)ergic fiber plexus in layer 1 of the developing rodent somatosensory cortex. Incertocortical axons contact the distal apical dendrites of postmigratory cortical pyramidal cells. Although recent work has shown that these GABAergic incertocortical fibers are likely to provide widespread fast synaptic excitation of pyramidal cells in layers 2-6 during peak periods of cortical synaptogenesis, little is known about the mechanisms by which these axons project to the neocortex and are confined to layer 1. Here we characterize organotypic slice co-cultures in which a region of embryonic diencephalon containing the ZI is maintained adjacent to a region of embryonic somatosensory cortex. Diencephalic explants from transgenic mice expressing enhanced green fluorescent protein (EGFP) enabled direct visualization of diencephalocortical connections. Isochronic co-cultures exhibited diencephalocortical fiber ingrowth immunoreactive for both GABA and the presynaptic vesicle-associated protein synaptophysin that was restricted to neocortical layer 1. This pattern of lamina-specific diencephalocortical ingrowth occurred irrespective of placement of the afferent explant, and persisted in the absence of action potential activity and GABA(A) receptor activation. Heterochronic co-cultures containing older cortex demonstrated that the cortical explants remain permissive for lamina-specific ingrowth through the first postnatal week. Organotypic slice cocultures provide a system in which to study the mechanisms underlying the layer 1-specific ingrowth of extrinsic GABAergic inputs to the perinatal neocortex.

Evidence for functional localization of the proenkephalin-processing enzyme, prohormone thiol protease, to secretory vesicles of chromaffin cells.

The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephalin-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.

Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers.

Early generated layers of neocortex are important factors in forming the subsequent architecture of the cerebral cortex. To further explore the role of early generated cortex, we disrupted formation of an early generated cohort of cells by intraperitoneal injections of the mitotic inhibitor methylazoxymethanol (MAM) into pregnant ferrets timed to coincide with generation of subplate neurons in the ventricular zone. Our studies demonstrate that if early development of the neocortex is interrupted by injection of MAM during embryogenesis (on embryonic day 24 or 28; E24 or E28), a distinct laminar pattern fails to form properly in the parietal cortex. A reduced number of MAP2-positive cells were observed in the region of the subplate when compared with the number of MAP2-positive cells found in normal animals. Interference with the superficial neocortical layers that form later during development (on embryonic day 33) by appropriately timed MAM injections does not result in a severely disrupted laminar pattern. The interrupted laminar pattern that arises after early MAM injections coincides with distorted radial glial cells (identified by immunoreactivity to the intermediate filament protein, vimentin), which occur after early, but not late, MAM injections. Further analysis suggests that interference with early development of neocortex leads to premature differentiation of radial glial cells into astrocytes, as demonstrated by the presence of glial fibrillary acidic protein (GFAP). Experiments involving injections of the thymidine analog, bromodeoxyuridine (BRDU), demonstrated that 4 days after E24 MAM injection cells are generated and migrate into the thin cortical plate. By E38, however, cells continue to be generated in animals treated with MAM on E24 but do not reach their normal positions in the cortical plate. In addition, immunoreactivity using the CR50 antibody, which identifies presumptive Cajal-Retzius cells present in layer 1, demonstrates that the CR50-positive cells, normally precisely located in the outer portion of layer 1, are distributed in disarray throughout the thickness of the neocortex and intermediate zone in early MAM-treated animals, but not in those treated with MAM injections later during gestation. These findings are consistent with the idea that early generated layers are important in providing factors that maintain the environment necessary for subsequent neuronal migration and formation of neocortical layers.

Histogenesis of ferret somatosensory cortex.

The ferret has emerged as an important animal model for the study of neocortical development. Although detailed studies of the birthdates of neurons populating the ferret visual cortex are available, the birthdates of neurons that reside in somatosensory cortex have not been determined. The current study used bromodeoxyuridine to establish when neurons inhabiting the somatosensory cortex are generated in the ferret; some animals also received injections of [3H]thymidine. In contrast to reports of neurogenesis in ferret visual cortex, most neurons populating the somatosensory cortex have been generated by birth. Although components of all somatosensory cortical layers have been produced at postnatal day 0, the layers are not distinctly formed but develop over a period of several weeks. A small number of neurons continue to be produced for a few days postnatally. The majority of cells belonging to a given layer are born over a period of approximately 3 days, although the subplate and last (layer 2) generated layer take somewhat longer. Although neurogenesis of the neocortex begins along a similar time line for visual and somatosensory cortex, the neurons populating the visual cortex lag substantially during the generation of layer 4, which takes more than 1 week for ferret visual cortex. Layer formation in ferret somatosensory cortex follows many established principles of cortical neurogenesis, such as the well-known inside-out development of cortical layers and the rostro-to-caudal progression of cell birth. In comparison with the development of ferret visual cortex, however, the generation of the somatosensory cortex occurs remarkably early and may reflect distinct differences in mechanisms of development between the two sensory areas.

Development of local connections in ferret somatosensory cortex.

Ferrets have become recognized as a useful and interesting model for study of neocortical development. Because of their immaturity at birth, it is possible to study very early events in the ontogeny of the brain. We used living slices of ferret somatosensory cortex to study the formation and development of intrinsic elements within the neocortex. A small number of fixed, hemisected brains injected with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were also used. The slices were obtained from ferret kits aged postnatal day (P)1 to P62 and maintained in a chamber; each slice received injections of fluorescent-labeled dextrans. The injections were made at different ages in several distinct sites, which included the proliferative ventricular zone, the intervening white matter (or intermediate zone), and different sites of developing cortex, including the deeper cortical plate, which incorporated the subplate in young animals, and more superficial cortical sites depending on the age of the animal. Several animals also received injections into the ventrobasal thalamus. Injections into young animals (P1-7) produced a dominant radial pattern that extended from the ventricular zone into the cortex. Injections into the ventricular zone labeled many cells that appeared morphologically like radial glia as well as presumptive neurons. Although the predominant pattern was radial, injections in the ventricular zone often produced tangentially oriented cells and horizontally arranged fibers at the outer edge of the proliferative zone. These cells and fibers may provide a substrate for tangential dispersion of neurons within the neocortex. More superficial injections within the slice labeled lines of cells that appeared to be stacked upon one another in a radial pile in the cortex; the cortical plate received very few lateral projections. Data obtained from more mature slices indicated that although the overall pattern of staining remained radial, the precise character of the pattern changed to include more lateral spread into surrounding cortex, which eventually refined and developed into distinct patches by P28, when the overall cortical architecture appeared adult like. The data involving thalamocortical connections were more limited, but they indicated that the thalamus projects precisely to the somatosensory cortex in a point-to-point fashion from the earliest date studied (P0) and that the ventrobasal nucleus terminates upon the somatosensory cortex in a patchy manner during the early postnatal days of development. This study of the development of the somatosensory cortex confirms the ubiquitous nature of column-like connections throughout the neocortex and provides a novel view of the radial nature of early neocortical maturation.

Stimulation of "prohormone thiol protease" (PTP) and [Met]enkephalin by forskolin. Blockade of elevated [Met]enkephalin by a cysteine protease inhibitor of PTP.

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.

Fos-like immunoreactivity in the brain of homozygous diabetes insipidus Brattleboro and normal Long-Evans rats.

Water deprivation induces the production of the transcription factor Fos in neurons of the neurohypophysial system. These neurons, which are located primarily in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), produce the antidiuretic hormone vasopressin. The present immunocytochemical study has analyzed the distribution of Fos in brain regions involved in osmoregulation and compared the extent of Fos immunoreactivity (Fos-IR) in vasopressin-deficient Brattleboro and normal Long-Evans rats under stimulated and non-stimulated conditions. Rats were osmotically challenged by means of a single intraperitoneal injection of 1.5 M/L NaCl. Since Fos may be induced by the stress of handling of animals, non-injected and isotonic saline-injected rats were used as controls. Faint nuclear Fos immunostaining was found in the organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus (MnPO), subfornical organ (SFO), and SON of non-injected and isotonic saline-injected Brattleboro but not Long-Evans rats. Hypertonic saline injection specifically induced Fos-IR in neurons located in the SFO, OVLT, MnPO, PVN, SON, hypothalamic accessory nuclei (including the nucleus circularis), and arcuate hypothalamic nucleus (Arc) in both Long Evans and Brattleboro rats. No differences in distribution of the induced immunostaining were found between the strains. Stress of handling and (isotonic saline) injection induced Fos-IR in the lateral septal nuclei, central amygdaloid nuclei, medial amygdaloid nucleus, medial preoptic area, the bed nucleus of the stria terminalis, cingulate- and piriform cortex, the lateral hypothalamic area, ventromedial hypothalamic nucleus, and the habenular nucleus. The data are consistent with a role for Fos in the regulation of vasopressin gene expression during acute hyperosmotic stimulation. In addition, this study demonstrated that during chronic osmotic stimulation, as experienced by homozygous Brattleboro rats, Fos-IR is limited but apparently present constantly and that it increased in these animals following acute osmotic challenge. Our observations suggest that c-fos regulatory controls in homozygous Brattleboro rats are different from those in Long-Evans rats.