Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, we generated a monoclonal antibody, designated PC3.1, that labels a subset of neurons confined to the lateral cortex in the rat (Arimatsu et al., 1992). The PC3.1 antibody binds a 26 kDa protein, referred to as latexin, which is encoded by the lxn gene (Hatanaka et al., 1994
; Jin et al., 1997
). An independent biochemical study has revealed that latexin can act as an inhibitor against carboxypeptidase A (Normant et al., 1995
). Immunohistochemical analyses established that latexin-expressing neurons are restricted primarily to layer VI and to a lesser extent to layer V within the lateral cortex, including the secondary somatosensory (SII) and visceral sensory (Vi) areas, but are rarely found in the dorsomedial cortex, including the primary somatosensory (SI) and the primary motor (MI) areas (Arimatsu et al., 1994
, 1999a
). A substantial number of latexin-immunostained neurons exhibit the morphology of modified pyramidal neurons, including the atypically oriented pyramids. Double-immunofluorescence labeling experiments have shown that ~90% of these latexin-immunoreactive neurons are also glutamate immunoreactive. Thus, it seems likely that the majority of cortical latexin-expressing neurons are excitatory projection neurons (Arimatsu et al., 1999a
).
Taking advantage of the region-specific distribution of latexin-expressing neurons, we demonstrated cortical specification early in development (Arimatsu et al., 1992). We showed that the capacity in vitro to generate latexin-immunoreactive neurons in specific regions of the cerebral wall on either embryonic day (E) 13 or E16 matched well with the tangential distribution of latexin-immunoreactive neurons in the adult cortex (Arimatsu and Ishida, 1998
). This provides convincing evidence that the cerebral wall is prepatterned early in development by elements intrinsic to the cortex rather than by connectional interactions between cortical and extracortical structures. More recently, to address the precise timing of cortical specification at the cellular level, we monitored latexin expression in developing cortical cells under specific conditions in vitro (Arimatsu et al., 1999b
). Compared with progenitor cells derived from the lateral cortex, far fewer progenitors from the dorsal cortex became latexin-immunoreactive neurons under the same environmental conditions, indicating early establishment of cortical specification at the progenitor cell level. Furthermore, it was shown that the probability of postmitotic cells (derived from the lateral cortex) becoming latexin-expressing neurons could be reduced by environmental signals.
The functional specificity of cortical areas depends primarily on afferent and efferent connections as well as on local area and lamina-specific neurochemical features. Previously, O'Leary and colleagues showed, by heterotopic transplantation experiments, that area-specific projections of layer V pyramidal neurons were appropriate to the transplant's regional locale in the host cortex rather than to its site of origin (O'Leary and Stanfield, 1989). However, later experiments demonstrated that target specificity and functional properties of transplants could not be completely respecified following the same or analogous transplantation paradigms (Barth and Stanfield, 1994
; Ebrahimi-Gaillard et al., 1994
). Moreover, recent analyses of mice deficient in certain developmentally regulated genes suggested that genetic elements directly contribute to the specification of cortical circuitry (Hebner et al., 1998; Weimann et al., 1998
). Thus, it remains to be determined when and to what extent genetic and epigenetic elements contribute to the specification events for cortical efferent projections. Since cortical latexin-expressing neurons exhibit predominantly pyramidal-like morphologies, are glutamate immunoreactive and are probably long-projecting neurons, we have speculated that these neurons might contribute to specific cortical efferent pathways (Arimatsu et al., 1999a
). If this is the case, then it should be possible to analyze the specific formation of cortical circuitry using latexin as a molecular tag for the pathway.
The first aim of the present study was to examine the ultrastructure of latexin-expressing neurons by immunoelectron microscopy to ascertain whether or not they have features common to pyramidal neurons. The second goal determined the connectional target(s) of latexin-expressing neurons with combined retrograde tracing and immunofluorescence techniques to examine whether latexin is expressed in a specific category of cortical efferent neurons. Finally, we have examined the connectional specificity formed in vitro between cortical latexin-expressing neurons and their normal targets using organotypic slice cultures.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wistar rats were maintained under a 12 h/12 h light/dark photocycle. Timed-pregnant rats were obtained by overnight mating. Days on which vaginal plugs were identified were designated as E0. Birthdays usually occurred on E21 (= P0).
Pre-embedding Immunoelectron Microscopy
Adult male rats were perfused with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) under an anesthesia overdose with sodium pentobarbital (50 mg/kg). Coronal slices (1 mm thick) of the brain were postfixed with 4% paraformaldehyde (3 h at 4°C), equilibrated with 25% sucrose in phosphate-buffered saline (PBS, pH 7.4), frozen with liquid nitrogen and then thawed in PBS. Subsequent 50 µm thick sections were made with a vibratome and treated sequentially with (i) 1% sodium borohydride (30 min), (ii) 5% normal goat serum in PBS (30 min), (iii) anti-latexin monoclonal antibody [PC3.1, mouse IgG1 (Arimatsu et al., 1992); 20 µg/ml, 2 days at 4°C]; (iv) goat anti-mouse IgG (Cappel, 1:25 dilution; 3 h), and (v) mouse peroxidase antiperoxidase (Jackson, 1:500 dilution; 2 h). All immunochemicals [steps (iii)(v)] were dissolved in 5% normal goat serum in PBS. The bound antibody was visualized with 0.01% 3,3'-diaminobenzidine4HCl and 0.01% hydrogen peroxide in 50 mM TrisHCl, pH 7.6 (20 min). The immunoperoxidase-labeled sections were treated with 0.1% osmium tetroxide (10 min) and embedded in Epon 812. Ultrathin sections were cut and observed with a JEM-1200EX electron microscope (JEOL, Japan).
Mapping of Latexin-immunoreactive and Retrogradely Labeled Neurons
Retrograde Labeling
A retrograde tracer, fluoro-gold (Fluorochrome; 2% in H2O, 0.5 µl volume), was pressure injected stereotaxically with a glass micropipette (o.d. 150200 µm) into a region which involves the SI barrel field (n = 3), MI (n = 3), SII and Vi (n = 2), the ventroposterior and posterior thalamic nuclei (n = 3), or the dorsal striatum (n = 3) of adult rats under deep anesthesia with sodium pentobarbital (4050 mg/kg). For the SI, MI and SII/Vi, the tracer was injected at two depths from the dural surface (0.8 and 1.6 mm). Four days following the fluoro-gold injection, the rats received an overdose injection of sodium pentobarbital (50 mg/kg) and were perfused with 250 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed and coronal slices (2 mm thick) from each brain were postfixed with the same fixative (1620 h at 4°C). They were equilibrated in 25% sucrose in PBS, and frozen in OCT compound (Miles). Serial coronal sections were cut on a cryostat at a thickness of 1020 µm, collected in ice-cold PBS, mounted onto gelatin/chrome alum-subbed glass slides, and stored until use at 20°C.
Double-immunofluorescence Labeling
The sections were incubated sequentially with (i) 5% normal goat serum in PBS containing 0.1% Triton X-100, (ii) PC3.1 monoclonal antibody (10 µg/ml, 16 h at 4°C), (iii) rhodamine-conjugated goat anti-mouse IgG (Chemicon, AP181R; 1:50 dilution, 2 h), (iv) rabbit anti-fluoro-gold polyclonal antibody (Chemicon; 1:2000 dilution, 2 h), and finally (v) fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Chemicon, AP182F; 1:50 dilution, 1 h). All immunochemicals [steps (ii)(v)] were dissolved in 5% normal goat serum in PBS containing 0.1% Triton X-100.
Data analysis
The locations of latexin-immunoreactive and fluoro-gold-labeled neuronal profiles were mapped using a fluorescence light microscope (Zeiss Axioskop, Germany) equipped with a motor-driven scanning stage and a KS400 image processing/analysis system (Zeiss, Germany).
Analysis of Axonal Connections Formed In Vitro
Organotypic Slice Culture
Newborn pups (P0 or P1) and E16 fetuses were used. Brains were removed and kept in ice-cold PBS. Transverse slices of the forebrain were cut to a thickness of 400 µm using a vibratome. Portions of the cerebral wall corresponding to the SI and SII (P0 or P1), and a portion of the thalamus containing the ventroposterior and posterior nuclei (E16) were dissected out according to maps of the developing rat brain (Altman and Bayer, 1995). The SII slices were cocultured with either the SI slice or the thalamic slice on collagen-coated membranes of Millicell CM inserts (Millipore, PICM03050) placed in wells of six-well plates. The white matter side of the SII slice was placed immediately adjacent to that of the SI slice or the thalamic slice. The cocultures were maintained at 37°C in 5% CO2/95% air with 1.3 ml of a serum-containing medium (45% Dulbecco's modified Eagle's medium, GIBCO; 45% Ham's F12 medium, GIBCO; 5% heat-inactivated horse serum, GIBCO; 5% precolostrum newborn calf serum, Mitsubishi Chemical) which filled the well under the membrane. The medium was replaced once a week.
1',1-Dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI) Labeling
After 1315 days in vitro, a small crystal of the lipophilic fluorescent dye DiI (Molecular Probe) was placed at multiple sites within the SI or thalamic slices through the tip of a glass micropipette (o.d. 150200 µm). Twenty-four hours following the DiI placement, the cocultures were fixed with 4% paraformaldehyde (2 days at 4°C). After the SI and thalamic slices were removed from the cocultures, the remaining SII slices were equilibrated in 25% sucrose, embedded in OCT compound, cryosectioned serially (10 µm), and thaw-mounted onto gelatin-coated slides.
Immunofluorescence of Cultured Cells
Sections of slice cultures were stained for latexin immunoreactivity using a rabbit antiserum raised against the synthetic 10mer peptide containing the carboxy-terminal sequences of latexin (Hatanaka et al., 1994; Takiguchi-Hayashi et al., 1998
). After incubation with the antibody (1:1000 dilution) in PBS containing 5% normal goat serum (without Triton X-100), the sections were treated with the FITC-conjugated donkey anti-rabbit IgG (1:50 dilution). The total number of DiI-labeled neurons was counted in every fourth section under the fluorescence microscope, as well as the total number of latexin-immunoreactive neurons and double-labeled DiI-filled and latexin-immunoreactive neurons.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Latexin-immunoreactive neurons were readily identified at the ultrastructural level by a conspicuous peroxidase reaction product (Fig. 1A). Although we did not attempt to determine precisely the subcellular localization of the latexin immunoreactivity because of potential diffusion artifacts of the pre-embedding immunoelectron microscopic method employed, we were able to characterize certain ultrastructural features of the latexin-immunoreactive neurons. Of the 30 latexin-immuno-reactive neurons in layer VI of the SII, all exhibited a round or oval nucleus (Fig. 1A,B
). A total of 106 axosomatic synapses on 24 latexin-immunoreactive neuronal profiles were solely symmetrical (Fig. 1C,D
), although numerous asymmetrical synapses were found on the surface of the latexin-immunopositive dendritic spines (Fig. 1E
). Latexin-immunoreactive axonal terminals formed asymmetrical synapses on dendrites of unknown origin in layer VI of the SI (Fig. 1F
). These observations are compatible with previous data defining the ultrastructural features of cortical pyramidal neurons (Colonnier, 1981
).
|
Axonal connections of latexin-immunoreactive neurons were analyzed using combined retrograde-tracing and immunofluorescence techniques. Since previous studies demonstrated that major efferent neurons in layer VI of the SII project ipsilaterally to the principal thalamic relay nuclei (Wise, 1975; Jones, 1984
), or to the SI and MI (Koralek et al., 1990
; Reep et al., 1990
; Fabri and Burton, 1991
; Paperna and Malach, 1991
), we examined latexin-immunoreactive neurons in the SII (and in the Vi located ventrally to the SII) (Zilles and Wree, 1995
) in animals that had received a fluoro-gold injection into either the SI, MI or the lateral thalamus. We also examined latexin immunoreactivity in animals that had received a fluoro-gold injection into the ipsilateral striatum or the contralateral SII and Vi to ascertain if certain components of latexin-expressing neurons contribute to either corticostriatal (Lévesque et al., 1996
) or callosal projections (Wise and Jones, 1978
; Ivy and Killackey, 1981
; Koralek et al., 1990
).
SI Injections
In agreement with previous reports (Tracey and Waite, 1995), injections of fluoro-gold into the SI barrel field labeled neurons in layers II/IIIVI of the ipsilateral SII and Vi (Figs 2A and 3A
). Among these corticocortical associative neurons, the majority in layer VI were latexin-immunoreactive, though a substantial number of tracer-filled latexin-negative neurons were also found in sublayer VIb of the SII. In agreement with our previous immunoperoxidase observations (Arimatsu et al., 1999a
), the majority of latexin-expressing neurons that were labeled with fluoro-gold resembled the atypically oriented pyramidal, fusiform, or other pyramidal-like neurons (Tömböl, 1984
; Miller, 1988
; de Lima, 1990
; Bueno-López, 1991
), characteristics that collectively correspond to the modified pyramidal cells of the infragranular layers' (DeFelipe and Fariñas, 1992
). In contrast, virtually no fluoro-gold-labeled neurons were labeled with the anti-latexin antibody in layer II/III of the SII and Vi. These immunonegative neurons generally resembled typical pyramids with an apical dendrite extending vertically toward the pial surface.
|
|
As in the case of the SI injections, numerous fluoro-gold-labeled neurons colocalized with latexin immunofluorescence in the infragranular layers of the ipsilateral SII and Vi (Fig. 2B). Again virtually all the fluoro-gold-labeled neurons in layer II/III were latexin-negative.
Thalamic Injections
Retrogradely labeled neurons were observed almost exclusively in the infragranular layers within the ipsilateral SII and Vi following fluoro-gold placement involving the ventroposterior and posterior thalamic nuclei (Figs 2C and 3B). The fluoro-gold-labeled neurons predominantly exhibited the morphology of typical pyramidal neurons. Despite extensive examination at various rostrocaudal levels (10 sections from two rats), these thalamic-projecting neurons were completely segregated from latexin-immunoreactive neurons within the same layers.
Striatal Injections
Following placement of fluoro-gold in the dorsal part of the striatum, numerous fluoro-gold-labeled neurons were found in layer II/III through the upper part of layer VI in the SII and Vi, but rare fluoro-gold-labeled neurons colocalized with latexin in the infragranular layers of the ipsilateral SII and Vi (Fig. 2D).
SII/Vi Injections
When the fluoro-gold injection involved the SII and the Vi as well, retrogradely labeled neurons were found contralaterally in layer II/III through the upper part of layer VI of the SII and Vi (Fig. 2E). Although callosal neurons were abundant in layer V and upper layer VI, fluoro-gold-labeled neurons and latexin-immunoreactive neurons were completely segregated.
Axonal Connections of Latexin-expressing Neurons Formed In Vitro
When slices of the developing SII were cocultured next to those of the SI and the thalamus, axons emanating from the SII slices grew into cocultured slices of either the SI or the thalamus (Fig. 4A,B). Thus, an average of 2031 ± 719 (SEM, n = 6) and 979 ± 111 (n = 6) retrogradely labeled neurons were found in the SII slices following the placement of DiI into the SI and thalamic slices respectively. However, latexin-expressing neurons grown in vitro avoided thalamic slices while innervated prominently the correct SI target. Out of the DiI-labeled neurons, 79.0 ± 34.3 and 4.3 ± 1.6 were latexin-immunoreactive in the SII slices cocultured with the SI and thalamic slice respectively. As shown in Figure 5
, the proportion of double-labeled latexin-immunoreactive DiI-filled neurons was much greater in the SII slices cocultured with the SI slice (3.92 ± 0.63%) compared to that cocultured with the thalamic slice (0.42 ± 0.14%; P < 0.001, Student's t-test).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most data about the morphological and neurochemical characteristics of synaptic inputs on pyramidal neurons have been obtained primarily from typical, rather than modified, pyramidal neurons. There could, however, be significant differences between the synaptic inputs to different populations of pyramidal and pyramidal-like neurons, but the extent of such differences has not been fully understood (DeFelipe and Fariñas, 1992). Previous electron microscopic analyses established that typical pyramidal neurons receive solely symmetrical (Gray's type II) synaptic contacts on their cell bodies and have a round or oval nucleus, whereas nonpyramidal neurons receive both symmetrical and asymmetrical axosomatic synapses and have a nucleus with irregular contours (Colonnier, 1981
; Peters and Jones, 1984
). Although the morphology of numerous latexin-immunostained neuronal profiles resembled modified rather than typical pyramids (Arimatsu et al., 1999a
) (see also Fig. 3A
), the present immunoelectron microscopic examination demonstrates that they display the ultrastructural features common to pyramidal neurons. This is consistent with the previous Golgi electron microscopic observation that some inverted and pyramidal-like neurons in layer V and VI of the rat visual cortex exhibited the same synaptic organization as typical pyramidal neurons (Parnavelas et al., 1977
). Furthermore, in layer VI of the SI, the target area of associative neurons in layer VI of the SII (Zhang and Deschênes, 1998
), latexin-immunoreactive axonal terminals were observed to form asymmetrical (Gray's type I) synaptic contacts, an indicator of excitatory synaptic sites (Colonnier, 1981
; DeFelipe and Fariñas, 1992
). These observations are compatible with our previous assumption, based on glutamate immunoreactivity, that latexin-expressing neurons are predominantly long-projecting excitatory neurons (Arimatsu et al., 1999a
).
Axonal Connections of Latexin-expressing Neurons In Vivo
The present retrograde-tracing experiments in vivo demonstrate that numerous latexin-immunoreactive neurons are indeed long-projecting neurons. It was previously established that a major population of layer VI efferent neurons have corticothalamic projections (Jones, 1984), and that a substantial population of layer VI neurons represents corticocortical associative neurons (Fabri and Burton, 1991
). We have shown in the present study that numerous latexin-immunoreactive neurons in the infra-granular layers of SII and Vi project to the ipsilateral SI and MI and are completely segregated from corticothalamic neurons in the same layers. They are also completely, or nearly completely, segregated from callosal and corticostriatal neurons. These results are in line with data showing that the majority of corticothalamic neurons have the morphology of typical pyramidal neurons (Katz, 1987
; Zhang and Deschênes,1997
) and that corticocortical associative neurons in the infragranular layers exhibit a variety of morphological features, including those of modified pyramidal neurons (Bueno-López et al., 1991
; Zhang and Deschênes, 1997
). Based on previous data that ~3% of SI-projecting neurons in either the SII or Vi are immunoreactive for glutamic acid decarboxylase, the synthetic enzyme of
-aminobutyric acid (GABA) (Fabri and Manzoni, 1996
) and that <1% of the latexin-immunoreactive neurons are GABA-immunoreactive (Arimatsu et al., 1999a
), an extremely minor component, if any, of latexin-expressing neurons would be inhibitory projecting neurons. Thus, it is now apparent that layer VI efferent neurons in the SII and Vi are composed of two distinct major populations: (i) corticothalamic excitatory neurons not expressing latexin with typical pyramidal morphology, and (ii) corticocortical excitatory associative neurons expressing latexin with a modified pyramidal morphology. Additionally, within upper layer VI, there is a minor neuronal population, the callosal projection neurons without latexin expression (see Fig. 2E
). These features demonstrate a distinct cortical organization in which a molecular phenotype is linked tightly with a specific neuronal connectivity and morphology. In line with the present data, Gasper et al. (Gasper et al., 1995) have shown in the rat frontal cortex that, despite some overlap, the expression of the D1 and D2 dopamine receptor genes is specific for different categories of cortical efferent neurons: D1 receptor mRNA is expressed in corticocortical, corticothalamic and corticostriatal neurons, while D2 receptor mRNA is expressed in corticocortical and corticostriatal neurons. Neither is expressed in corticospinal or corticopontine neurons. Similarly, expression of nonphosphorylated neurofilament protein was also shown to be associated with certain corticocortical projections in monkeys (Hof et al., 1996
).
Axonal Connections of Latexin-expressing Neurons Formed In Vitro
While it has been suggested that early cortical progenitors are multipotent and committed to infragranular neurons during their final cell cycle (McConnell and Kaznowski, 1991), it remains unknown when and how the distinctive target specificities between corticothalamic and corticocortical neurons in layer VI are determined. Given the intimate correlation between molecular phenotype and neuronal connectivity, one may predict certain cooperative mechanisms that underlie the differentiation of developing cortical cells to become latexin-expressing neurons and to form their specific efferent projections. We have previously shown that the region-specific distribution of latexin-expressing neurons is substantiated during early corticogenesis by the restriction of the developmental potential of dorsal progenitor cells, but that the phenotype of competent lateral cortical cells can be regulated by later environmental cues (Arimatsu et al., 1999b
). The time period when cell fate is susceptible to environmental cues apparently overlaps that of migration of prospective latexin-expressing neurons, which is consistent with the idea that cortical specification involves progressive cell fate determination toward a molecular phenotype (Levitt et al., 1993
; Edlund and Jessell, 1999
). Thus we speculate that the target specificity of latexin-expressing neurons would also be established progressively during corticogenesis. In efforts to determine this, it is notable that the target specificity of latexin-expressing neurons (corticocortical versus corticothalamic) can be established even in slice cultures in vitro. Previous coculture experiments demonstrated that slices from the superior colliculus or the pons were innervated by pyramidal neurons located in layer V, whereas thalamic slices received their inputs predominantly from pyramidal neurons located in layer VI (Yamamoto et al., 1989
, 1992
; Bolz et al., 1990
; Heffner et al., 1990
). Our findings from the present coculture experiment further revealed that pyramidal neurons of different categories in perinatal layer VI are already specified to grow correctly towards their normal targets. Since layer VI corticocortical neurons can be identified unambiguously by the expression of latexin, rather than their relative locations in slice cultures, they may provide a unique model system to analyze genetic and epigenetic elements regulating specific formation of corticocortical associative pathways.
![]() |
Notes |
---|
Address correspondence to Y. Arimatsu, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 1948511, Japan. Email: yasu{at}libra.ls.m-kagaku.co.jp.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arimatsu Y (1994) Latexin: a molecular marker for regional specification in the cerebral cortex. Neurosci Res 20:131135.[ISI][Medline]
Arimatsu Y, Ishida M (1998) Early patterning of the rat cerebral wall for regional organization of a neuronal population expressing latexin. Dev Brain Res 106:7178.[ISI][Medline]
Arimatsu Y, Miyamoto M, Nihonmatsu I, Hirata K, Uratani Y, Hatanaka Y, Takiguchi-Hayashi K (1992) Early regional specification for a molecular neuronal phenotype in the rat neocortex. Proc Natl Acad Sci USA 89:88798883.[Abstract]
Arimatsu Y, Nihonmatsu I, Hirata K, Takiguchi-Hayashi K (1994) Cogeneration of neurons with a unique molecular phenotype in layers V and VI of widespread lateral neocortical areas in the rat. J Neurosci 14:20202031.[Abstract]
Arimatsu Y, Kojima M, Ishida M (1999a) Area and lamina-specific organization of a neuronal subpopulation defined by expression of latexin in the rat cerebral cortex. Neuroscience 88:93105.[ISI][Medline]
Arimatsu Y, Ishida M, Takiguchi-Hayashi K, Uratani Y (1999b) Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons. Development 126:629638.
Barth TM, Stanfield BB (1994) Homotopic, but not heterotopic, fetal cortical transplants can result in functional sparing following neonatal damage to the frontal cortex in rats. Cereb Cortex 4:271278.[Abstract]
Bolz J, Novak N, Götz M, Bonhoeffer T (1990) Formation of target specific neuronal projections in organotypic slice cultures from rat visual cortex. Nature 346:359362.[ISI][Medline]
Bueno-López JL, Reblet C, López-Medina A, Gómez-Urquijo SM, Grandes P, Gondra J, Hennequet L (1991) Targets and laminar distribution of projection neurons with inverted morphology in rabbit cortex. Eur J Neurosci 3:415430.[ISI][Medline]
Colonnier M (1981) The electron-microscopic analysis of the neuronal organization of the cerebral cortex. In: The organization of the cerebral cortex (Schmit FO, Worden FG, Adelman G, Dennis SG, eds), pp. 125152. Cambridge, MA: MIT Press.
DeFelipe J, Fariñas I (1992) The pyramidal neuron of the cerebral cortex: morphological and chemical characteristics of the synaptic inputs. Progr Neurobiol 39:563607.[ISI][Medline]
de Lima AN, Voigt T, Morrison JH (1990) Morphology of the cells within the inferior temporal gyrus that project to the prefrontal cortex in the macaque monkey. J Comp Neurol 296:159172.[ISI][Medline]
Ebrahimi-Gaillard A, Guitet J, Garnier C, Roger M (1994) Topographic distribution of efferent fibers originating from homotopic or heterotopic transplants: heterotopically transplanted neurons retain some of the developmental characteristics corresponding to their site of origin. Dev Brain Res 77:271283.[ISI][Medline]
Edlund T, Jessell TM (1999) Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system. Cell 96:211224.[ISI][Medline]
Fabri M, Burton H (1991) Ipsilateral cortical connections of primary somatosensory cortex in rats. J Comp Neurol 311:405424.[ISI][Medline]
Fabri M, Manzoni T (1996) Glutamate decarboxylase immunoreactivity in corticocortical projecting neurons of rat somatic sensory cortex. Neuroscience 72:435448.[ISI][Medline]
Gaspar P, Block B, LeMoine C (1995) D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons. Eur J Neurosci 7:10501063.[ISI][Medline]
Hatanaka Y, Uratani Y, Takiguchi-Hayashi K, Omori A, Sato K, Miyamoto M, Arimatsu, Y (1994) Intracortical regionality represented by specific transcription for a novel protein, latexin. Eur J Neurosci 6:973982.[ISI][Medline]
Heffner CD, Lumsden AGS, O'Leary DDM (1990) Target control of collateral extension and directional axon growth in the mammalian brain. Science 247:217220.[ISI][Medline]
Hevner RF, Miyashita E, Martin GR, Rubenstein JLR (1998) Lack of thalamocortical connections in mutants affecting cortical (Tbr-1) or thalamic (Gbx-2) gene expression. Soc Neurosci Abst 24:30.9.
Hof PR, Ungerleider LG, Webster MJ, Gattass R, Adams MM, Sailstad CA, Morrison JH (1996) Neurofilament protein is differentially distributed in subpopulations of corticocortical projection neurons in the macaque monkey visual pathways. J Comp Neurol 376:112127.[ISI][Medline]
Ivy GO, Killackey HP (1981) The ontogeny of the distribution of callosal projection neurons in the rat parietal cortex. J Comp Neurol 195:367389.[ISI][Medline]
Jin M-h, Uratani Y, Arimatsu Y (1997) Mapping to mouse chromosome 3 of the gene encoding latexin (Lxn) expressed in neocortical neurons in a region-specific manner. Genomics 39:419421.[ISI][Medline]
Jones EG (1984) Laminar distribution of cortical efferent cells. In: Cerebral cortex (Peters A, Jones EG, eds), Vol. 1, pp. 521552. New York: Plenum Press.
Katz LC (1987) Local circuitry of identified projection neurons in cat visual cortex brain slices. J Neurosci 7:12231249.[Abstract]
Kennedy H, Dehay C (1993) Cortical specification of mice and men. Cereb Cortex 3:171186.[Abstract]
Koralek KA, Olavarria J, Killackey HP (1990) Areal and laminar organization of corticocortical projections in the rat somatosensory cortex. J Comp Neurol 299:133150.[ISI][Medline]
Lévesque M, Gagnons S, Parent A, Deschênes M (1996) Axonal arborization of corticostriatal and corticothalamic fibers arising from the second somatosensory area in the rat. Cereb Cortex 6:759570.[Abstract]
Levitt P, Ferri RT, Barbe MF (1993) Progressive acquisition of cortical phenotypes as a mechanism for specifying the developing cerebral cortex. Perspect Dev Neurobiol 1:6574.[Medline]
Levitt P, Barbe MF, Eagleson KL (1997) Patterning and specification of the cerebral cortex. Annu Rev Neurosci 20:124.[ISI][Medline]
McConnell SK, Kaznowski CE (1991) Cell cycle dependence of laminar determination in developing neocortex. Science 254:282285.[ISI][Medline]
Miller MW (1988) Maturation of rat visual cortex: IV. The generation, migration, morphogenesis, and connectivity of atypically oriented pyramidal neurons. J Comp Neurol 274:387405.[ISI][Medline]
Normant E, Martres M-P, Schwartz J-C, Gros C (1995) Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor. Proc Natl Acad Sci USA 92:1222512229.[Abstract]
O'Leary DDM, Stanfield BB (1989) Selective elimination of axons extended by developing cortical neurons is dependent on regional locale: experiments utilizing fetal cortical transplants. J Neurosci 9:22302246.[Abstract]
O'Leary DDM, Schlaggar B, Tuttle R (1994) Specification of neocortical areas and thalamocortical connections. Annu Rev Neurosci 17:419439.[ISI][Medline]
Paperna T, Malach R (1991) Patterns of sensory intermodality relationships in the cerebral cortex of the rat. J Comp Neurol 308:432456.[ISI][Medline]
Parnavelas JG, Sullivan K, Lieberman AR and Webster KE (1977) Neurons and their synaptic organization in the visual cortex of the rat. Cell Tiss Res 183:499517.[ISI][Medline]
Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. North Ryde: Academic Press.
Peters A, Jones EG (1984) Classification of cortical neurons. In: Cerebral cortex (Peters A, Jones EG, eds), Vol. 1, pp. 107121. New York: Plenum Press.
Rakic P (1988) Specification of cerebral cortical areas. Science 241:170176.[ISI][Medline]
Reep RL, Goodwin GS, Corwin JV (1990) Topographic organization in the corticocortical connections of medial agranular cortex in rats. J Comp Neurol 294:262280.[ISI][Medline]
Takiguchi-Hayashi K, Sato M, Sugo N, Ishida M, Sato K, Uratani Y, Arimatsu Y (1998) Latexin expression in smaller diameter primary sensory neurons in the rat. Brain Res 801:920.[ISI][Medline]
Tömböl T (1984) Layer VI cells. In: Cerebral cortex (Peters A, Jones EG, eds), Vol. 1, pp. 479519. New York: Plenum Press.
Tracey and Waite (1995) Somatosensory system. In: The rat nervous system, 2nd edn (Paxinos G, ed.), pp. 649685. San Diego: Academic Press.
Weimann JM, Levin ME, Zhang YA, McConnell SK (1998) Otx1 is required for target selection by subcortically projecting layer 5 neurons. Soc Neurosci Abstr 24:414.2.
Wise SP (1975) The laminar organization of certain afferent and efferent fiber systems in the rat somatosensory cortex. Brain Res 90:139142.[ISI][Medline]
Wise SP, Jones EG (1978) Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex. J Comp Neurol 175:187208.
Yamamoto N, Kurotani T, Toyama K (1989) Neural connections between the lateral geniculate nucleus and visual cortex in vitro. Science 245:192194.[ISI][Medline]
Yamamoto N, Yamada K, Kurotani T, Toyama K (1992) Laminar specificity of extrinsic cortical connections studied in coculture preparations. Neuron 9:217228.[ISI][Medline]
Zhang Z-W, Deschênes M (1997) Intracortical axonal projections of lamina VI cells of the primary somatosensory cortex in the rat: a single-cell labeling study. J Neurosci 17:63656379.
Zhang Z-W, Deschênes M (1998) Projections to layer VI of the posteromedial barrel field in the rat: a reappraisal of the role of corticothalamic pathways. Cereb Cortex 8:428436.[Abstract]
Zilles K, Wree A (1995) Cortex: areal and laminar structure. In: The rat nervous system, 2nd edn (Paxinos G, ed.), pp. 649685. San Diego: Academic Press.