Structural features of mossy cells in the hamster dentate gyrus, with special reference to somatic thorny excrescences

We have recently revealed that large multipolar neurons, presumed mossy cells in the hamster dentate gyrus (DG), were calretinin (CR)-immunoreactive (IR) at the ventral level, although these neurons were CR-negative at the dorsal level. In the present study, we confirmed this identification with several methods and analyzed structural features of hamster mossy cells in detail. Golgi impregnationi and intracellular Lucifer yellow labeling studies revealed that mossy cells in the hamster dentate hilus had extraordinarily prominent thorny excrescences on their somata as well as on their proximal dendrites. Mossy cells exhibited dorsoventral differences in their structural features; proximal dendrites of single mossy cells were fewer, and thorny excrescences were larger and more complicated at the dorsal level than at the ventral level. Electron microscopic serial section three-dimensional reconstructions revealed that somatic thorny excrescences consisted of large and complicated spines, which received numerous asymmetrical synapses from mossy fiber terminals. In addition, our confocal laser scanning microscopic observations also revealed many glutamic acid decarboxylase-immunoreactive punctae abutting the mossy cell somata and dendrites. Our present and previous observations revealed the structural features of hamster mossy cells and their differences along the dorsoventral axis and further indicated that mossy cells were prominently different in their chemical and morphological features among species.     

Mossy Cells of the Rat Dentate Gyrus are lmmunoreactive for Calcitonin Gene-related Peptide (CGRP)

The neuropeptide calcitonin gene-related peptide (CGRP) was localized in the hippocampus and dentate gyrus of the rat by immunocytochemistry at the light and electron microscopic levels. Without colchicine treatment only faint neuropil labelling was found in the inner molecular layer of the dentate gyrus. Following colchicine treatment, a large number of neurons with numerous complex spines along the proximal dendrites were visualized in the hilus of the dentate gyrus, particularly in the ventral areas, and, in addition, staining of the inner molecular layer became stronger. Several CA3c pyramidal cells located adjacent to the hilar region in the ventral hippocampus also appeared to be faintly positive, although in most cases only their axon initial segments were labelled. Outside this region, the subicular end of the CA1 subfield contained occasional CGRP-positive non-pyramidal cells. The hilar CGRP-positive neurons were negative for parvalbumin, calretinin, cholecystokinin and somatostatin, whereas most of them were immunoreactive for GluR2/3 (the AMPA-type glutamate receptor known to be expressed largely by principal cells). Correlated electron microscopy showed that the spines along the proximal dendritic shafts indeed correspond to thorny excrescences engulfed by large complex mossy terminals forming asymmetrical synapses. Pre-embedding immunogold staining demonstrated that CGRP immunoreactivity in the inner molecular layer was confined to axon terminals that form asymmetrical synapses, and the labelling was associated with large dense-core vesicles. The present data provide direct evidence that CGRP is present in mossy cells of the dentate gyrus and to a lesser degree in CA3c pyramidal cells of the ventral hippocampus. These CGRP-containing principal cells terminate largely in the inner molecular layer of the dentate gyrus, and may release the neuropeptide in conjunction with their 'classical' neurotransmitter, glutamate.     

Stereologic estimation of hippocampal GluR2/3- and calretinin-immunoreactive hilar neurons (presumptive mossy cells) in two mouse models of temporal lobe epilepsy

Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate‐injected hippocampus, there was an 80% and 85% reduction of GluR2/3‐ and GluR2/3/calretinin‐positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3‐, but only a 20% loss of GluR2/3‐calretinin‐immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed. Significance: These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.     

Distribution of nonprincipal neurons in the rat hippocampus, with special reference to their dorsoventral difference

In the present study we examined the distribution of chemically identified subpopulations of nonprincipal neurons in the rat hippocampus, focusing on the dorsoventral differences in their distributions. The subpopulations analyzed were those immunoreactive for parvalbumin, calretinin, nitric oxide synthase, somatostatin, calbindin D28K, vasoactive intestinal polypeptide and cholecystokinin. Using a confocal laser scanning light microscope, we could confirm that the penetration of each immunostaining, except that of calbindin D28K, was complete throughout 50 μm thick sections under our immunostaining conditions. We counted numbers of immunoreactive somata according to the ‘disector' principle, measured areas of hippocampal subdivisions and the thickness of sections, and estimated the approximate numerical densities of these subpopulations, especially for those neurons immunoreactive for nitric oxide synthase, calretinin, somatostatin and parvalbumin. Generally speaking, neurons immunoreactive for parvalbumin showed no significant dorsoventral differences in the numerical densities in any of the subdivisions of the hippocampus, whereas the numerical densities of somata immunoreactive for calretinin, nitric oxide synthase and somatostatin were significantly larger in ventral levels than at dorsal levels of the hippocampus. The numerical density of somatostatin neurons was significantly larger in ventral levels than in dorsal levels of the dentate gyrus, and, although not prominent, of the CA1 region. That of nitric oxide synthase positive neurons was significantly larger in ventral levels than in dorsal levels of the CA3 region as well as of the DG but not of the CA1 region. The numerical density of calretinin positive neurons was larger in ventral levels than in dorsal levels of all hippocampal subdivisions. The present study also revealed that dorsal and ventral levels of the hippocampus differ from each other in the composition of their nonprincipal neurons. ©1997 Elsevier Science B.V. All rights reserved.     

Cellular distributions of AMPA glutamate receptor subunits in fetal ventral mesencephalon transplants in the dopamine-depleted striatum of a rat

To demonstrate the cellular distributions of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits (GluR1, GluR2/3, and GluR4) in the intrastriatal grafts of a rat model of Parkinson’s disease, immunocytochemistry was performed in 6-hydroxydopamine rats with intrastriatal transplants of fetal ventral mesencephalon (VM). In the fetal VM (at embryonic day 15) in which the tyrosine hydroxylase (TH) immunoreactivity was intensely observed, no GluR subunit immunoreactivity was detected. Within the intrastriatal fetal VM grafts containing TH-positive cells, a large number of cells immunoreactive for GluR1 and GluR2/3 were observed. However, the GluR1- and GluR2/3-positive cells tended to locate homogeneously within the grafts and were composed of various cell sizes and shapes, mainly medium-sized and aspiny cells. Weak GluR4-positive cells were seen in the grafts, although in some cases the staining was too faint to see any immunoreactive cells at all. Double immunostaining revealed that a part of TH-positive cells in the grafts was also immunopositive for GluR1 or GluR2/3. Both dopaminergic neurons and nondopaminergic neurons in the VM transplants appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including GluR1 and GluR2/3 and, to a lesser extent, GluR4.     

Type II glucocorticoid receptor immunoreactivity in the mossy cells of the rat and the mouse hippocampus

Hippocampal principal neurons, granule and pyramidal cells, are known to express type II glucocorticoid receptors (GR) and it is believed that glucocorticoids (GC) mediate at least some of their effects through GR. Under conditions of severe stress and trauma, these principal cells are vulnerable to damage and this mechanism may be exacerbated by GR. The mossy cell, an excitatory dentate gyrus neuron, is also damaged following trauma, with over 50% reported loss in rats after kainate-induced seizures. However, it has not been determined if GC play any role in protecting or exacerbating damage to this important hippocampal cell type. In the present study, we have undertaken an evaluation of the presence of GR in mossy cells of the rat and mouse utilizing an immunocytochemical double-labeling technique. To identify mossy cells in the rat, we utilized an antibody to the glutamate receptor subunit 2/3 (GluR2/3). In addition to GluR2/3 antibodies, in the mouse, an antibody to the calcium-binding protein, calretinin (CR), to identify mossy cells was also employed. Our results show that GR immunoreactivity (IR) was colocalized with GluR2/3-IR in approximately 90% of the rat and the mouse mossy cells. In addition, GR-IR was identified in the CR-IR mossy cells in the mouse hippocampus, whereas the CR-IR interneurons of rat and mouse were negative for GR-IR. The presence of GR on mossy cells may indicate the ability of GC to mediate cellular activity of these cells.     

Ionic currents underlying rhythmic bursting of ventral mossy cells in the developing mouse dentate gyrus

The electrophysiological properties of mossy cells were examined in developing mouse hippocampal slices using whole-cell patch-clamp techniques, with particular reference to the dorsoventral difference. Dorsal mossy cells exhibited a higher spontaneous excitatory postsynaptic potential (EPSP) frequency and larger maximal EPSP amplitude than ventral mossy cells. On the other hand, the blockade of synaptic inputs with glutamatergic and GABAergic antagonists disclosed a remarkable dorsoventral difference in the intrinsic activity: none (0/27) of the dorsal mossy cells showed intrinsic bursting, whereas the majority (35/47) of the ventral mossy cells exhibited intrinsic rhythmic bursting. To characterize the ionic currents underlying the rhythmic bursting of mossy cells, we used somatic voltage-clamp recordings in the subthreshold voltage range. Ventral bursting cells possessed both hyperpolarization-activated current (Ih) and persistent sodium current (INaP), whereas dorsal and ventral nonbursting cells possessed Ih but no INaP. Blockade of Ih with cesium did not affect the intrinsic bursting of ventral mossy cells. In contrast, the blockade of INaP with tetrodotoxin or phenytoin established a stable subthreshold membrane potential in ventral bursting cells. The current-voltage curve of ventral bursting cells showed a region of tetrodotoxin-sensitive negative slope conductance between -55 mV and a spike threshold ( approximately -45 mV). On the other hand, no subthreshold calcium conductances played a significant role in the intrinsic bursting of ventral mossy cells. These observations demonstrate the heterogeneous electrophysiological properties of hilar mossy cells, and suggest that the subthreshold INaP plays a major role in the intrinsic rhythmic bursting of ventral mossy cells.     

Hilar mossy cells share developmental influences with dentate granule neurons

Mossy cells are the major class of excitatory neurons in the dentate hilus. Although mossy cells are involved in a range of physiological and pathological conditions, very little is known about their ontogeny. To gain insight into this issue, we first determined the developmental stage at which mossy cells can be reliably identified with the molecular markers calretinin and GluR2/3 and found that hilar mossy cells were first identifiable around the end of the 1st postnatal week. Birthdating studies combined with staining for these markers revealed that the appearance of mossy cells coincided with the first wave of dentate granule cell production during mid-gestation. Since mossy cells are born as the first granule cells are produced and it is believed that mossy cells originate from the neuroepithelium adjacent to the dentate progenitor zone, we examined to what extent the development of mossy cells is controlled by the same molecular pathways as that of granule cells. To do this, we analyzed the production of mossy cells in Lef1 and NeuroD mutant animals, in which granule cell production is disrupted during precursor proliferation or neuronal differentiation, respectively. The production of mossy cells was almost entirely lost in both mutants. Collectively, these data suggests that hilar mossy cells, unlike CA subfield pyramidal cells, are influenced by many of the same developmental cues as dentate granule cells.     

Differential expression of calretinin and metabotropic glutamate receptor mGluR1alpha defines subsets of unipolar brush cells in mouse cerebellum

The unipolar brush cell (UBC) is a type of glutamatergic interneuron in the granular layer of the cerebellum. The UBC brush and a single mossy fiber (MF) terminal contact each other within a cerebellar glomerulus, forming a giant synapse. Many UBCs receive input from extrinsic MFs, whereas others are innervated by intrinsic mossy terminals formed by the axons of other UBCs. In all mammalian species so far examined, the vestibulocerebellum is enriched of UBCs that are strongly immunoreactive for the calcium binding protein calretinin (CR) in both the somatodendritic and axonal compartment. UBCs have postsynaptic ionotropic glutamate receptors and extrasynaptic metabotropic glutamate receptors that immunocytochemically highlight their somatodendritic compartment and brush, respectively. In this study on the mouse cerebellum, we present evidence that immunoreactivities to CR and mGluR1alpha define two distinct UBC subsets with partly overlapping distributions in lobule X (the nodulus). In sections double-labeled for CR and mGluR1alpha, the patterns of distributions of CR(+)/mGluR1alpha(-) UBCs and CR(-)/mGluR1alpha(+) UBCs differed along the mediolateral and dorsoventral axes of the folium. Moreover, mGluR1alpha(+) UBCs outnumbered CR(+) UBCs. Both UBC subsets were mGluR2/3, GluR2/3, and NMDAR1 immunoreactive. The different distribution patterns of the two UBC subsets within lobule X suggest that expression of CR or mGluR1alpha by UBCs may be afferent-specific and related to the terminal fields of different vestibular MF afferents.     

Expression of c‐fos in hilar mossy cells of the dentate gyrus in vivo

Granule cells (GCs) of the dentate gyrus (DG) are considered to be quiescent—they rarely fire action potentials. In contrast, the other glutamatergic cell type in the DG, hilar mossy cells (MCs) often have a high level of spontaneous activity based on recordings in hippocampal slices. MCs project to GCs, so activity in MCs could play an important role in activating GCs. Therefore, we investigated whether MCs were active under basal conditions in vivo, using the immediate early gene c‐fos as a tool. We hypothesized that MCs would exhibit c‐fos expression even if rats were examined randomly, under normal housing conditions. Therefore, adult male rats were perfused shortly after removal from their home cage and transfer to the laboratory. Remarkably, most c‐fos immunoreactivity (ir) was in the hilus, especially temporal hippocampus. C‐fos‐ir hilar cells co‐expressed GluR2/3, suggesting that they were MCs. C‐fos‐ir MCs were robust even when the animal was habituated to the investigator and laboratory where they were euthanized. However, c‐fos‐ir in dorsal MCs was reduced under these circumstances, suggesting that ventral and dorsal MCs are functionally distinct. Interestingly, there was an inverse relationship between MC and GC layer c‐fos expression, with little c‐fos expression in the GC layer in ventral sections where MC expression was strong, and the opposite in dorsal hippocampus. The results support the hypothesis that a subset of hilar MCs are spontaneously active in vivo and provide other DG neurons with tonic depolarizing input. © 2013 Wiley Periodicals, Inc.     

GluR1-immunopositive interneurons in rat neocortex

Recent in vitro studies suggest that inhibitory interneurons in cortex may express the GluR1 glutamate receptor subunit in the absence of GluR2, leading to calcium-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) channels. We performed a study of rat somatic sensory cortex to confirm and extend these observations, using quantitative immunocytochemistry for multiple antigens. A morphologically distinct subpopulation of nonpyramidal neurons in neocortex was intensely immunoreactive for GluR1. Electron microscopic analysis of these cells revealed somatic staining for GluR1, mainly in the rough endoplasmic reticulum. Dendritic staining was concentrated at the synaptic active zone and in the adjacent subsynaptic cytoplasm. Double immunostaining revealed that the large majority of intensely GluR1-positive cells contained γ-aminobutyric acid or its synthetic enzyme, glutamic acid decarboxylase, but little or no GluR2. Thus, AMPA receptors on a subpopulation of inhibitory interneurons in cortex are likely to be calcium permeable. This calcium permeability is likely to influence functional properties of these neurons; it may underlie the high levels of calcium-binding proteins they contain; and may render them liable to excitotoxic injury. © 1996 Wiley-Liss, Inc.     

Chrna2‐OLM interneurons display different membrane properties and h‐current magnitude depending on dorsoventral location

The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2‐cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike‐frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization‐activated current (I_h) compared to ventral OLM cells. Immunohistochemical examination indicates the h‐current to correspond to hyperpolarization‐activated cyclic nucleotide‐gated subunit 2 (HCN2) channels. Computational studies suggest that I_h in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.     

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: correlation of cell loss with mossy fiber sprouting

Mossy fiber sprouting and the genesis of ectopic granule cells contribute to reverberating excitation in the dentate gyrus of epileptic brain. This study determined whether the extent of sprouting after status epilepticus in rats correlates with the seizure-induced degeneration of GluR2-immunoreactive (GluR2+) hilar neurons (presumptive mossy cells) and also quantitated granule cell-like GluR2-immunoreactive hilar neurons. Stereological cell counting indicated that GluR2+ neurons account for 57% of the total hilar neuron population. Prolonged pilocarpine-induced status epilepticus killed 95% of these cells. A smaller percentage of GluR2+ neurons (74%) was killed when status epilepticus was interrupted after 1-3.5 h with a single injection of phenobarbital, and the number of residual GluR2+ neurons varied among animals by a factor of 6.2. GluR2+ neurons were not necessarily more vulnerable than other hilar neurons. In rats administered phenobarbital, the extent of recurrent mossy fiber growth varied inversely and linearly with the number of GluR2+ hilar neurons that remained intact (P=0.0001). Thus the loss of each GluR2+ neuron was associated with roughly the same amount of sprouting. These findings support the hypothesis that mossy fiber sprouting is driven largely by the degeneration of and/or loss of innervation from mossy cells. Granule cell-like GluR2-immunoreactive neurons were rarely encountered in the hilus of control rats, but increased 6- to 140-fold after status epilepticus. Their number did not correlate with the extent of hilar cell death or mossy fiber sprouting in the same animal. The morphology, number, and distribution of these neurons suggested that they were hilar ectopic granule cells.     

Distribution of calbindin, parvalbumin, and calretinin immunoreactivity in the reticular thalamic nucleus of the marmoset: evidence for a medial leaflet of incertal neurons

The placement of the reticular thalamic nucleus (RTN) between the dorsal thalamus and the cortex and the inhibitory nature of reticulothalamic projections has led to suggestions that it "gates" the flow of sensory information to the cortex. The New World diurnal monkey, the marmoset, Callithrix jacchus is emerging as an important "model primate" for the study of sensory processing. We have examined the distribution of Nissl-stained somata and calbindin, parvalbumin, and calretinin immunoreactivity in the ventral thalamus for comparison with other species. Cells were labeled using standard immunohistochemistry, ExtraAvidin-HRP, and diaminobenzidine reaction products. The RTN is constituted by a largely homogeneous population of parvalbumin immunoreactive cells with respect to size and orientation. Calbindin and calretinin immunoreactive cells were only found along the medial edge of the RTN adjacent to the external medullary lamina of the dorsal thalamus and laterally near the ventral RTN. These cells were considered to be part of the zona incerta (ZI). The marmoset ZI could be subdivided into dorsal and ventral regions on the basis of its immunoreactivity to calcium binding proteins. Both the ZI and nucleus subthalamicus Luysi contained scattered calbindin and calretinin immunoreactive cells with well-defined dendritic processes. These cells were clearly different to cells in the dorsal thalamus. Parvalbumin immunoreactive cells in RTN, ZI, and subthalamic nucleus were on average larger than neurons positive for the other calcium binding proteins. Future studies reporting the afferent and efferent projections to the RTN must view their results in terms of the close apposition of RTN and ZI somata.     

The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat

The localization of serotonin_2A (5-HT_2A) receptors in the adult rat spinal cord and dorsal root ganglia was examined by using a polyclonal antibody that recognizes the C-terminus peptides of the mouse 5-HT_2A receptor. Positive cell bodies of 5-HT_2A receptor were found in several regions of the spinal cord. Generally, large-to-intermediate sized neuronal cell bodies were intensely immunolabeled. Motoneurons in the ventral horn were the most intensely labeled. Dot-like immunoreactive profiles were located beneath the cell membrane of motoneurons. Neuronal somata in the intermediolateral nucleus of the thoracic spinal cord were moderately labeled. The immunoreactivity in the dorsal horn was weak. A considerable number of glial cell bodies in the white matter were immunostained. The majority of both small and large sized neurons were 5-HT_2A immunopositive in the dorsal root ganglion.     

Calretinin-Containing Neurons in Rat Cerebellar Granule Cell Cultures

Using an antiserum against calretinin, a calcium-binding protein, we discovered two distinct neuronal cell types that stain intensely in enriched cerebellar granule cells. One neuronal cell type resembles unipolar brush cells, whereas the other resembles Lugaro cells. During early culture times, these calretinin-positive neurons are most numerous but represent less than one percent of the total neuronal population. In cultured cells, calretinin mRNA levels peak at day three in vitro, followed by a rapid decline to undetectable levels by day six in vitro. However, calretinin-immunoreactive neurons are observed up to 29 days in vitro. Excitotoxic concentrations of glutamate receptor agonists failed to elicit an excitotoxic response on the intensely staining calretinin-positive neurons, whereas greater than 95% of the cerebellar granule cells were susceptible to the excitotoxic actions of the glutamate receptor agonists. To distinguish between the two possibilities that calretinin-positive neurons either do not express glutamate receptors or they are not susceptible to the excitotoxic effects of glutamate receptor agonists, we performed immunocytochemistry using glutamate receptor antibodies to detect the presence of receptor protein. We found that the AMPA/kainate glutamate receptor (GluR₂R₃) colocalized with calretinin, suggesting that calretinin-immunoreactive neurons express the AMPA/kainate receptor; cerebellar granule cells, which are known to express this receptor, were also immunoreactive for the GluR₂R₃ receptor.     

Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression

To better understand the organization and evolution of the dorsal thalamus, we have made a first approach to analyze the possible histogenetic compartments of the mammalian dorsal thalamus using mouse embryos. For that, we have analyzed the expression of the proneural gene Math4a and the protein calretinin. Our results suggest the existence of rostrodorsal, caudoventral, and ventral compartments in the embryonic dorsal thalamus of the mouse, which partly parallel the dorsoventral histogenetic tiers postulated in the dorsal thalamus of sauropsids. The rostrodorsal compartment of the mouse dorsal thalamus is characterized by expression of Math4a, and it appears to include sensory and motor thalamic nuclei projecting to the dorsal pallium (isocortex). This compartment appears equivalent to the lemnothalamus proposed by Butler in tetrapods based on hodological grounds. The caudoventral and ventral compartments of the mouse dorsal thalamus lack expression of Math4a in the mantle, but they are characterized by several populations of calretinin-immunorective neurons that show projections to the claustroamygdaloid region in the ventrolateral pallium. More studies will be needed to analyze if the compartments proposed in this study represent true histogenetic units, and to find homologous developmental fields in all vertebrates.     

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of lungfishes

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.