Calretinin-immunoreactive Cells Research Articles

Neurochemical organization of the nucleus paramedianus dorsalis in the human

We have characterized the neurochemical organization of a small brainstem nucleus in the human brain, the nucleus paramedianus dorsalis (PMD). PMD is located adjacent and medial to the nucleus prepositus hypoglossi (PH) in the dorsal medulla and is distinguished by the pattern of immunoreactivity of cells and fibers to several markers including calcium-binding proteins, a synthetic enzyme for nitric oxide (neuronal nitric oxide synthase, nNOS) and a nonphosphorylated neurofilament protein (antibody SMI-32). In transverse sections, PMD is oval with its long axis aligned with the dorsal border of the brainstem. We identified PMD in eight human brainstems, but found some variability both in its cross-sectional area and in its A–P extent among cases. It includes calretinin immunoreactive large cells with oval or polygonal cell bodies. Cells in PMD are not immunoreactive for either calbindin or parvalbumin, but a few fibers immunoreactive to each protein are found within its central region. Cells in PMD are also immunoreactive to nNOS, and immunoreactivity to a neurofilament protein shows many labeled cells and fibers. No similar region is identified in atlases of the cat, mouse, rat or monkey brain, nor does immunoreactivity to any of the markers that delineate it in the human reveal a comparable region in those species. The territory that PMD occupies is included in PH in other species. Since anatomical and physiological data in animals suggest that PH may have multiple subregions, we suggest that the PMD in human may be a further differentiation of PH and may have functions related to the vestibular control of eye movements.

Populations of hippocampal inhibitory neurons express different levels of cytochrome c

Cytochrome c (CC) immunoreactivity was quantified in functionally distinct rat hippocampal inhibitory neuron populations using double immunocytochemistry and laser scanning confocal microscopy to measure the CC expression level as well as the amount of mitochondria within the cells, which is a sign of neuronal activity. The CC signal showed a similar distribution to cytochrome c oxidase histochemical staining. Strongly stained somata, dendrites and axon terminal clouds were dispersed over the low intensity neuropil staining. The staining was granular and electron microscopic investigation confirmed that the signal was localized in mitochondria. Intensively labeled neurons, showing the morphological features of inhibitory cells, were most frequently found in the principal cell layers, stratum oriens of the CA1-3 areas, stratum lucidum and hilus. These neurons contained not only a higher number of mitochondria than the principal cells but the intensity of the mitochondrial staining was evidently stronger. Among the examined interneuronal populations, parvalbumin-immunoreactive neurons were intensively labeled for CC. Calbindin D28k- (CB), somatostatin- and cholecystokinin-labeled cells showed heterogeneous CC levels, whereas calretinin-immunoreactive cells never showed a strong CC signal. CB cells in stratum oriens and alveus layers, lucidum and the hilus were strongly labeled for CC. CB cells in such regions are known to project to the medial septum and contain somatostatin. We have demonstrated that the CA1 interneurons that project to the medial septum (hippocampo-septal neurons) express a high level of CC. Thus, similar to the parvalbumin-containing basket and axo-axonic cells, the hippocampo-septal neurons potentially have a high average activity level.

Mapping glutamate responses in immunocytochemically identified neurons of the mouse retina

The mammalian retina contains as many as 50-60 unique cell types, many of which have been identified using various neurochemical markers. Retinal neurons express N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) receptor subunits in various mixtures, densities, and spatial distributions. Ionotropic glutamatergic drive in retinal neurons can be mapped using a cation channel permeant guanidinium analog called agmatine (1-amino-4-guanidobutane; AGB). This alternative approach to physiologically characterize neurons in the retina was introduced by Marc (1999, J Comp Neurol 407:47-64, 407:65-76), and allows the simultaneous mapping of responses of glutamate receptor-gated channels from an entire population of neurons. Unlike previous AGB studies, we colocalized AGB with various macromolecular markers using direct and indirect immunofluorescence to characterize the glutamate agonist sensitivities of specific cell types. Activation with NMDA, AMPA, and KA resulted in AGB entry into neurons in a dose-dependent manner and was consistent with previous receptor subunit localization studies. Consistent with the various morphological phenotypes encompassed by the calbindin and calretinin immunoreactive cells, we observed various functional phenotypes revealed by AGB labeling. Not all calbindin or calretinin immunoreactive cells showed ligand-evoked AGB permeation. A small proportion either did not possess functional glutamate receptors, required higher activation thresholds, or express functional channels impermeable to AGB. AMPA and KA activation of bipolar cells resulted in AGB permeation into the hyperpolarizing variety only. We also studied the glutamate ligand-gating properties of 3[alpha1-3]-fucosyl-N-acetyl-lactosamine (CD15) immunoreactive cells and show functional responses consistent with receptor subunit gene expression patterns. CD15-immunoreactive bipolar cells only responded to AMPA but not KA. The CD15 immunoreactive amacrine cells demonstrated an identical selectivity to AMPA activation, but were also responsive to NMDA. Finally, localization of AGB secondary to glutamate receptor activation was visualized with a permanent reaction product.

Regional difference in corticotropin-releasing factor immunoreactivity in mossy fiber terminals innervating calretinin-immunoreactive unipolar brush cells in vestibulocerebellum of rolling mouse Nagoya

Unipolar brush cells (UBCs), a class of interneurons in the vestibulocerebellum, play roles in amplifying excitatory inputs from vestibulocerebellar mossy fibers. This study aimed to clarify whether corticotropin-releasing factor (CRF)-positive mossy fiber innervation of calretinin (CR)-positive UBCs was altered in rolling mouse Nagoya (RMN). The distribution and the number of CR-positive UBCs in the vestibulocerebellum were not different between RMN and control mice. Double immunofluorescence revealed that some CRF-positive mossy fiber terminals were in close apposition to CR-positive UBCs. In the lobule X of vermis, such mossy fiber terminals were about 5-fold greater in number in RMN than in controls. In contrast, the number of CRF-positive mossy fiber terminals adjoining CR-positive UBCs in the flocculus was not significantly different between RMN and controls. The results suggest increased number of CRF-positive mossy fiber terminals innervating CR-positive UBCs in the lobule X but not in the flocculus of RMN. CRF may alter CR-positive UBC-mediated excitatory pathways in the lobule X of RMN and may disturb functions of the lobule X such as cerebellar adaptation for linear motion of the head.

Expression of calcium-binding proteins in the proliferative zones around the corticostriatal junction of rabbits during pre- and postnatal development

Herein we asked whether cells expressing calcium-binding proteins around the corticostriatal junction are of pallial or subpallial origin. Brains of rabbit embryos between embryonic day E18 and E28 and postnatal day 0–P22 were immunoreacted with monoclonal antibodies raised against calretinin, calbindin and parvalbumin. At E18–E21, calbindin- and calretinin-immunoreactive cells were seen in distinct proliferative zones in the vicinity of the corticostriatal junction. Whereas calbindin-immunoreactive neurons were in the ventricular zone of the ventral pallium (the medial wall of the lateral ventricular angle), calretinin-immunoreactive cells were, nearby, in the subventricular zone of the subpallium at the lateral edge of the lateral ganglionic eminence. From E25 to P22, both calbindin- and calretinin-immunoreactive cells appeared in the pallial ventricular and subventricular zones around the lateral ventricular angle. Some of these cells resembled migratory neuroblasts. Parvalbumin-immunoreactive cells appeared at P5–P10, albeit they were almost negligible in the proliferative zones around the corticostriatal junction and the lateral ventricular angle. The results suggest that a number of the calbindin-expressing neurons that are generated in mid-gestation and postnatally are of pallial origin. They also indicate that only a few of the late-generated calretinin-immunoreactive cells may have a pallial source. The origin of the parvalbumin-immunoreactive cells was not ascertained in the present study.

Immunoreactivity for calcium-binding proteins defines subregions of the vestibular nuclear complex of the cat

The vestibular nuclear complex (VNC) is classically divided into four nuclei on the basis of cytoarchitectonics. However, anatomical data on the distribution of afferents to the VNC and the distribution of cells of origin of different efferent pathways suggest a more complex internal organization. Immunoreactivity for calcium-binding proteins has proven useful in many areas of the brain for revealing structure not visible with cell, fiber or Golgi stains. We have looked at the VNC of the cat using immunoreactivity for the calcium-binding proteins calbindin, calretinin and parvalbumin. Immunoreactivity for calretinin revealed a small, intensely stained region of cell bodies and processes just beneath the fourth ventricle in the medial vestibular nucleus. A presumably homologous region has been described in rodents. The calretinin-immunoreactive cells in this region were also immunoreactive for choline acetyltransferase. Evidence from other studies suggests that the calretinin region contributes to pathways involved in eye movement modulation but not generation. There were focal dense regions of fibers immunoreactive to calbindin in the medial and inferior nuclei, with an especially dense region of label at the border of the medial nucleus and the nucleus prepositus hypoglossi. There is anatomical evidence that suggests that the likely source of these calbindin-immunoreactive fibers is the flocculus of the cerebellum. The distribution of calbindin-immunoreactive fibers in the lateral and superior nuclei was much more uniform. Immunoreactivity to parvalbumin was widespread in fibers distributed throughout the VNC. The results suggest that neurochemical techniques may help to reveal the internal complexity in VNC organization.

Prenatal exposure to cocaine selectively disrupts the development of parvalbumin containing local circuit neurons in the medial prefrontal cortex of the rat

Exposure to cocaine in utero can result in cognitive deficits potentially through a disruption in the inhibitory processes of the frontal cortex. One potential mechanism is through alterations in the inhibitory local circuit neurons containing the calcium-binding protein, parvalbumin. Parvalbumin-immunostaining primarily identifies 2 types of local circuit neurons: larger, rounder, axo-somal basket cells and smaller, more-spindle shaped, axo-axonic chandelier cells. Both are thought to have critical impact on the excitatory/inhibitory balance due to the proximal site of projection on pyramidal neurons. Calretinin, another calcium-binding protein, identifies a distinct sub-population of inhibitory local circuits that impinges more distally on the dendritic arbor and serves as a control population for this study. Here, we examine local circuit neurons containing either parvalbumin or calretinin in adolescent male rats (approximately 45 days old) exposed to saline or cocaine (3 mg/kg, intravenous twice a day during embryonic days 10 to 20). Prenatal cocaine exposure caused select changes in the parvalbumin, but not calretinin, containing cells in the frontal cortex. Specifically, prenatal cocaine exposure is associated with a 50% reduction in spindle-shaped parvalbumin-immunoreactive cells potentially indicating a select loss of chandelier cells or a shift to a rounder shape. Additionally, a reduction in the number of dendrites of parvalbumin-immunoreactive cells in rats exposed to cocaine in utero was noted. Other measures of both parvalbumin- and calretinin-immunoreactive cells were unchanged, including total number of cells, distribution by depth, and sizes of cells. These changes to the excitatory/inhibitory balance in the frontal cortex may contribute to the cognitive deficits associated with prenatal cocaine exposure.

Calretinin/PSA-NCAM immunoreactive granule cells after hippocampal damage produced by kainic acid and DEDTC treatment in mouse

There is a dramatic increase in the number of lightly immunoreactive calretinin cells in the granular layer of the dentate gyrus of the mouse hippocampus 1 day after excitotoxic injury using kainic acid combined with the zinc chelator diethyldithiocarbamate. At 7 days after treatment, these cells are strongly immunoreactive for calretinin and for the polysialated form of the glycoprotein neural cell adhesion molecule (PSA-NCAM). The reexpression of calretinin and PSA-NCAM after treatment corresponds well with the loss of input from the damaged hilar mossy cells. These cells could be considered immature granule cells since they are immunoreactive to markers for immature cells such as PSA-NCAM, and are not immunoreactive to calbindin D28k and neuronal nuclear specific protein NeuN (present in mature granule cells), or GABA (present in interneurons). Ultrastructural analysis of these cells indicates that they are immature. Labelling of cell proliferation with 5-bromo-2′-deoxyuridine (BrdU) shows that by day 1 no calretinin immunoreactive cell of the dentate gyrus corresponds to newly generated cells. By day 7 only 6% of the calretinin immunoreactive cells in the dentate gyrus are marked for BrdU. Our data indicate that the CR/PSA-NCAM immunoreactive cells of the dentate gyrus, in spite of their immature characteristics, are not the products of reactive neurogenesis. These cells could represent a reservoir of pre-existing not completely differentiated granule cells that react to damage.

Immunocytochemical localization of calretinin in the superficial layers of the cat superior colliculus.

We localized calretinin-immunoreactive (IR) fibers and cells in the superior colliculus (SC) of the cat and studied the distribution and effect of enucleation on the distribution of this protein. Calretinin was localized with antibody immunocytochemistry. A dense plexus of anti-calretinin-IR fibers was found within the upper part of the superficial gray layer. Almost all of the labeled fibers were small diameter fibers with few varicosities. Monocular enucleation produced an almost complete reduction of calretinin-IR fibers in the SC contralateral to the enucleation. Furthermore, many calretinin-IR cells appeared in the contralateral SC. The newly appeared cells had small- to medium-sized vertical fusiform, oval or round, or stellate cell bodies. Two-color immunofluorescence revealed that no cells in the superficial layers expressed both calretinin and GABA. Many retinal ganglion cells were labeled after injections of retrograde axonal transport horseradish peroxidase (HRP) in the superficial layers. However, no large cells were double-labeled with calretinin and HRP. More than 95% of the double-labeled cells were small cells (<15 microm). Based on the retinal ganglion cell size, we believe that the vast majority of calretinin-IR retinocollicular fibers in cat SC are small gamma type cells that have W type physiologies.

Selective deficits in prefrontal cortical GABAergic neurons in schizophrenia defined by the presence of calcium-binding proteins

Background: Postmortem studies have provided evidence for abnormalities of the γ-aminobutyric acid (GABA)-ergic system in schizophrenia, including deficits of GABA-containing interneurons. The calcium-binding proteins parvalbumin, calbindin, and calretinin can be used as markers for specific subpopulations of cortical GABAergic interneurons. Methods: Following our previous observation of a reduction in the density of parvalbumin- but not calretinin-immunoreactive cells in the prefrontal cortex (Brodmann area 10) in schizophrenia, we have quantified the laminar density of neurons immunoreactive for the calcium-binding proteins parvalbumin, calbindin, and calretinin in a further prefrontal cortical region (Brodmann area 9) in patients with schizophrenia, bipolar disorder, major depression, and in matched control subjects (each group n = 15). Results: Initial statistical analysis revealed reductions in the total cortical density of parvalbumin- and calbindin- but not calretinin-immunoreactive neurons in schizophrenia relative to control subjects. Further analysis comparing individual laminar densities between groups indicated that, following correction for multiple comparisons, only a reduction in calbindin-immunoreactive neurons in cortical layer II in the schizophrenic group attained statistical significance. Conclusions: These findings suggest that deficits of specific GABAergic neurons, defined by the presence of calcium-binding proteins, are present in schizophrenia. Trends toward similar reductions are observed in bipolar disorder.

Calretinin and FMRFamide immunoreactivity in the nervus terminalis of prenatal tree shrews ( Tupaia belangeri)

The distribution and development of FMRFamide- and calretinin-immunoreactive neurons were investigated in the nervus terminalis of prenatal tree shrews from gestation day 19 onwards. The first FMRFamide-immunoreactive cells were observed medially in the olfactory epithelium on gestation day 20. From gestation day 23 onwards, the migrating nervus terminalis ganglion cells showed FMRFamide calretinin immunoreactivity. The distribution pattern of FMRFamide- and calretinin-immunoreactive cells was similar along the migratory route and in the ganglion of the terminal nerve. However, most probably calretinin and FMRFamide were expressed in separate neuronal populations. For the first time in a mammal, FMRFamide and calretinin are reported to occur in the migrating perikarya and neuronal processes of the nervus terminalis during prenatal development. The results suggest (i) an early activation of the rostral FMRFamide-immunoreactive migratory stream comparable to that described for the GnRH-immunoreactive part of the terminal nerve in other mammals and possibly (ii) an involvement of calretinin in mechanisms of cell migration and outgrowth of neuronal processes in the terminal nerve during the studied period.

Excitatory and inhibitory neurons express c-Fos in barrel-related columns after exploration of a novel environment

Recent work has shown that behaviorally meaningful sensory information processing is accompanied by the induction of several transcription factors in the barrel cortex of rodents. It is now generally accepted that stimulus–transcription coupling is an important step in the sequence of events leading to long-term plastic changes in neuronal structure and function. Nevertheless, so far few data are available as to what types of neurons are involved in such a genomic response. Here, we determined the morphological and neurochemical identity of neurons in rat barrel cortex showing a c-Fos-immunoreactive nucleus after exploration of an enriched environment. Double stainings of c-Fos and glial fibrillary acidic protein excluded astrocytes as a possible cell type expressing this transcription factor. By morphological phenotyping with intracellular Lucifer Yellow injections, it was found that a large majority were probably excitatory pyramidal cells, but inhibitory interneurons were also found to contain c-Fos-immunoreactive nuclei. By neurochemical phenotyping of GABAergic interneurons with specific antibodies, a significant induction was found, in a layer-dependent manner, for the populations of glutamic acid decarboxylase-, parvalbumin-, calbindin- and vasoactive intestinal polypeptide-immunoreactive neurons but not for calretinin-immunoreactive cells in experimental compared to control columns. From these data we conclude that thalamic afferents effectively drive cortical excitatory as well as inhibitory intracortical circuits. Thus, the adaptations of receptive field properties of cortical neurons after different manipulations of the sensory periphery are likely to be caused by plastic changes in excitatory and inhibitory networks.

Cell dynamics of calretinin-immunoreactive neurons in the rostral migratory stream after ibotenate-induced lesions in the forebrain.

It is now apparent that adult neurogenesis is taking place during life in the olfactory bulb (OB) of the rodent brain. In the olfactory nervous system, the precursor cells of the subventricular zone are known to continually proliferate, migrate through the rostral migratory stream (RMS) and differentiate into the bulbar neurons. The RMS, consisting of heterogeneous cell populations of the neural and neuronal precursor cells, is the unique forebrain structure that provides a long-distance migratory route for the precursor cells. The present study was undertaken to examine whether neuronal regeneration, focusing on calretinin-immunoreactive (+) cells, may proceed in the RMS following lesions induced by an excitotoxin. Two days after ibotenate injections, massive degeneration of calretinin (+) cells occurred in the RMS and its adjacent forebrains. Thereafter, calretinin (+) cells gradually increased in the RMS and reached above their control value 2 weeks after ibotenate injections. Removal of the OB also produced a marked increase in calretinin (+) cells in the RMS. Autoradiographic experiments using (3)H-thymidine showed that calretinin (+) cells were continually generated in the RMS and underwent neuronal turnover within 8 weeks in a normal condition. The results indicate that, in terms of calretinin (+) cells, neuronal differentiation and replacement is continually taking place within the RMS, and that the RMS is capable of repopulating those cells which were injured by ibotenate.

GABAergic neuronal subtypes in the human frontal cortex — development and deficits in schizophrenia

Recent studies have provided evidence for a deficit of GABA-containing interneurons in the frontal cortex in schizophrenia. That this deficit might be brought about during early foetal or neonatal life is a hypothesis consistent with the substantial indications for a neurodevelopmental aetiology of the disease. GABAergic neurons can be defined by the presence of one of three types of calcium binding proteins, which are thought to have neuroprotective properties. We have undertaken an investigation into the postnatal ontology of these neuronal subtypes and find that calretinin expression is relatively constant and present from before birth, calbindin expression is also present early but redistributes in the cortex over the first months of life, while parvalbumin-immunoreactivity is not observed until between 3 and 6 months of age. Investigation of frontal cortical tissue taken post mortem from a series of schizophrenic patients and matched control subjects revealed that parvalbumin-, but not calretinin-immunoreactive cells are significantly diminished in schizophrenia. These observations support the hypothesis that GABAergic deficits in schizophrenia may stem from toxic events occurring during cortical development which selectively target immature neurons before protection by parvalbumin is conferred.

Expression of calbindin and calretinin in the human ganglionic eminence

The conspicuous ganglionic eminence representing a part of the telencephalic proliferative zone contains neuroblasts of the striatum. Recently it has been found to contribute significantly to the class of interneurons in the cerebral cortex. Subpopulations of cortical interneurons contain calretinin and calbindin. The expression of calretinin and calbindin in the ganglionic eminence and adjacent areas has been investigated immunocytochemically during fetal development using the brains of 10 infants ranging in age from 16 and 26 weeks gestation. Between 16 and 20 weeks gestation, numerous calretinin-immunreactive nerve cells are found in the ganglionic eminence, particularly in the mantle and the intermediate zones. The number of calretinin-immunoreactive cells decreases gradually from 21 weeks gestation onwards. Larger calbindin-immunoreactive cells are seen in the ganglionic eminence, and their number increases from 20 weeks gestation in the mantle zone. These results may indicate that calretinin-immunoreactive precursor cells, found in the ganglionic eminence, tangentially migrate toward the cortex. Moreover, the mantle zone displaying a specific calretinin and calbindin immunolabeling may represent an intermediate target for outgrowing axons. The findings are discussed with regard to central nervous system complications in preterm infants involving the ganglionic eminence.

Distribution of calcium binding proteins, sodium channels and persistent sodium current in the rat ventral respiratory group

The ventral respiratory group (VRG) is important in generating both the breathing rhythm and motor pattern. The VRG is generally subdivided into 4 regions, the Botzinger complex, preBotzinger Complex (pBc), rostral VRG, and caudal VRG based on neuron discharge patterns and axonal projections. Anatomical markers provide a potential bridge between the physiological analysis of the respiratory system and the neurochemical elements that are the ultimate building blocks of this system. An example of this approach is the coincidence of NK1 receptors with neurons in the pBc complex [1]. VRG labeling by antibodies to calcium binding proteins (parvalbumin, calbindin, and calretinin) in some respects complements the NK1 immunoreactivity. Parvalbumin has been suggested to label VRG neurons [2]. We have verified this by demonstrating that parvalbumin positive neurons project to the ipsilateral and contralateral VRG as well as to the phrenic nucleus. We have also found that a prominent gap in the column of VRG related parvalbumin cells [2] likely corresponds to the pBc since parvalbumin cells are rare in this zone and never co-localize with NK1 receptors. Calbindin and calretinin immunoreactive cells are scattered in the pBc and rostral VRG but rare in the Botzinger complex. Calbindin neurons are intermingled, but not colocalized with pBc NK1 cells. Calretinin is not colocalized with NK1, except for a small population of cells at the caudal ventral edge of the pBc, which likely corresponds to NK1 bulbospinal neurons [3]. Finally, preliminary evidence indicates glycine immunoreactivity in some parvalbumin neurons within the Botzinger complex region. In addition to this compartmentalized distribution of calcium binding proteins within the VRG, preliminary evidence is consistent with a differential distribution of Na channel alpha subunits. A slowly inactivating persistent Na current is postulated to underlie the pacemaker activity seen in a subset of pBc neurons [4]. Within the CNS at least 5 different Na channel alpha subunits have been identified, termed Nav1.1, 1.2, 1.3, 1.5 and 1.6. Of these Nav1.6 has been most strongly linked to persistent Na current. Immunohistochemical examination of Nav1.2, 1.3 and 1.6 demonstrated Nav1.2 is widely expressed in the VRG including some NK1 neurons. Nav1.3 was present in cranial motoneurons. In neurons acutely dissociated from the pBc of 1–15 day old rats, whole-cell voltage clamp recordings were used to analyze transient and persistent Na currents and single-cell RT-PCR was used to probe for Nav1.1, 1.2 and 1.6 mRNA. Whole-cell recordings were made using an external solution containing 50 mM NaCl. Slow ramp depolarization from -80 to +30 mV revealed a TTX-sensitive, persistent Na current. The current kinetics were dependent upon the ramp speed. A slow ramp (100 mV/s), elicited an inward non-inactivating current in 42% (10 of 24) of neurons sampled from the pBc. These results are consistent with a role for persistent Na current in regulation of the subthreshold behavior, including pacemaker activity. Moreover, single-cell RT-PCR revealed the presence of Nav1.6 in 40 of 72 cells (55%). The Nav1.1 subunit mRNA was present in 47 of 82 neurons (57%) and was co-expressed with Nav1.6 in 28% of cells. Preliminary findings on Nav1.2 mRNA are consistent with the immunohistochemistry with it present in 7 of 18 (38%) pBc neurons.

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.

Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis

Previously we showed that large multipolar cells immunoreactive for calretinin and subunits 2 and 3 of amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptors (GluR2/3) clustered in the ventral hilus of the mouse dentate gyrus and revealed that they were mossy cells. Although such large calretinin immunoreactive cells were not seen in the dorsal hilus, our Golgi study revealed the presence of mossy cells in the dorsal hilus. As we observed large intensely GluR2/3 immunoreactive cells in the dorsal hilus, we suggested that these calretinin negative but intensely GluR2/3 positive large cells in the dorsal hilus were also mossy cells. In the present study we confirmed this identification with several methods. The extracellular tracer labeling studies revealed that all of 47 mossy cells identified morphologically were intensely GluR2/3 positive but calretinin negative, whereas none of 22 non-mossy hilar neurons were intensely GluR2/3 positive. Electron microscopically most of intensely GluR2/3 positive somata and dendritic processes showed the characteristic ultrastructural features of mossy cells. Furthermore, the fimbria–fornix–hippocampal commissure transection procedures induced the calretinin expression in some of these dorsal GluR2/3 immunoreactive cells. On the basis of these observations, we concluded that the vast majority of intensely GluR2/3 immunoreactive large cells in the mouse dorsal hilus were mossy cells. Then we evaluated the presumed difference in the distribution of mossy cells along the dorsoventral axis by the disector. The numerical density of mossy cells was about 1.4 times larger at the ventral level than at the dorsal level, indicating that the dorsoventral difference in the distribution of mossy cells in the mouse hilus was far smaller than that previously speculated.

Calcium-binding proteins and GABA reveal spatial segregation of cell types within the developing lateral superior olivary nucleus of the ferret.

Chemical characteristics of developing neurons in the superior olivary complex of the ferret were analyzed using immunohistochemical methods. The present report of calcium-binding proteins in the developing and adult superior olivary complex shows distinct distribution patterns for parvalbumin, calbindin, and calretinin in the lateral superior olivary nucleus (LSO) of the developing ferret that correspond to distribution patterns for different projection cell types and neurotransmitters. In the neonate, there was an initial complementary distribution of calcium-binding proteins between the shell and core of the body of the developing LSO. Parvalbumin and calbindin-immunoreactive cells were present in the shell, whereas calretinin-immunoreactive cells were restricted to the core of the LSO. Gamma amino butyric acid (GABA), but not glycine, immunoreactive cells were distributed similarly in the shell of the LSO in the neonate. There were, in addition, reciprocal medial-to-lateral gradients of parvalbumin and calbindin-immunoreactive cells in the LSO shell of the neonate. These complementary patterns in the LSO were transient, however, and by the end of the second postnatal week, each calcium-binding protein differed markedly in its cellular distribution in the superior olive, including the LSO. GABA-immunoreactive cells also were restricted transiently to the shell of the LSO in neonates. The radial segregation of transient calcium-binding expression in LSO cells was orthogonal to the medial-to-lateral axis in the LSO and, therefore, parallels fibrodendritic layers and presumed isofrequency planes of the LSO. The early postnatal segregation of calcium-binding proteins in the isofrequency axis was congruent with the gradients of contralateral and ipsilateral projection cell types in adult LSO. It seems likely that developmental mechanisms regulate expression of calcium-binding protein and neurotransmitter phenotypes and that these mechanisms operate in development within the isofrequency axis as well as along the tonotopic axis of this auditory nucleus.

Cholinergic, but not the rod pathway-related glycinergic (AII), amacrine cells contain calretinin in the rat retina

Double-label immunocytochemistry was carried out on cryostat sections of rat retina to test for the presence of calretinin in cholinergic starburst and the rod pathway-related glycinergic (AII) amacrine cells. All cholinergic cells contained calretinin, but calretinin-immunoreactive cells were much more numerous in both the inner nuclear and ganglion cell layers than the cholinergic cells. Glycinergic AII amacrine cells have been found to contain calretinin in cat, monkey and rabbit retinas. Since AII amacrine cells in rat can be selectively labeled with antibodies against parvalbumin, in a second experiment we attempted to colocalize these proteins. We found that calretinin- and parvalbumin-immunoreactive neurons belonged to distinct amacrine cell populations permitting the conclusion that, in the rat retina, AII amacrine cells do not contain calretinin. The results indicate that even those amacrine cells of the mammalian retina that are highly conserved with respect to morphology and transmitter content, may differ with respect to other neurochemical characteristics, such as their calcium-binding proteins.