MAB1568 Sigma-AldrichAnti-Calretinin Antibody, clone 6B8.2

Retinogenesis is a precisely controlled developmental process during which different types of neurons and glial cells are generated under the influence of intrinsic and extrinsic factors. Three transcription factors, Foxn4, RORβ1 and their downstream effector Ptf1a, have been shown to be indispensable intrinsic regulators for the differentiation of amacrine and horizontal cells. At present, however, it is unclear how Ptf1a specifies these two cell fates from competent retinal precursors. Here, through combined bioinformatic, molecular and genetic approaches in mouse retinas, we identify the Tfap2a and Tfap2b transcription factors as two major downstream effectors of Ptf1a. RNA-seq and immunolabeling analyses show that the expression of Tfap2a and 2b transcripts and proteins is dramatically downregulated in the Ptf1a null mutant retina. Their overexpression is capable of promoting the differentiation of glycinergic and GABAergic amacrine cells at the expense of photoreceptors much as misexpressed Ptf1a is, whereas their simultaneous knockdown has the opposite effect. Given the demonstrated requirement for Tfap2a and 2b in horizontal cell differentiation, our study thus defines a Foxn4/RORβ1-Ptf1a-Tfap2a/2b transcriptional regulatory cascade that underlies the competence, specification and differentiation of amacrine and horizontal cells during retinal development.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

As the first neural element in the auditory pathway, neurons in the spiral ganglion shape the initial coding of sound stimuli for subsequent processing. Within the ganglion, type I and type II neurons form divergent and convergent innervation patterns, respectively, with their hair cell sensory receptors, indicating that very different information is gathered and conveyed. Layered onto these basic innervation patterns are structural and electrophysiological features that provide additional levels of processing multifaceted sound stimuli. To understand the nature of this additional complexity of signal coding, we characterized the distribution of calretinin and calbindin, two regulators of intracellular calcium that serve as markers for neuronal subpopulations. We showed in acute preparations and in vitro that calretinin and calbindin staining levels were heterogeneous. Immunocytochemical analysis of colocalization further showed that high levels of staining for the two molecules rarely overlapped. Although varied amounts of calbindin and calretinin were found within each tonotopic location and neuronal type, some distinct subdistributions were noted. For example, calretinin levels were highest in neurons innervating the midcochlea region, whereas calbindin levels were similar across the entire ganglion. Furthermore, we noted that apical type II neurons, identified by antiperipherin labeling, had significantly lower levels of calretinin and higher levels of calbindin. We also established that the endogenous firing feature of onset tau of the subthreshold response showed a pattern related to quantified calretinin and calbindin staining levels. Taken together, our results suggest an additional dimension of complexity within the spiral ganglion beyond that currently categorized.

Topoisomerase IIbeta (Top2b) is an enzyme that modulates DNA supercoiling by catalyzing the passage of DNA duplexes through one another. It is ubiquitously expressed in postmitotic cells and known to function during the development of neuromuscular junctions in the diaphragm and the proper formation of laminar structure in the cerebral cortex. However, due to the perinatal death phenotype of the traditional constitutive and brain-specific Top2b knockout mice, the precise in vivo function of Top2b, especially during postnatal neural development, remains to be determined. Using both the constitutive and retina-specific knockout mouse models, we showed that Top2b deficiency resulted in delayed neuronal differentiation, degeneration of the plexiform layers and outer segment of photoreceptors, as well as dramatic reduction in cell number in the retina. Genome-wide transcriptome analysis by RNA sequencing revealed that genes involved in neuronal survival and neural system development were preferentially affected in Top2b-deficient retinas. Collectively, our findings have indicated an important function of Top2b in proper development and the maintenance/survival of postmitotic neurons in the retina.

A 6-year-old female rabbit was presented to a veterinary clinic, and the result of ultrasound examination suggested a tumor in the uterine tube. Subsequently, both ovaries and uterus were surgically removed. In gross, a single large cyst in the right ovary and enlargement of the left uterine tube were observed. Histological examination revealed that the cyst had developed in the hilus of the ovary and was lined by single-layered cuboidal cells. In the left uterine tube, a tumor composed of epithelial cells arranged in tubular structures and pleomorphic cells between the tubular structures was observed. Immunohistochemically, the epithelial cells of the cyst were positive for pan-cytokeratin, cytokeratin 18, CD10, E-cadherin, calretinin and estrogen receptor; the tumor cells of the left uterine tube were positive for pan-cytokeratin, cytokeratin 18, E-cadherin, vimentin, calretinin and estrogen receptor. From these results, the cyst was diagnosed as cystic rete ovarii, and the tumor was diagnosed as adenoma of the uterine tube. This case is the first to demonstrate cystic rete ovarii and uterine tube adenoma in rabbits.

The rodent barrel cortex has been established as an ideal model for studying the development and plasticity of a neuronal circuit. The barrel cortex consists of barrel and septa columns, which receive various input signals through distinct pathways. The lemniscal pathway transmits whisker-specific signals to homologous barrel columns, and the paralemniscal pathway transmits multi-whisker signals to both barrel and septa columns. The integration of information from both lemniscal and paralemniscal pathways in the barrel cortex is critical for precise object recognition. As the main target of the posterior medial nucleus (POm) in the paralemniscal pathway, layer 5a (L5a) pyramidal neurons are involved in both barrel and septa circuits and are considered an important site of information integration. However, information on L5a neurons is very limited. This study aims to explore the cellular features of L5a neurons and to provide a morphological basis for studying their roles in the development of the paralemniscal pathway and in information integration.1. We found that the calcium-binding protein calretinin (CR) is dynamically expressed in L5a excitatory pyramidal neurons of the barrel cortex, and L5a neurons form a unique serrated pattern similar to the distributions of their presynaptic POm axon terminals. 2. Infraorbital nerve transection disrupts this unique alignment, indicating that it is input dependent. 3. The formation of the L5a neuronal alignment develops synchronously with barrels, which suggests that the lemniscal and paralemniscal pathways may interact with each other to regulate pattern formation and refinement in the barrel cortex. 4. CR is specifically expressed in the paralemniscal pathway, and CR deletion disrupts the unique L5a neuronal pattern, which indicates that CR may be required for the development of the paralemniscal pathway.Our results demonstrate that L5a neurons form a unique, input-dependent serrated alignment during the development of cortical barrels and that CR may play an important role in the development of the paralemniscal pathway. Our data provide a morphological basis for studying the role of L5a pyramidal neurons in information integration within the lemniscal and paralemniscal pathways.

The activity of excitatory neurons is controlled by a highly diverse population of inhibitory interneurons. These cells show a high level of physiological, morphological and neurochemical heterogeneity, and play highly specific roles in neuronal circuits. In the mammalian hippocampus, these are divided into 21 different subtypes of GABAergic interneurons based on their expression of different markers, morphology and their electrophysiological properties. Ideally, all can be marked using an antibody directed against the inhibitory neurotransmitter GABA, but parvalbumin, calbindin, somatostatin, and calretinin are also commonly used as markers to narrow down the specific interneuron subtype. Here, we describe a journey to find the necessary immunological reagents for studying GABAergic interneurons of the mouse hippocampus. Based on web searches there are several hundreds of different antibodies on the market directed against these four markers. Searches in the literature databases allowed us to narrow it down to a subset of antibodies most commonly used in publications. However, in our hands the most cited ones did not work for immunofluorescence stainings of formaldehyde fixed tissue sections and cultured hippocampal neurons, and we had to immunostain our way through thirteen different commercial antibodies before finally finding a suitable antibody for each of the four markers. The antibodies were evaluated based on signal-to-noise ratios as well as if positive cells were found in layers of the hippocampus where they have previously been described. Additionally, the antibodies were also tested on sections from mouse spinal cord with similar criteria for specificity of the antibodies. Using the antibodies with a high rating on pAbmAbs, an antibody review database, stainings with high signal-to-noise ratios and location of the immunostained cells in accordance with the literature could be obtained, making these antibodies suitable choices for studying the GABAergic system.

The claustrum is a telencephalic structure which consists of dorsal segment adjoining the insular cortex and a ventral segment termed also endopiriform nucleus (END). The dorsal segment (claustrum) is divided into a dorsal and ventral zone, while the END is parcellated into dorsal, ventral and intermediate END. The claustrum and the END consist of glutamatergic projection neurons and GABAergic local interneurons coexpressing calcium binding proteins. Among neurons expressing calcium binding proteins the calretinin (CR)-immunoreactive interneurons exert specific functions in neuronal circuits, including disinhibition of excitatory neurons. Previous anatomical data indicate extensive and reciprocally organized claustral projections with cerebral cortex. We asked if the distribution of cells immunoreactive for CR delineates anatomical or functional subdivisions in the claustrum and in the END. Both segments of the claustrum and all subdivisions of the END contained CR immunoreactive neurons with varying distribution. The ventral zone of the claustrum exhibited weak labeling with isolated cell bodies and thin fibers and is devoid of immunoreactive puncta. Within the medial margin of the intermediate END we noted a group of strongly positive neurons. Cells immunoreactive for CR in all subdivisions of the claustrum and END were bipolar, multipolar and oval with smooth, beaded aspiny dendrites. Small number of CR-immunoreactive neurons displayed thin dendrites which enter to adjoining structures. Penetration of dendrites was reciprocal. These results show an inhomogenity over the claustrum and the END in distribution and types of CR immunoreactive neurons. The distribution of the CR-immunoreactive neurons respects the anatomical but not functional zones of the claustral complex.

DTNBP1 (dystrobrevin-binding protein 1), which encodes dysbindin-1, is one of the leading susceptibility genes for schizophrenia. Both dysbindin-1B and -1C isoforms are decreased, but the dysbindin-1A isoform is unchanged in schizophrenic hippocampal formation, suggesting dysbindin-1 isoforms may have distinct roles in schizophrenia. We found that mouse dysbindin-1C, but not dysbindin-1A, is localized in the hilar glutamatergic mossy cells of the dentate gyrus. The maturation rate of newborn neurons in sandy (sdy) mice, in which both dysbindin-1A and -1C are deleted, is significantly delayed when compared with that in wild-type mice or with that in muted (mu) mice in which dysbindin-1A is destabilized but dysbindin-1C is unaltered. Dysbindin-1C deficiency leads to a decrease in mossy cells, which causes the delayed maturation of newborn neurons. This suggests that dysbindin-1C, rather than dysbindin-1A, regulates adult hippocampal neurogenesis in a non-cell autonomous manner.

The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR(+)) cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR(+) interneurons originate in the caudal ganglionic eminence (CGE) from Gsx2(+) progenitors, but in humans it has been suggested that a subpopulation of CalR(+) cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ). The progenitors for cortically generated CalR(+) subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs) that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2(+) cells, not only in the ventral telencephalon (GE) as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR(+) neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh), an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR(+) cortical interneurons.