Calcium-binding Protein calbindin-D28k Research Articles

Immunocytochemical Localization of Calbindin D28K, Calretinin, and Parvalbumin in Bat Superior Colliculus.

The purpose of this study was to investigate the localization of cells containing the calcium-binding proteins (CBPs) calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the superior colliculus (SC) of the bat using immunocytochemistry. CB-immunoreactive (IR) cells formed a laminar tier within the upper superficial gray layer (SGL), while CR-IR cells were widely distributed within the optic layer (OL). Scattered CR-IR cells were also found within the intermediate gray, white, and deep gray layers. By contrast, PV-IR cells formed a laminar tier within the lower SGL and upper OL. Scattered PV-IR cells were also found throughout the intermediate layers, but without a specific laminar pattern. The CBP-IR cells varied in size and morphology: While most of the CB-IR cells in the superficial layers were small round or oval cells, most CR-IR cells in the intermediate and deep layers were large stellate cells. By contrast, PV-IR cells were small to large in size and included round or oval, stellate, vertical fusiform, and horizontal cells. The average diameters of the CB-, CR-, and PV-IR cells were 11.59, 17.17, and 12.60 μm, respectively. Double-immunofluorescence revealed that the percentage of co-localization with GABA-IR cells was 0.0, 0.0, and 10.27% of CB-, CR-, and PV-IR cells, respectively. These results indicate that CBP distribution patterns in the bat SC are unique compared with other mammalian SCs, which suggest functional diversity of these proteins in visually guided behaviors.

Alterations of Ca2+-responsive proteins within cholinergic neurons in aging and Alzheimer's disease

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D28K (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca2+-responsive proteins Ca2+/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated μ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of μ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca2+-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.

Expression of the calcium binding proteins Necab-1,-2 and -3 in the adult mouse hippocampus and dentate gyrus

The family of EF-hand calcium binding proteins is composed of more than 250 members. In search for other neuronal markers, we studied the expression pattern of Necab-1, -2 and -3 in the Ammons horn of adult mice at the gene- and protein levels using in-situ hybridization and immunohistochemistry. The genes for the three Necab's were expressed in specific, non-overlapping areas of the hippocampus. A minority of the Necab-positive interneurons were GABA-ergic, and they virtually never coexpressed one of the classical calcium binding proteins (calretinin, calbindin D-28k and parvalbumin). Necab's are promising new neuronal markers in the brain.

Immunohistochemical distribution of calretinin and calbindin (D‐28k) in the brain of the cladistian Polypterus senegalus

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes.

Selegiline (deprenyl) decreases calbindin-D28k expression in cortical neurons of rats socially deprived during the post-weaning period

Preclinical studies indicate that selegiline (deprenyl), frequently used in some neurodegenerative diseases, exert protective effects on central nervous system neurons of individuals exposed to social isolation (SI). Furthermore, it has been suggested that SI produces neuronal dysfunction due in part to an excessive intracellular Ca2+ overload. Since the main intracellular Ca2+ buffering mechanism involves changes in the calcium-binding protein calbindin-D28k (CB), and that CB neuronal expression can increase in response to Ca2+ transients, we hypothesized that chronic selegiline administration in early socially isolated animals could minimize cell CB expression as an indirect indicator of protective mechanism against Ca2+ overload. In the present study male rats were weaned at postnatal day 21 (P21) and randomly assigned to social deprivation (SI) or control (SC) environments for 30 days (P21–51). SI animals were further subdivided in two experimental groups: socially deprived-saline (SI-SAL) and socially isolated-selegiline (SI-SEL) for additional 30 days (P52–82). Medial frontal CB immunoreactivity (CB-ir) neurons were quantitatively and qualitatively analyzed. The results obtained indicate that neocortical cells of adult rats submitted to early SI show a significant increase in the number of CB-ir neurons per cortical field, while selegiline treatment significantly reduces this parameter.

SUN11602, a Novel Aniline Compound, Mimics the Neuroprotective Mechanisms of Basic Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF) offers some measure of protection against excitotoxic neuronal injuries by upregulating the expression of the calcium-binding protein calbindin-D28k (Calb). The newly synthesized small molecule 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) mimics the neuroprotective effects of bFGF, and thus, we examined how SUN11602 exerts its actions on neurons in toxic conditions of glutamate. In primary cultures of rat cerebrocortical neurons, SUN11602 and bFGF prevented glutamate-induced neuronal death. This neuroprotection, which occurred in association with the augmented phosphorylation of the bFGF receptor-1 (FGFR-1) and the extracellular signal-regulated kinase-1/2 (ERK-1/2), was abolished by pretreatment with PD166866 (a FGFR-1 tyrosine kinase-specific inhibitor) and PD98059 (a mitogen-activated protein kinase [MAPK]/[ERK-1/2] kinase [MEK] inhibitor). In addition, SUN11602 and bFGF increased the levels of CALB1 gene expression in cerebrocortical neurons. Whether this neuroprotection was linked to Calb was investigated with primary cultures of cerebrocortical neurons from homozygous knockout (Calb(-/-)) and wild-type (WT) mice. In WT mice, SUN11602 and bFGF increased the levels of newly synthesized Calb in cerebrocortical neurons and suppressed the glutamate-induced rise in intracellular Ca(2+). This Ca(2+)-capturing ability of Calb allowed the neurons to survive severe toxic conditions of glutamate. In contrast, Calb levels remained unchanged in Calb(-/-) mice after exposure to SUN11602 or bFGF, and due to a loss of function of the gene, these neurons were no longer resistant to toxic conditions of glutamate. These findings indicated that SUN11602 activated a number of cellular molecules (FGFR-1, MEK/ERK intermediates, and Calb) and consequently contributed to intracellular Ca(2+) homeostasis as observed in the case of bFGF.

In vivo AAV-mediated expression of calbindin-D28K in rat basal forebrain cholinergic neurons

The cholinergic neurons of the basal forebrain (BFCNs) in human and non-human primates are rich in the calcium binding protein calbindin-D28k (CB). We have shown a selective loss of CB from BFCNs in the course of normal aging, which appears to predispose these neurons to tangle formation and degeneration in Alzheimer's disease. Our previous preliminary investigation demonstrated that rodent BFCNs are devoid of CB. Here we confirm that rat choline acetyltransferase-rich BFCNs are devoid of CB immunoreactivity. We then describe a method for adeno-associated viral vector (AAV) induced expression of CB in rat BFCNs in vivo. We constructed AAV vectors bearing the CB gene under the control of the CMV promoter, or neuron-specific enolase (NSE) promoter, to bias expression in neurons. Both vectors resulted in CB expression in mouse neuronal cultures, and in rat brain following injections. AAV–NSE–CB resulted in more robust expression in neurons. Injections of 10μl of AAV–NSE–CB in the BFCNs component located within the internal segment of globus pallidus and internal capsule resulted in expression of CB in 84% of BFCNs. Expression was optimum at 14 days. Injections of AAV–NSE–LacZ resulted in robust β-galactosidase expression, but no CB immunoreactivity. Our results show that use of NSE promoter leads to high expression of genes in neurons and that the BFCNs can be targeted for expression of genes that are differentially expressed in the rodent and primate brains. These findings have important implications for gene replacement therapy in human BFCNs.

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca2+-dependent fashion. Molecular docking and conformational sampling studies of the Ca2+-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding. Structured summary of protein interactionsCalbindin-D28K and Caspase-3bind by isothermal titration calorimetry(View interaction)

Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G-protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor-mediated cytoprotection following exposure to glutamate at a toxic level (25 μm) was determined by assessing cell viability, apoptosis, and intracellular free Ca(2+) levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium-binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor β (ERβ): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.

Two types of periglomerular cells in the olfactory bulb of the macaque monkey (Macaca fascicularis)

The olfactory bulb (OB) of mammals is the brain region that receives the sensory information coming from the olfactory epithelium. The entrance of the olfactory information occurs in spherical structures of neuropil named olfactory glomeruli and is modulated by a population of interneurons known as periglomerular cells (PG). It has been demonstrated that there are two types of PG in the OB of some macrosmatic mammals, including rats and mice. Type 1 PG (PG-1) receive synapses from the olfactory nerve, whereas type 2 PG (PG-2) do not receive synapses from the olfactory axons. To date, the presence of the two types of PG has not been investigated in microsmatic mammals. In this context, we analyze the presence of PG-1 and PG-2 in the OB of the long-tailed macaque (Macaca fascicularis). For that, we used the enzyme tyrosine hydroxylase, the neuronal isoform of the enzyme nitric oxide synthase and the calcium-binding proteins calbindin D-28k and calretinin as neurochemical markers. Our results demonstrate that the OB of the macaque contains PG-1 and PG-2. A subpopulation of PG-1 expresses tyrosine hydroxylase and another expresses the neuronal isoform of nitric oxide synthase. In addition, a subpopulation of PG-2 expresses calbindin D-28k and another expresses calretinin. Double immunofluorescence demonstrates that there is no colocalization of two markers in the same PG. These results mimic those found in macrosmatic animals. The presence of two types of PG in the glomerular circuits seems to be a key principle for the organization of the OB of mammals.

Changes in the distribution of calbindin D28-k, parvalbumin, and calretinin in the hippocampus of the circling mouse

The circling (cir) mouse strain, a murine model of deafness caused by a spontaneous mutation, exhibits characteristic behaviors of circling and hyperactivity. In an induced-noise paradigm, cir mice display a significant loss in their spatial orientation abilities, and this has been suggested to be due at least in part to changes in calcium homeostasis. Auditory information is transferred from the cochlear nucleus to the hippocampus, where it is processed to modulate motor and sensory activity. Such a pathway could be affected at the cellular level by alterations in neurotransmission, including alterations that involve Ca2+. However, there have been no studies in a hearing deficit model examining the concomitant molecular alterations in the hippocampus. Thus, in the present study we used immunohistochemistry to compare the distribution of the calcium-binding proteins (CaBPs) calbindin D-28k, parvalbumin, and calretinin in the hippocampi of heterozygous (+/cir), homozygous (cir/cir), and wild-type (+/+) mice. The expression of the CaBPs in various hippocampal subfields appeared to be significantly lower in cir mice (+/− and −/−) than in +/+ mice. Such a decrease in CaBP expression in cir/cir mice would alter calcium homeostasis, which in turn could affect the connection of the tri-synaptic circuit of the hippocampus as well as the cortical region. A decrease in CaBPs and the probable resultant glutamate-mediated excitability could contribute to the functional changes that lead to the characteristic behavioral features of cir mice.

Suppression of Calbindin-D28k Expression Exacerbates SCA1 Phenotype in a Disease Mouse Model

Spinocerebellar ataxia type1 (SCA1) is an autosomal dominant neurological disorder caused by the expansion of a polyglutamine tract in the mutant protein ataxin-1. The cerebellar Purkinje cells (PCs) are the major targets of mutant ataxin-1. The mechanism of PC death in SCA1 is not known; however, previous work indicates that downregulation of specific proteins involved in calcium homeostasis and signaling by mutant ataxin-1 is the probable cause of PC degeneration in SCA1. In this study, we explored if targeted deprivation of PC specific calcium-binding protein calbindin-D28k (CaB) exacerbates ataxin-1 mediated toxicity in SCA1 transgenic (Tg) mice. Using behavioral tests, we found that though both SCA1/+ and SCA1/+: CaB null (-/+) double mutants exhibited progressive impaired performance on the rotating rod, a simultaneous enhancement of exploratory activity, and absence of deficits in coordination, the double mutants were more severely impaired than SCA1/+ mice. With increasing age, SCA1/+ mice showed a progressive loss in the expression and localization of CaB and other PC specific calcium-binding and signaling proteins. In double mutants, these changes were more pronounced and had an earlier onset. Gene expression profiling of young mice exhibiting no behavior or biochemical deficits revealed a differential expression of many genes common to SCA1/+ and CaB-/+ lines, and unique to SCA1/+: CaB-/+ phenotype. Our study provides further evidence for a critical role of CaB in SCA1 pathogenesis, which may help identify new therapeutic targets to treat SCA1 or other cerebellar ataxias.

The effect of different maternal deprivation paradigms on the expression of hippocampal glucocorticoid receptors, calretinin and calbindin-D28k in male and female adolescent rats

Maternal deprivation (MD) is a well-established protocol used to investigate neurobiological changes that are associated with the etiology of and vulnerability to stress-related diseases in animal models. The resulting psychophysiological effects, the timing and duration of these adverse stimuli, and the method by which they exert their effects on the animals remain unclear. This study characterized differences in the hippocampal expression of glucocorticoid receptors (GRs) and the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) in male and female rats that underwent different MD paradigms during the stress hyporesponsive period (SHRP). Both GRs and the two calcium-binding proteins were much more abundant in females than in males. MD paradigms had a significant effect on CALR and CALB expression in both males and females but affected GR levels only in males. Additionally, expression of the two calcium-binding proteins in the hippocampus responded differently to MD-induced stress, especially in females. Taken together, these results indicate that females are able to modulate their response to stress better than males.

Theta-burst transcranial magnetic stimulation alters cortical inhibition.

Human cortical excitability can be modified by repetitive transcranial magnetic stimulation (rTMS), but the cellular mechanisms are largely unknown. Here, we show that the pattern of delivery of theta-burst stimulation (TBS) (continuous versus intermittent) differently modifies electric activity and protein expression in the rat neocortex. Intermittent TBS (iTBS), but not continuous TBS (cTBS), enhanced spontaneous neuronal firing and EEG gamma band power. Sensory evoked cortical inhibition increased only after iTBS, although both TBS protocols increased the first sensory response arising from the resting cortical state. Changes in the cortical expression of the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) indicate that changes in spontaneous and evoked cortical activity following rTMS are in part related to altered activity of inhibitory systems. By reducing PV expression in the fast-spiking interneurons, iTBS primarily affected the inhibitory control of pyramidal cell output activity, while cTBS, by reducing CB expression, more likely affected the dendritic integration of synaptic inputs controlled by other classes of inhibitory interneurons. Calretinin, the third major calcium-binding protein expressed by another class of interneurons was not affected at all. We conclude that different patterns of TBS modulate the activity of inhibitory cell classes differently, probably depending on the synaptic connectivity and the preferred discharge pattern of these inhibitory neurons.

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.

Borders, extent, and topography of human perirhinal cortex as revealed using multiple modern neuroanatomical and pathological markers

Despite rapidly increasing interests in specific contributions of different components of human medial temporal lobe (MTL) to memory and memory impairments in normal aging and in many abnormal conditions such as Alzheimer's disease and Pick's disease, few modern neuroanatomical studies are available about the borders, extent, and topography of human perirhinal areas 35 and 36, which are important components of the MTL memory system. By a combined use of several cellular, neurochemical, and pathological markers, which mainly include neuronal nuclear antigen, calcium-binding proteins (parvalbumin and calbindin-D28k), nonphosphorylated neurofilament protein (SMI-32), Wisteria floribunda agglutinin, and abnormally phosphorylated tau (AT8), this study has revealed that the borders of human perirhinal areas 35 and 36 are significantly different from those defined with conventional Nissl staining. In general, areas 35 and 36 occupy the ventromedial temporopolar and rhinal sulcal regions, the collateral sulcal region, and the anterior two-thirds of fusiform gyrus or occipitotemporal gyrus. Furthermore, the precise borders, extent, and topography of human areas 35 and 36 and adjoining entorhinal cortex were marked at different anteroposterior levels of the MTL with reference to variations of rhinal and collateral sulci and other useful landmarks. These findings would provide reliable neuroanatomical base for the great and yet rapidly increasing number of neuroimaging studies of the human MTL structures in healthy and many abnormal conditions.

Evaluation of Long-Term Upregulation of Calbindin D28K as a Preventive Approach for Ischaemic Stroke

Buffering of intracellular calcium peaks following an acute ischaemic stroke protects neurons from necrotic and apoptotic cell death. However, the need for on-demand delivery of protective agents due to the short therapeutic window in stroke therapy makes it difficult to apply a calcium-buffering strategy in patients. We investigated the effects of a preventive upregulation of the calcium-binding protein Calbindin D28K as a potential approach for patients at a high risk of ischaemic stroke. We overexpressed Calbindin D28K in the striatal and cortical region in mice using an adeno-associated viral vector (AAV) for 12 weeks, and then assessed neuroprotective effects after MCAO. In contrast to studies showing a neuroprotective effect of shortly induced Calbindin D28K overexpression, we found no increased survival of neurons overexpressing Calbindin D28K for 12 weeks.We suggest that neuronal calcium metabolism adapts to higher Calbindin D28K levels after long-term overexpression. This potentially preventive approach to protect from ischaemic stroke does not have clinical applicability.

Calcium-binding protein immunoreactivity characterizes the auditory system of Gekko gecko.

Geckos use vocalizations for intraspecific communication, but little is known about the organization of their central auditory system. We therefore used antibodies against the calcium-binding proteins calretinin (CR), parvalbumin (PV), and calbindin-D28k (CB) to characterize the gecko auditory system. We also examined expression of both glutamic acid decarboxlase (GAD) and synaptic vesicle protein (SV2). Western blots showed that these antibodies are specific to gecko brain. All three calcium-binding proteins were expressed in the auditory nerve, and CR immunoreactivity labeled the first-order nuclei and delineated the terminal fields associated with the ascending projections from the first-order auditory nuclei. PV expression characterized the superior olivary nuclei, whereas GAD immunoreactivity characterized many neurons in the nucleus of the lateral lemniscus and some neurons in the torus semicircularis. In the auditory midbrain, the distribution of CR, PV, and CB characterized divisions within the central nucleus of the torus semicircularis. All three calcium-binding proteins were expressed in nucleus medialis of the thalamus. These expression patterns are similar to those described for other vertebrates.

Tecto-reticulospinal pathway (TRS)와 dorsal lateral geniculate nucleus (dLGN)에서 역행성이동추적물질 이용 햄스터 상구에서 GluR1-, GluR4- 면역반응 신경세포 연구

본 연구자들의 앞선 연구에서는 햄스터 상구에서 AMPA 수용체의 아형인 GluR1과 GluR4의 분포와 눈 적출 이후 이들 수용체의 분포를 면역세포화학적 방법으로 연구하였다. 또한, 표지한 GluR1과 GluR4를 칼슘결합단백질인 calbindin D28K, calretinin, parvalbumin과 GABA로 표지하여 비교하였다. 본 연구에서 역행성이동추적물질(retrograde tracer)인 호스래디시퍼옥시다아제(horseradish peroxidase, HRP)를 상구의 각 주요 상행로와 하행로에 주입함으로써 GluR1- 면역반응 신경세포들과 GluR4- 면역반응 신경세포들이 투사신경세포(projection neurons)임을 밝혀내었다. Tecto-reticulospinal pathway (TRS)와 dorsal lateral geniculate nucleus (dLGN)으로 HRP 를 주입한후, 햄스터들은 회복을 위해 48시간 동안 살려둔 뒤 관류(perfusion)하였다. GluR- 면역반응 처리된 절편들은 역행성 표지된 신경세포를 지님을 확인하였다. HRP를 주입하였더니 단지 적은 수의 GluR1- 면역반응 신경세포들이 TRS (1.4%)와 dLGN (2.6%)으로 투사되었고, 반면에 많은 수의 GluR4- 면역반응 신경세포들이 TRS (32.7%)로 투사되었다. 사이/투사 신경세포(inter/projection neurons)들은 GluR 아단위들의 분류된 분포와 차별화된 양상을 보였고 이들의 이러한 분포는 칼슘결합단백질들과 GABA와는 겹쳐지지 않았으며, 일전에 발표했던 시각적 행동 반응에서 안구 적출 후 수용체 아단위들의 기능적 다양화와는 차별화된 양상을 보였다. We recently reported the distributions of AMPA (<TEX>$\alpha$</TEX>-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor subtypes glutamate receptors (GluR) 1 and GluR4 in the superior colliculi (SC) of hamsters with antibody immunocytochemistry and the effect of enucleation on these distributions. We also compared these labelings to those of calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. In the present study, we investigated whether the GluR1- and GluR4-immunoreactive (IR) neurons are interneurons or projection neurons by injection of the retrograde tracer horseradish peroxidase (HRP) into one of each major ascending and descending pathways of the SC. HRP injections were made into a tecto-reticulospinal pathway (TRS) and dorsal lateral geniculate nucleus (dLGN). Animals were then allowed to recover and to survive for 48 hr before perfusion. Sections containing retrograde-labeled neurons were then treated for GluR-immunoreactivity. HRP injections proved that only a small population of the GluR1-IR cells project into TRS (1.4%) and dLGN (2.6%). However, a large subpopulation of GluR4-IR cells project into TRS (32.7%). The differential compositions of inter/projection neurons, along with our previous studies on the separate distribution of the GluR subunits, its differential co-localization with calcium-binding proteins and GABA, and differential reactions to enucleations, strongly imply the functional variety of the receptor subunits in visual behavior responses.

Anatomical changes in the primary visual cortex of the congenitally blind Crx−/− mouse

Mutations in the human cone-rod homeobox ( Crx) gene are associated with retinal dystrophies such as Leber Congenital Amaurosis (LCA), characterized by complete or near complete absence of vision from birth. The photoreceptors of Crx−/− mice lack outer segments, and therefore cannot capture light signals through rods and cones, thus resulting in a lack of normal retinal ganglion cell activity from birth. Using specific antibodies to subsets of neurons and markers of activity, we examined the impact of this absence of sensory input on the development of the primary visual cortex (V1) in early postnatal Crx−/− mice, before wiring of the visual system is complete, and in adulthood. We revealed that Crx−/− mice did not exhibit gross anatomical differences in V1; however, they exhibited significantly fewer calcium-binding protein (parvalbumin and calbindin-D28k) expressing interneurons, as well as reduced nonphosphorylated neurofilament expression in V1. These results reveal that the Crx mutation and lack of light stimulation through the photoreceptor pathway regulate the development and phenotype of different neuronal populations in V1 but not its general morphology. We conclude, therefore, that photoreceptor-mediated visual input during development is crucial for the normal postnatal development and maturation of subsets of cortical neurons.