Calbindin Research Articles

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed.

Botulinum toxin type A induces changes in the chemical coding of substance P-immunoreactive dorsal root ganglia sensory neurons supplying the porcine urinary bladder.

Botulinum toxin (BTX) is a potent neurotoxin which blocks acetylcholine release from nerve terminals, and therefore leads to cessation of somatic motor and/or parasympathetic transmission. Recently it has been found that BTX also interferes with sensory transmission, thus, the present study was aimed at investigating the neurochemical characterization of substance P-immunoreactive (SP-IR) bladder-projecting sensory neurons (BPSN) after the toxin treatment. Investigated neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against SP, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase activating polypeptide (PACAP), neuronal nitric oxide synthase (nNOS), galanin (GAL), calbindin (CB), and somatostatin (SOM). In the control group (n = 6), 45% of the total population of BPSN were SP-IR. Nearly half of these neurons co-expressed PACAP or CGRP (45% and 35%, respectively), while co-localization of SP with GAL, nNOS, SOM or CB was found less frequently (3.7%, 1.8%, 1.2%, and 0.7%, respectively). In BTX-treated pigs (n = 6), toxin-injections caused a decrease in the number of SP-IR cells containing CGRP, SOM or CB (16.2%, 0.5%, and 0%, respectively) and a distinct increase in these nerve cells immunopositive to GAL (27.2%). The present study demonstrates that BTX significantly modifies the chemical phenotypes of SP-IR BPSN.

Ectopic transgene expression in the retina of four transgenic mouse lines.

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR- and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression.

Immunocytochemical localization of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin in bat visual cortex.

It is a common misconception that bats are blind, and various studies have suggested that bats have visual abilities. The purpose of this study was to investigate the cytoarchitecture of calbindin D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunoreactive (IR) neurons in the bat visual cortex using immunocytochemistry. The highest density of CB- and PV-IR neurons was located in layer IV of the visual cortex. The majority of CB- and PV-IR neurons were characterized by a stellate or round/oval shape. CR-IR neurons were predominantly located in layers II/III, and the cells were principally round/oval in shape. Two-color immunofluorescence revealed that 65.96%, 24.24%, and 77.00% of the CB-, CR-, and PV-IR neurons, respectively, contained gamma-aminobutyric acid (GABA). We observed calcium-binding protein (CBP)-IR neurons in specific layers of the bat visual cortex and in specific cell types. Many of the CBP-IR neurons were GABAergic interneurons. These data provide useful clues to aid in understanding the functional aspects of the bat visual system.

Developmental Characteristics of Neurons in the Intramural Ganglia of the Small Intestine Containing Different Types of Calcium-Binding Proteins

Immunohistochemical and morphometric methods were used to study the locations, relative contents, and cross-sectional areas of calbindin (CB)- and calretinin (CR)-immunopositive neurons in the intramural ganglia of the intermuscular plexus of the rat duodenum (n = 37) during postnatal ontogeny (days 1, 10, 20, 30, and 60 and 1 and 2 years of age). CB- and CR-immunopositive neurons were detected in all the rats studied, from neonatal to aged. Proportions of CR-immunopositive neurons increased over the first 10 days of life and then showed no significant changes, including in aged animals. Proportions of CB-containing neurons increased to reach a maximum by day 20 of life, decreased insignificantly to day 30, and then remained without significant change. The mean sizes of CB- and CR-immunopositive neurons were significantly greater than the mean cross-sectional area of immunonegative neurons in all age groups.

Neurons of visual thalamic nuclei projecting to telencephalon express different types of calcium-binding proteins: A combined immunocytochemical and tracer study

In turtles (Testudo horsfieldi, Emys orbicularis), immunoreactivity to calbindin (CB), parvalbumin (PV), calretinin (CR) and co-localization of CB and PV were studied in neurons of the visual thalamic nuclei (Rot, GLd) projecting to the telencephalon using a combination of immunohistochemical and tracer methods. The prevalence of CB-immunoreactive (-ir) neurons in Rot, CB-ir and CR-ir neurons in GLd, and a smaller number of PV-ir neurons in both nuclei was shown. Double immunofluorescent labeling revealed that within both nuclei PV and CB are colocalized in most PV-ir and fewer CB-ir neurons. After injection of horseradish peroxidase into the Rot and GLs telencephalic projection fields, retrograde labeling was found in corresponding thalamic projection neurons immunoreactive to all the three proteins. After introduction of the fluorescent tracer Fluo-gold into the same telencephalic regions, retrograde labeling was detected in Rot and GLd neurons immunoreactive only to PV and CB as well as in neurons with colocalization of both proteins. These findings provide further evidence that in turtles the CB component prevails in the rotundo-telencephalic pathway while the CB/CR component is dominant in the geniculotelencephalic pathway. The role of functional specialization in segregation of neurons expressing distinct types of calcium-binding proteins is postulated.

The role of gastro-intestinal tract in the calcium absorption

Calcium is associated with various functions of clinical importance. Its unique distribution;low intracellular and high extracellular concentration, is crucial for the neuro-muscular function. Calcium is also indispensable for the vascular contraction and blood coagulation. Thus, circulating calcium concentration must be strictly maintained within a narrow range, for which parathyroid hormone(PTH), vitamin D, and calcitonin contribute. Food-derived protein-bound calcium must be first released in the acidic condition. Thus, gastric acid is essential for the effective calcium absorption. Intestinal calcium absorption occurs via both active transport and passive transport. For the former, such molecules as transient receptor potential vanilloid type 6(TRPV6), calbindin 9k, and Ca²⁺-ATPase contribute. In the adult, calcium absorption rate is approximately 30% under the ordinary condition. Lower calcium intake is associated with increased calcium absorption and decreased urinary excretion. In the Dietary Reference Intakes for Japanese, calcium requirement is determined based on factorial method. Recommended Dietary Allowance(RDA)for calcium ranges from 600-800 mg/day for adult. However, the average calcium intake is far lower than Estimated Average Requirement(EAR). Thus, an effort to increase the calcium intake, rather than considering the detailed calcium absorption rate, is most essential in Japan.

Changes in Calbindin-Containing Neurons in the Posterior Horn of the Gray Matter of the Spinal Cord and the Sensory Ganglion of a Spinal Nerve in White Rats after Sensory Deprivation

Immunohistochemical studies in Wistar rats (n = 4) addressed the effects of capsaicin on the morphometric and structural characteristics of 28-kDa calbindin (CB)-containing neurons in the posterior horn of the gray matter of segment TII of the spinal cord (SC) and the second thoracic spinal nerve sensory ganglion (SNSG). Capsaicin was given to adult animals as three doses with intervals of 24 h, totaling 125 mg/kg, and specimens were collected on day 14. Administration of capsaicin decreased the proportion of CB-immunopositive (CB-IP) neurons in the SNSG (by 60%) and in laminae I, II, and III of the posterior horn (by 8%, 18%, and 15%, respectively); mean CB-IP neuron size increased as a result of intracellular edema. Deafferentation led to the development of consistent morphometric and structural changes in CB-IP neurons both in the SNSG and in the posterior horn of the gray matter of the SC, with central chromatolysis, and nuclear and cytoplasmic vacuolization, providing evidence of hydropic dystrophy. The irreversibility of these changes in SNSG neurons and laminae I, II and V of the posterior horn of the SC was evidenced by deformation of nuclei, lysis of nucleoli, and decreases in the numbers of CB-containing neurons, along with signs of neuronophagia, with the formation of residual nodules at the sites of dead cells.

Calbindin-Immunoreactive Interneurons in the Intermediate Zone and Ventral Horn of the Gray Matter of the SC in White Rats

The aim of the present work was to study the topography and structure of 28-kDa calbindin (CB)-containing interneurons in the ventral horn and intermediate zone of the gray matter at levels TII and LIV of the spinal cord (SC). Experiments were performed in adult female Wistar rats (n = 4) using immunohistochemical and morphometric methods. Cryostat transverse sections of the SC of thickness 14 μm were studied. The intermediate zone of the SC was found to contain two subpopulations of CB-immunoreactive (IR) interneurons in lamina VII in both segments: sympathetic preganglionic neurons and “partition” cells. The ventral horn also contained two subpopulations of CB-IR interneurons: Renshaw cells in lamina IX in TII and lamina VII in LIV and large interneurons in lamina VIII in TII and lamina VII in LIV. The only differences between segments were in the larger numbers of Renshaw cells and partition cells in LIV. The mean cross-sectional areas of CB-IR Renshaw cells and autonomic preganglionic neurons were greater in TII, while large interneurons of the ventral horn and partition cells were larger in LIV.

Chronic maternal morphine alters calbindin D-28k expression pattern in postnatal mouse brain.

The distribution pattern of calbindin (CB)-D28k-expressing neurons results to be altered in several brain regions of chronic morphine exposed adult mice. In this study, the influence of chronic maternal exposure to morphine on the distribution pattern of CB-D28k-expressing neurons in the brain of mouse offspring was investigated. Females of CD-1 mice were daily administered with saline or morphine for 7 days before mating, during the whole gestation period, and until 21 day post-partum. Their offspring were sacrificed on postnatal day 18, and the brains were examined by histology using cresyl violet and by immunohistochemistry using a rabbit polyclonal anti-CB-D28k antibody. Histology revealed no significant differences in the distribution pattern and the number of neurons between the offspring forebrain of the control group of mice and the two groups of mice treated with different doses of morphine. However, immunohistochemical analysis revealed that the number of CB-D28k-immunoreactive neurons remarkably decreased in the cingulate cortex, in the layers II-IV of the parietal cortex and in all regions of the hippocampus, while it increased in the layers V-VI of the parietal cortex and in the subicular region of the offspring brain of morphine treated mice. Overall, our findings demonstrate that maternal exposure to morphine alters the pattern of CB-D28k-expressing neuron pattern in specific regions of murine developing brain, in a layer- and dose-dependent way, thus suggesting that these alterations might represent a mechanism by which morphine modifies the functional aspects of developing brain.

Editorial: At the top of the interneuronal pyramid-calretinin expressing cortical interneurons.

Editorial: At the top of the interneuronal pyramid-calretinin expressing cortical interneurons.

Calcium-binding protein immunoreactivity in Gudden's tegmental nuclei and the hippocampal formation: differential co-localization in neurons projecting to the mammillary bodies.

The principal projections to the mammillary bodies arise from just two sites, Gudden’s tegmental nuclei (dorsal and ventral nuclei) and the hippocampal formation (subiculum and pre/postsubiculum). The present study sought to compare the neurochemical properties of these mammillary body inputs in the rat, with a focus on calcium-binding proteins. Neuronal calretinin (CR) immunoreactivity was sparse in Gudden’s tegmental nuclei and showed no co-localization with neurons projecting to the mammillary bodies. In contrast, many of the ventral tegmental nucleus of Gudden cell that project to the mammillary bodies were parvalbumin (PV)-positive whereas a smaller number of mammillary inputs stained for calbindin (CB). Only a few mammillary body projection cells in the dorsal tegmental nucleus of Gudden co-localized with PV and none co-localized with CB. A very different pattern was found in the hippocampal formation. Here, a large proportion of postsubiculum cells that project to the mammillary bodies co-localized with CR, but not CB or PV. While many neurons in the dorsal and ventral subiculum projected to the mammillary bodies, these cells did not co-localize with the immunofluorescence of any of the three tested proteins. These findings highlight marked differences between hippocampal and tegmental inputs to the rat mammillary bodies as well as differences between the medial and lateral mammillary systems. These findings also indicate some conserved neurochemical properties in Gudden’s tegmental nuclei across rodents and primates.

Determinants of different deep and superficial CA1 pyramidal cell dynamics during sharp-wave ripples.

Sharp-wave ripples represent a prominent synchronous activity pattern in the mammalian hippocampus during sleep and immobility. GABAergic interneuronal types are silenced or fire during these events, but the mechanism of pyramidal cell (PC) participation remains elusive. We found opposite membrane polarization of deep (closer to stratum oriens) and superficial (closer to stratum radiatum) rat CA1 PCs during sharp-wave ripples. Using sharp and multi-site recordings in combination with neurochemical profiling, we observed a predominant inhibitory drive of deep calbindin (CB)-immunonegative PCs that contrasts with a prominent depolarization of superficial CB-immunopositive PCs. Biased contribution of perisomatic GABAergic inputs, together with suppression of CA2 PCs, may explain the selection of CA1 PCs during sharp-wave ripples. A deep-superficial gradient interacted with behavioral and spatial effects to determine cell participation during sleep and awake sharp-wave ripples in freely moving rats. Thus, the firing dynamics of hippocampal PCs are exquisitely controlled at subcellular and microcircuit levels in a cell type-selective manner.

Distinction in the immunoreactivities of two calcium-binding proteins and neuronal birthdates in the first and higher-order somatosensory thalamic nuclei of mice: Evolutionary implications.

Comparative embryonic studies are the most effective way to discern phylogenetic changes. To gain insight into the constitution and evolution of mammalian somatosensory thalamic nuclei, we first studied how calbindin (CB) and parvalbumin (PV) immunoreactivities appear during embryonic development in the first-order relaying somatosensory nuclei, i.e., the ventral posteromedial (VPM) and posterolateral (VPL) nuclei, and their neighboring higher-order modulatory regions, including the ventromedial or ventrolateral nucleus, posterior, and the reticular nucleus. The results indicated that cell bodies that were immunoreactive for CB were found earlier (embryonic day 12 [E12]) in the dorsal thalamus than were cells positive for PV (E14), and the adult somatosensory thalamus was characterized by complementary CB and PV distributions with PV dominance in the first-order relaying nuclei and CB dominance in the higher-order regions. We then labeled proliferating cells with [(3) H]-thymidine from E11 to 19 and found that the onset of neurogenesis began later (E12) in the first-order relaying nuclei than in the higher-order regions (E11). Using double-labeling with [(3) H]-thymidine autoradiography and CB or PV immunohistochemistry, we found that CB neurons were born earlier (E11-12) than PV neurons (E12-13) in the studied areas. Thus, similar to auditory nuclei, the first and the higher-order somatosensory nuclei exhibited significant distinctions in CB/PV immunohistochemistry and birthdates during embryonic development. These data, combined with the results of a cladistic analysis of the thalamic somatosensory nuclei, are discussed from an evolutionary perspective of sensory nuclei.

Distribution and Morphology of Calcium-Binding Proteins Immunoreactive Neurons following Chronic Tungsten Multielectrode Implants.

The development of therapeutic approaches to improve the life quality of people suffering from different types of body paralysis is a current major medical challenge. Brain-machine interface (BMI) can potentially help reestablishing lost sensory and motor functions, allowing patients to use their own brain activity to restore sensorimotor control of paralyzed body parts. Chronic implants of multielectrodes, employed to record neural activity directly from the brain parenchyma, constitute the fundamental component of a BMI. However, before this technique may be effectively available to human clinical trials, it is essential to characterize its long-term impact on the nervous tissue in animal models. In the present study we evaluated how chronic implanted tungsten microelectrode arrays impact the distribution and morphology of interneurons reactive to calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV) across the rat’s motor cortex. Our results revealed that chronic microelectrode arrays were well tolerated by the nervous tissue, with recordings remaining viable for up to 6 months after implantation. Furthermore, neither the morphology nor the distribution of inhibitory neurons were broadly impacted. Moreover, restricted microglial activation was observed on the implanted sites. On the whole, our results confirm and expand the notion that tungsten multielectrodes can be deemed as a feasible candidate to future human BMI studies.

Calcium-binding proteins in focal cortical dysplasia.

SummaryObjectiveAlterations in γ‐aminobutyric acid (GABA)‐ergic cortical neurons have been reported in focal cortical dysplasia (FCD)Ia/IIIa, a malformation of cortical development associated with drug‐resistant epilepsy. We compared numbers of neurons containing calcium‐binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR) and densities of respective fibers in lateral temporal lobe surgical specimens of 17 patients with FCD with 19 patients who underwent anterior temporal lobe resection due to nonlesional temporal lobe epilepsy (non‐FCD) as well as with 7 postmortem controls.Methods PV‐, CB‐, and CR‐immunoreactive (IR) neurons were quantitatively investigated with use of two‐dimensional cell counting and densitometry (reflecting mainly IR fibers) in cortical layers II, IV, and V.ResultsNumbers of PV‐IR neurons, ratios of PV‐containing to Nissl‐stained neurons (correcting for eventual cell loss), and densities of PV‐IR were higher in layer II of the cortex of FCD compared to non‐FCD patients. Similarly, densities of CB‐IR and CR‐IR were also higher in layers II and V, respectively, of FCD than of non‐FCD patients. Comparison with postmortem controls revealed significant higher cell numbers and fiber labeling for all three calcium‐binding proteins in FCD cortex, whereas numbers of Nissl‐stained neurons did not vary between FCD, non‐FCD, and postmortem controls. In non‐FCD versus postmortem controls, ratios of calcium‐binding protein‐IR cells to Nissl‐stained neurons were unchanged in most instances except for increased CB/Nissl ratios and CB‐IR densities in all cortical layers.SignificanceIncreased numbers of PV neurons and fiber labeling in FCD compared to nondysplastic epileptic temporal neocortex and postmortem controls may be related to cortical malformation, whereas an increased number of CB‐IR neurons and fiber labeling both in FCD and non‐FCD specimens compared with postmortem controls may be associated with ongoing seizure activity. The observed changes may represent increased expression of calcium‐binding proteins and thus compensatory mechanisms for seizures and neuronal loss in drug‐resistant epilepsy.

Calbindin-D28k immunoreactivity in the mice thoracic spinal cord after space flight

AbstractThe aim of the work was to analyse changes in the location and morphological characteristics of calbindin (CB)-immunoreactive (IR) neurons of the thoracic spinal cord of C57BL/6N male mice after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). Space flight induced multidirectional changes of the number and morphological parameters of CB-positive neurons. The number of IR neurons increased in laminae I (from 10 to 17 neurons per section), II (from 42 to 67 cells per section) and IX (from two neurons per segment to two neurons per section), but CB disappeared in neurons of lamina VIII. Weightlessness did not affect the number of CB-IR neurons in laminae III–V and VII, including preganglionic sympathetic neurons. The cross-sectional area of CB-IR neurons decreased in lamina II and VII (group of partition cells) and increased in laminae III–V and IX. After a space flight, few very large neurons with long dendrites appeared in lamina IV. The results obtained give evidence about substantial changes in the calcium buffer system and imbalance of different groups of CB-IR neurons due to reduction of afferent information under microgravity.

Laboratory procedures update on Hirschsprung disease.

The detection of ganglion cells in rectal biopsies of infants or toddlers with severe constipation is routinely performed by pediatric pathologists in many institutions. Hirschsprung disease (HD) is defined by the lack of ganglion cells (aganglionosis). The early recognition and the prompt implementation of surgical procedures obviously protect infants affected with HD from potential life-threatening conditions, including enterocolitis and debilitating constipation. Image-based and non-image-based clinical techniques and some laboratory tests have been reevaluated along the years, but often fragmentarily. Immunohistochemical markers have been increasingly used in pathology laboratories to detect ganglion cells and nerve fibers. Recently, calretinin, a vitamin D-dependent calcium-binding protein with expression in ganglion cells and nerves, has been described as an adjunctive or primary diagnostic test in HD. The aim of the present study was to systematically summarize and update laboratory procedures targeting ganglion cells in rectal biopsies. Procedures and tests have been reviewed and values of specificity and sensitivity have been calculated according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Contrast enema has the lowest sensitivity and specificity of all of the 3-index investigations under the lens: contrast enema, anorectal manometry, and biopsy with histology. The latter procedure seems to have the highest sensitivity and specificity. Acetylcholinesterase staining on fresh-frozen material has been found to have slightly higher rates of sensitivity and specificity when compared with hematoxylin and eosin only. Calretinin staining may be supportive for the diagnosis, although some cases with false-positivity may be of some concern. Hematoxylin and eosin with or without acetylcholinesterase remains the criterion standard according to our PRISMA-based data. In our opinion, the number of false-positive results with potential overtreatment may limit the increasing advocacy for calretinin staining. Both the "primum non nocere" dictum and the "loss aversion heuristic" need to be satisfied harmoniously by preventing harm from unnecessary surgery.

Cochleotopy of human cochlear nucleus

IntroductionThe human peripheral auditory pathway in infants is a relatively less explored domain in auditory research. In the present study, the cochlea, auditory nerve (AN) and the cochlear nucleus (CN) were examined using immunohistochemistry and light and electron-microscopy. MethodsThe study was performed on specimens retrieved from a 40-day-old infant who could not survive after corrective surgery for congenital cyanotic heart disease. The donation was done in accordance with the protocol approved by the Human Ethics Committee, All India Institute of Medical Sciences (AIIMS), New Delhi, India. ResultsThe AN revealed degeneration of nerve fascicles originating from the spiral ganglion neurons of the middle and basal turns of the cochlea. The proximal and middle segments of the nerve showed varying degrees of degenerating fibers with macrophages whereas the most distal part of the nerve showed dilated endoneurial capillaries without any degeneration. On electron-microscopy, the nerve revealed features of acute myelinopathy. The differential perisomatic synaptophysin immunoreactivity was observed in the CN. The unaffected neurons showed strong microtubule-associated protein 2 (MAP-2) immunoreactivity, while those denervated revealed decreased staining. The VCN showed varying degree of parvalbumin (PV) and calbindin (CB) immunostaining with AN fibers and terminals showing strong immunoreactivity for PV. The AN revealed strong immunoreactivity for CB on the affected side. The spiral ganglion neurons did not reveal any pathological changes. DiscussionThe results provide a cochleotopic map of the human CN that maybe relevant in better understanding of the tonotopic organization and will be helpful during auditory implants in children.

GABA-Synthesizing Enzymes in Calbindin and Calretinin Neurons in Monkey Prefrontal Cortex.

Non-overlapping groups of cortical γ-aminobutyric acid-releasing (GABAergic) neurons are identifiable by the presence of calbindin (CB), calretinin (CR), or parvalbumin (PV). Boutons from PV neuron subtypes are also distinguishable by differences in protein levels of the GABA-synthesizing enzymes GAD65 and GAD67. Multilabel fluorescence microscopy was used to determine if this diversity extends to boutons of CB and CR neurons in monkey prefrontal cortex. CB and CR neurons gave rise to 3 subpopulations of GAD-containing boutons: GAD65+, GAD67+, and GAD65/GAD67+. Somatostatin and vasoactive intestinal peptide-expressing neurons, subtypes of CB and CR neurons, respectively, also gave rise to these distinct bouton subpopulations. At the transcript level, CB and CR neurons contained mRNA encoding GAD67-only or both GADs. Thus, the distinct subpopulations of CB/GAD+ and CR/GAD+ boutons arise from 2 unique subtypes of CB and CR neurons. The different CB and CR GAD-expressing neurons targeted the same projection neurons and neuronal structures immunoreactive for PV, CR, or CB. These findings suggest that GABA synthesis from CB/GAD67+ and CR/GAD67+ neurons would presumably be more vulnerable to disease-associated deficits in GAD67 expression, such as in schizophrenia, than neurons that also contain GAD65.