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PubMed1999

Synaptic and neurochemical features of calcitonin gene-related peptide containing neurons in the rat accessory optic nuclei.

Within the rodent visual system, calcitonin gene-related peptide (CGRP) is selectively expressed in neurons in the accessory optic nuclei (AON), including the dorsal terminal nucleus (DTN), lateral terminal nucleus (LTN) and medial terminal nucleus (MTN). To determine whether CGRP-immunoreactive neurons are involved in visual circuitry, electron microscopic preparations were analyzed from normal rats and rats with optic nerve transections. A co-localization analysis was also made because CGRP-labeled neurons had features of GABAergic neurons. Thus, sections were prepared for light microscopy to determine whether CGRP-containing neurons also had glutamate decarboxylase (GAD) and other markers for GABAergic neurons, such as calcium binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV). Electron microscopy of the DTN and LTN showed CGRP-labeled somata and dendrites that were postsynaptic to axon terminals forming asymmetric synapses. Many of these axon terminals degenerated following optic nerve transection indicating that retinal ganglion cells form synapses with CGRP-labeled neurons in the AON. In the DTN, LTN and MTN, CGRP-labeled axon terminals formed symmetric synapses with unlabeled somata as well as dendritic shafts and spines. Consistent with this type of synapse being GABAergic were the co-localization data showing that about 90% of the CGRP-labeled neurons co-localized GAD in the AON. Many CGRP-labeled neurons showed immunostaining for CR (40%) whereas only a few had labeling for CB (5%). No CGRP-labeled neurons had PV. These data show that CGRP-containing neurons receive direct retinal input and represent a subpopulation of GABAergic neurons which differentially co-express calcium-binding proteins.

Animals↗
PubMed1999

Calbindin-D 28kD immunofluorescence in ventral mesencephalic neurons labeled following injections of Fluoro-Gold in nucleus accumbens subterritories: inverse relationship relative to known neurotoxin vulnerabilities.

The shell and core of the nucleus accumbens exhibit different vulnerabilities to neurotoxins. Calcium binding proteins are reported to offer some neuroprotection against excitotoxicity by suppressing or buffering intracellular calcium. Differences in the distributions of the calbindin-D 28kD (CB) and calretinin (CR) might be related to the different vulnerabilities to neurotoxins of dopaminergic neurons in the ventral mesencephalon that project to the core and medial shell of the nucleus accumbens. To address this possibility, Fluoro-Gold (FG) was injected into accumbens subterritories and numbers of retrogradely labeled neurons in the ventral tegmental area containing CB and CR immunoreactivities (ir) were expressed as a percentage of total numbers of labeled neurons. The perikaryal diameters and lengths of the immunoreactive dendrites of FG labeled neurons were also measured. About 70% and 35% of retrogradely labeled cells observed following core and medial shell injections, respectively, exhibited CB immunoreactivity. Differences were not observed in the percentages of FG labeled cells exhibiting CR immunoreactivity following medial shell (13%) and core (15%) injections. The mean perikaryal diameters and median summed lengths of dendrites of retrogradely labeled neurons containing CB were smaller than in labeled neurons lacking CB following injections in both core and medial shell of the nucleus accumbens. The data indicate that the different 6-hydroxydopamine (6-OHDA) vulnerabilities of ventral mesencephalic dopaminergic neurons are not obviously related to the presence of CB and CR.

Animals↗
PubMed1999

Calretinin and calbindin-D28k in dopaminergic neurons of the rat midbrain: a triple-labeling immunohistochemical study.

We used triple-labeling immunohistochemistry in rat midbrain sections to identify dopaminergic neurons that contain either one or both of the calcium-binding proteins, calretinin (CR) and calbindin-D28k (CB). Midbrain dopaminergic neurons were immunohistochemically labeled for tyrosine hydroxylase (TH), CR, and CB. In the substantia nigra pars compacta (SNC), TH+/CR+/CB+ cells were clustered in two regions: the dorsal tier of the rostral SNC and the medial part of the intermediate SNC. The ventral tier of the rostral SNC mainly comprised both TH+/CR+/CB- and TH+/CR-/CB- cells. The lateral part of the intermediate SNC and the caudal SNC primarily consisted of TH+/CR-/CB- cells. Throughout the extent of the SNC, approximately half of the TH+ neurons were stained for neither CR nor CB, while the remaining TH+ populations were labeled for CR and/or CB. Throughout the ventral tegmental area, TH+/CR+/CB+ cells, TH+/CR+/CB- cells, TH+/CR-/CB+ cells, and TH+/CR-/CB- cells were found generally scattered, though the TH+/CR-/CB- cells were dominant in number. In the substantia nigra pars lateralis, interfascicular nucleus, and caudal linear nucleus, more than half of the TH+ cells were stained for both CR and CB. In the retrorubral field, two-thirds of the TH+ neurons contained neither protein. The present findings suggest that the SNC can be divided into subcompartments based on the distribution of dopaminergic neurons that contain calcium-binding proteins. Furthermore, because CR and CB likely contribute to calcium homeostasis by buffering intracellular calcium concentrations, midbrain dopaminergic neurons containing one or both of these calcium-binding proteins may have a higher calcium-buffering capacity than those lacking the two proteins.

Animals↗
PubMed1995

Hippocalcin in rat retina. Comparison with calbindin-D28k, calretinin and neurocalcin.

The post-natal developmental expression in rat retina of four calcium-binding proteins belonging to the calmodulin-troponin-C family was investigated by immunohistochemistry using anti-calbindin-D28k, anti-calretinin, anti-hippocalcin and anti-neurocalcin polyclonal antibodies on paraffin sections from Wistar rat retinae aged from post-natal days 1 (P1), 5 (P5), 10 (P10), 20 (P20) to adulthood (8 weeks). Immunoblot using anti-hippocalcin and homogenates proteins from retina, cerebellar cortex, hippocampus and cerebellum was also performed. Hippocalcin immunoreactivity in adult rat retina was demonstrated by both immunohistochemistry and Western blot. During post-natal development, calbindin-D28k, calretinin and neurocalcin immunoreactivity were detected at P1 in ganglion cells, whereas hippocalcin immunoreactivity was seen later at P5 in this cell layer. In the amacrine cell layer, neurocalcin immunoreactivity was detected at P5 and hippocalcin at P10. Calbindin-D28k was labelling the immature horizontal cell, calretinin was detected in nearly all ganglion cells and in some amacrine cells since P1. These three calcium-binding proteins do not seem to play a role in synaptogenesis which takes place later. We confirmed that calbindin-D28k appeared to be a good marker for horizontal cells. The presence of hippocalcin, a myristoylated calcium-binding protein belonging to the recovering subfamily and previously localized in few brain areas has been detected for the first time in retina.

Animals↗
PubMed1998

Identification of neurons that express stem cell factor in the mouse small intestine.

BACKGROUND & AIMS: Enteric neurons in the murine intestine express stem cell factor (SCF), which may provide an important signal in the development of the interstitial cells of Cajal (ICC). Our aim was to identify the subpopulation(s) of myenteric neurons that express SCF. METHODS: Myenteric plexus preparations from postnatal SCF-lacZ mice were processed for beta-galactosidase histochemistry followed by immunohistochemistry. RESULTS: Approximately 60% of the nitric oxide synthase-immunoreactive neurons, which projected to myenteric ganglia and to circular muscle, expressed SCF, and more than 80% of the calbindin-immunoreactive neurons, which projected exclusively to myenteric ganglia, expressed SCF. A small subpopulation of calretinin-immunoreactive neurons expressed SCF transiently. Many of the remainder of SCF-expressing neurons were choline acetyltransferase immunoreactive, but their projections are unknown. CONCLUSIONS: SCF-expressing neurons that project within the myenteric plexus may be an important source of SCF for the development of Kit-expressing ICC at this level. The only possible neuronal source of SCF for the ICC of the deep muscular plexus is a subpopulation of nitric oxide synthase-immunoreactive neurons.

Animals↗
PubMed2001

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

Acetylcholine↗
PubMed1993

Calretinin immunoreactivity in the magnocellular neurosecretory nuclei of the rat hypothalamus.

The distribution of calretinin-immunoreactivity in the magnocellular neurosecretory nuclei of the rat hypothalamus was studied using a polyclonal antibody and the avidin-biotin immunoperoxidase technique. Calretinin-immunoreactive neurons were observed in the supraoptic and paraventricular nuclei. Additionally, we detected for the first time, immunoreactive neurons located in the circularis and fornicals nuclei, and isolated positive neurons situated in the hypothalamic area located between the supraoptic and paracentricular nuclei. When these results were compared to those obtained in previous studies for another two calcium-binding proteins: calbindin D-28k and parvalbumin, two major differences may be concluded: a) a different distribution of calretinin, especially in the paraventricular nucleus, and b) an expression of calretinin lower than calbindin D-28k and higher than parvalbumin in the magnocellular hypothalamic nuclei. Of special interest is the fact that calretinin is one of the few markers which demonstrates predominantly parvicellular neurons in the paraventricular nucleus. Although the exact biochemical function of these three calcium-binding proteins remains unknown, their uneven and characteristic distributions strongly suggest that specific neuronal populations in the hypothalamus may use alternatively different calcium-sequestering molecules.

Animals↗
PubMed1997

An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative.

In the last 5 years the microwave oven has become a widely accepted apparatus for retrieving all kinds of antigens that are masked by prolonged formaldehyde fixation. However, it has so far not been possible to obtain good results for all the antigens we were interested in with just one method. This study offers a solution for this problem. We tested Tris buffered saline (TBS) pH 8-10 in order to see whether a microwave (MW) pretreatment in a solution with a high pH works on the antigens that have our particular interest: MAP-2, non-phosphorylated part of the neurofilament (SMI-32, SMI-311), phosphorylated part of the neurofilament (SMI-312) and the calcium binding proteins Calbindin D28-K, Parvalbumin and Calretinin. Furthermore we checked whether or not a lower temperature of 90 degrees C (instead of boiling the tissue) could be used to achieve the same good staining results. These tests showed that an MW pretreatment in TBS pH 9.0 at full power (boiling) gave the best results. Pretreatment at a lower temperature gave also good results for most (but not all) antibodies when the irradiation time was prolonged to 30 min. The optimal antigen retrieval method also improved the quality of staining with an antibody against neuropeptide Y (NPY), for which, until now, no antigen retrieval appeared to be necessary, even after a long duration of fixation.

Adolescent↗
PubMed2003

Cajal-Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin.

Cajal-Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal-Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal-Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal-Retzius cells (reelin(+)) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin(+), and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1(+) cells in the marginal zone were almost always reelin(+). The first Cajal-Retzius cells (Tbr1(+)/reelin(+)) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin(+), and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx(+)) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal-Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.

Animals↗
PubMed1997

Calcium-binding proteins in the substantia nigra and ventral tegmental area during development: correlation with dopaminergic compartmentalization.

The importance of calcium in neuronal function has been amply demonstrated in recent years. The discovery of a class of proteins within neurons which bind calcium, therefore, has proven to be a catalyst for the generation of theories and hypotheses regarding mechanisms of neurotoxicity in the CNS. In addition, the distribution of certain calcium-binding proteins changes during neural development, suggesting that they may play a role in organization or pattern generation. We have examined the ontogeny of three related calcium-binding proteins, calbindin-D28, parvalbumin and calretinin, with respect to the ventral and dorsal compartments or tiers of the dopaminergic population in the ventral midbrain. Single and dual-label immunocytochemistry was employed to map the distributions of calcium-binding proteins and tyrosine hydroxylase from E18 through adulthood. The results show that each of the three proteins exhibits a unique developmental sequence and compartment preference, with calbindin D28 clearly related to the later-developing dorsal tier, and parvalbumin and calretinin to the ventral tier of the dopaminergic ventral mesencephalon.

Aging↗
PubMed1997

Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum.

Three calcium-binding proteins, calretinin, calbindin D28K and parvalbumin, were immunohistochemically localized in the human cerebellum at different developmental stages. Cells positive for calretinin were not detected during early development of the cerebellum until 21 weeks of gestation at which stage weak staining was found in Purkinje and basket cells of the cortex and in neurons of the dentate nucleus. Both the number of positive cells and the intensity of immunoreactivities were found to increase as the cerebellum became more mature. Calbindin D28K immunoreactivity was, however, detected early in development at 14 weeks of gestation. Positive cells were found in Purkinje, basket, stellate and granule cells of the cerebellar cortex and in neurons of fastigial, globose, emboliform and dentate nuclei. The number of positive cells and the staining intensity for calbindin in both the cerebellar cortex and deep nuclei decreased at more advanced developmental stages. At 21-31 weeks of gestation, positive staining was restricted to Purkinje and basket cells of the cortex. Parvalbumin immunoreactivity was also observed early in development at 14 weeks of gestation. Positivity was found in Purkinje, basket and stellate cells of the cerebellar cortex and in neurons of all the deep nuclei, with the highest number of positive cells in the fastigial nucleus followed by emboliform, globose and dentate nuclei. As the cerebellum became more mature, both the number of positive cells and the staining intensity for parvalbumin decreased in the cortex and deep nuclei. The results of the present study showed that among the three calcium-binding proteins examined, strong immunoreactivities for calbindin D28K and parvalbumin were found inthe human cerebellum early in development at 14 weeks of gestation, but there was a decrease in both the intensity and number of positive cells at more advanced stages. In contrast, calretinin positive cells were not detected until 21 weeks of gestation and the immunoreactivity increased as the cerebellum became more mature. A possible correlation between the developmentally regulated expression of the calcium-binding proteins and expression of different neurotransmitters during development is discussed.

Abortion, Induced↗
PubMed2003

Distribution of NADPH-diaphorase in the superior colliculus of Cebus monkeys, and co-localization with calcium-binding proteins.

We examined the distribution of the enzyme dihydronicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the superior colliculus (SC) of the New World monkey Cebus apella, and the co-localization of this enzyme with the calcium-binding proteins (CaBPs) calbindin-D28K, parvalbumin and calretinin. Despite the intensely labeled neuropil, rare NADPH-d-positive cells were observed in the stratum griseum superficiale (SGS). Most of the labeled cells in the SC were found in the intermediate layers, with a great number also in the deeper layers. This pattern is very similar to that described in the opossum (Didelphis marsupialis) and in the cat, and different from the pattern found in the rat, which shows labeled cells mainly in the SGS. Cells doubly stained for NADPH-d and CaBPs were observed throughout the SC, although in a small number. Of the NADPH-d-positive cells, 20.3% were doubly labeled for NADPH-d and parvalbumin, 10.2% revealed co-localization with calretinin, and 5.6% with calbindin. The low number of double-stained cells for NADPH-d and the CaBPs indicates that these molecules must participate in different functional circuits within the SC.

Animals↗
PubMed1998

Calcium-binding proteins in primate cerebellum.

Single and double antigen localization procedures were used to study the distribution of the calcium-binding proteins calretinin, calbindin and parvalbumin in the cerebellum of the squirrel monkey (Saimiri sciureus). The immunostaining experiments have revealed that each of the three calcium-binding proteins occurred, either alone or in various combinations, in many neuronal types of the monkey cerebellum, including the Purkinje cells. Immunoreactivity for calbindin was detected in virtually all Purkinje cells, whereas immunoreactivity for calretinin and parvalbumin was encountered only in some subpopulations of Purkinje cells. In the vermal region, parvalbumin immunostaining appeared in the form of typical weak and strong alternating parasagittal bands. Calretinin immunoreactivity was found in virtually all neurons and fiber systems related to the granular layer, including the monodendritic cells, the granule cells and their parallel fibers, the Golgi and Lugaro cells and the mossy fibers. The Golgi cells also displayed calbindin and parvalbumin immunoreactivity. Parvalbumin was found to labeled both the climbing and mossy fibers, as well as the basket and stellate cells lying in the molecular layer. These results reveal that virtually all the different neuronal types in the primate cerebellum contain at least one of three calcium-binding proteins investigated in the present study. Furthermore, calretinin appears to be a particularly reliable molecular maker for all the neuronal elements associated with the granular layer in the primate cerebellum.

Animals↗
PubMed2001

Calbindin-D28k and calretinin immunoreactive neurons in the olfactory bulb of the musk shrew, Suncus murinus.

The distribution, morphological features, and postnatal development of calbindin-D28k (CB) and calretinin (CR) immunoreactive neurons in the main olfactory bulb (MOB) of the musk shrew, Suncus murinus, were studied by immunostaining to determine the degree of colocalization of CB and CR, and the relationship of CB and CR to neuron development in the MOB of animals of the order Insectivora. In adults, CB-positive neurons were identified as periglomerular and perinidal cells in the periglomerular region, as superficial short-axon cells in the external plexiform layer, and as four types of interneurons (Cajal, horizontal, Golgi, and bitufted cells) in the mitral cell, internal plexiform, and granule cell layers. CR-positive neurons were identified as projection neurons (tufted and mitral cells) and interneurons (periglomerular, perinidal, and granule cells). On postnatal days 1 and 3, CB-positive neurons revealed numerous processes finely arborized near the somata, and were morphologically unidentifiable. At the same time, CR-positive neurons were identified as young periglomerular and granule cells, and as migrating bipolar cells extending leading processes with growth cones in each layer of the MOB and the subependymal layer between the anterior lateral ventricle and the center of the MOB. On postnatal day 28, mature CB-positive and CR-positive interneurons were distributed in their corresponding layers, whereas migrating CR-positive bipolar cells were rarely detected. No cells colocalized CB and CR. The results suggest that perinidal cells in the shrew MOB may develop postnatally, together with glomerular and granule cells. We suggest that CB is associated with mechanisms of the outgrowth of neuronal processes, whereas CR is involved in mechanisms of cell migration and outgrowth of neuronal processes, in some types of neurons in the developing stage of the shrew MOB.

Age Factors↗
PubMed2003

Cholecystokinin activates specific enteric neurons in the rat small intestine.

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.

Animals↗
PubMedDate not supplied

Age-related changes of calbindin-D28k, calretinin, and parvalbumin mRNAs in the hamster brain.

Changes of three different cytosolic Ca2+ binding proteins, calbindin-D28k, calretinin, and parvalbumin mRNA expression in the brain of the hamster during aging were investigated by in situ hybridization using brains from hamsters aged 4, 9, 13, 19, to 24 months old. In cerebellum area, calbindin-D28k transcripts showed about 50% to 68% decrease in content in aged-hamster (19 and 24 months old) compared with young (4 months) and adult (9 months), whereas calretinin and parvalbumin mRNA expression remain unchanged throughout the ages examined. Calbindin-D28k gene expression was decreased during aging also in the hippocampus (approximately 60% reduction) and striatum (approximately 25%). In the same areas, striatum and hippocampus, calretinin and parvalbumin mRNA expression in the equivalent sections were not significantly changed with age. These data raise the possibility that CNS calbindin-D28k expression may be selectively down-regulated during aging. The statistically significant decrease of calbindin-D28k mRNA in the normal aging process also suggests and provides further support for the hypothesis that this calcium binding protein may have an important role in neuronal degeneration.

Aging↗
PubMedDate not supplied

Calretinin and calbindin-D28K in male rats during postnatal development.

Calcium-binding proteins play potentially important roles in neurogenesis and neuroprotective mechanism(s). Some evidence exists that brain calbindin-D28K (CALB) is regulated by androgens. In the present study, calretinin (CALRET) and CALB patterns were determined by Western analysis in the medial basal hypothalamus (MBH) from male rats along with assaying plasma testosterone levels during postnatal development. Testosterone levels were very low in 7-, 10-, and 30-day-old animals (approximately 0.5 ng/mL), increased in a stair-step fashion to peak levels at 90 days (approximately 3.8 ng/mL), then declined with increasing age to very low levels at 300 days of age (approximately 0.3 ng/mL). At 7 and 10 days, MBH CALRET and CALB levels were low; however, at Day 30 a significant twofold increased was observed. Thereafter, in 60-, 120-, 180-, and 300-day-old animals MBH CALRET and CALB levels were, in general, comparable to 30-day-old values. These findings suggest that there is not a clear correspondence between the androgen status in male rats and the calcium-binding proteins (CALRET & CALB) expressed in the MBH. Therefore, it appears that brain CALRET and CALB are regulated in a developmental fashion with significant increases in expression occurring around the 4th postnatal week.

Aging↗
PubMed2000

Co-existence of calcium-binding proteins in neurons of the medullary dorsal horn of the rat.

Double immunofluorescence histochemistry for calcium-binding proteins was performed in the caudal subnucleus of the rat spinal trigeminal nucleus; for calbindin D28k (CB) and calretinin (CR), for CB and parvalbumin (PV), and for CB and CR. CB-immunoreactive (-ir) neurons were seen 1.7 times more frequently than CR-ir neurons and 5.5 times more frequently than PV-ir neurons. About 70-90% of these neurons were distributed in substantia gelatinosa. Co-existence of CB and CR was indicated in 2.3% of CB-ir and 3.9% of CR-ir neurons. Co-existence of CB and PV was indicated in 1.0% of CB-ir and 5.5% of PV-ir neurons. Co-existence of CR and PV was indicated in 1.4% of CR-ir and 5.1% of PV-ir neurons. In these doubly immunostained neurons, 59.5-69.5% were observed in substantia gelatinosa, 5.9-17.8% in the marginal zone, and 12.7-31.0% in the magnocellular part.

Animals↗