Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala

Specific neuronal populations in the basolateral amygdala (ABL) exhibit immunoreactivity for distinct neuropeptides and calcium-binding proteins. In the present study, immunohistochemical techniques were used to analyze neurons in the rat ABL that contain cholecystokinin (CCK). Some pyramidal projection neurons in the anterior subdivision of the basolateral nucleus exhibited low levels of CCK immunoreactivity in rats that received injections of colchicine to interrupt axonal transport; staining was concentrated in the axon initial segments of these cells. High levels of CCK immunoreactivity were observed in two subpopulations of nonpyramidal interneurons in all nuclei of the ABL: (1) type L neurons (characterized by large somata and thick dendrites), and (2) type S neurons (characterized by small somata and thin dendrites). Dual-labeling immunofluorescence studies using confocal laser scanning microscopy revealed that many (30-40%) type L CCK+ interneurons exhibited immunoreactivity for calbindin (CB), but not for parvalbumin (PV), calretinin (CR), or vasoactive intestinal polypeptide (VIP). In contrast, there was extensive colocalization of CR and VIP with CCK in type S neurons, but no significant colocalization with CB or PV. In addition, the majority of CR and VIP interneurons exhibited colocalization of both neurochemicals. Collectively, the results of this and previous studies indicate that there are at least four distinct interneuronal subpopulations in the ABL: (1) PV+ neurons (the great majority of which are CB+); (2) SOM+ neurons (many of which are CB+ and NPY+); (3) large CCK+ neurons (some of which are CB+); and (4) small bipolar/bitufted neurons that exhibit various amounts of colocalization of CCK, VIP, and CR.     

Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala

There are discrete subpopulations of GABAergic interneurons in the basolateral amygdala (ABL) that contain particular neuropeptides or calcium-binding proteins (calbindin-D28k, parvalbumin (PV), or calretinin). The present study employed a dual-labeling immunofluorescence technique combined with confocal laser scanning microscopy to investigate the neurochemical characteristics of the interneuronal subpopulation containing somatostatin (SOM). The great majority of SOM+ neurons in the ABL exhibited GABA immunoreactivity (66-82% depending on the nucleus). These SOM+ neurons constituted 11-18% of the GABA+ population. There was also extensive colocalization of SOM with calbindin (CB) in all nuclei of the ABL, but no colocalization of SOM with parvalbumin, calretinin, or vasoactive intestinal polypeptide. In the basolateral nucleus more than 90% of SOM+ neurons also exhibited CB immunoreactivity, whereas in the lateral nucleus about two-thirds of SOM+ neurons contained significant levels of CB. These SOM/CB neurons constituted about one quarter of the CB+ population in the basolateral nucleus and about one third of the CB+ population in the lateral nucleus. These results, in conjunction with the findings of previous studies, indicate that there are at least three major subpopulations of GABAergic interneurons in the ABL: (i) SOM+ neurons (most of which also contain CB and/or neuropeptide Y); (ii) PV+ neurons (most of which also contain CB); and (iii) CR+ neurons (most of which also contain vasoactive intestinal polypeptide).     

Appearance of calretinin-immunoreactive neurons in the upper layers of the rat superior colliculus after eye enucleation

The effects of retinal deafferentation on a calcium-binding protein, calretinin, in the upper layers (superficial gray layer and optic nerve layer) of the rat superior colliculus were examined. In intact rats and on the ipsilateral side of unilaterally eye-enucleated rats, the superficial gray layer and optic nerve layer contained a few dispersed calretinin-immunoreactive cells. On the contralateral side to the enucleation, the number of immunostained cells in the superficial gray layer and optic nerve layer was increased. These findings suggest that retinal deafferentation results in an increase in contents of calretinin in some cell bodies within the upper layers of the superior colliculus.     

Feeding-related activity of glucose-and morphine-sensitive neurons in the monkey amygdala

Feeding-related neuronal activity of monkey amygdalar glucose-sensitive and morphine-sensitive cells was investigated during a task that required bar-pressing to obtain food. Both glucose-sensitive and morphine-sensitive cells, located mostly in the centromedial part of the amygdala, decreased firing during the bar-press period more often than insensitive cells. Naloxone attenuated the decrease in activity during the bar press period. The results suggest involvement of these glucose- and morphine-sensitive cells in the control of food acquisition behavior.     

Calretinin is largely localized to a unique population of striatal interneurons in rats

Previous studies have reported the presence of the calcium binding protein calretinin in neurons in the striatal part of the basal ganglia in rats and primates. In the present study, immunofluoroscence double-labeling techniques and immunofluorescence combined with retrograde labeling were used in rats to determine whether calretinin is found in any of the known types of striatal neurons. The results showed that a small fraction of the calretinin-containing neurons (< 10%) contain parvalbumin, but none of the calretinin-containing striatal neurons contained markers for the other tow major types of striatal interneurons (i.e., choline acetyltransferase-containing cholinergic neurons and somatostatin-containing neurons). Additionally, calretinin was not found in projection neurons, using either calbindin or DARPP32 as immunofluoroscent markers of striatal projections neurons in general, or using retrograde labeling to specifically identify either striatonigral or striatopallidal neurons. Thus, calretinin appears to be largely found in a unique population of striatal interneurons in rats. This population appears to be about one third the abundance of any of the previously identified populations of striatal interneurons.     

Decrease of GABA-immunoreactive neurons in the amygdala after electrical kindling in the rat

The present study was designed to investigate the effects of electrical kindling in vivo on GABA immunoreactivity (GABA-IR) of the lateral and basolateral amygdaloid nuclei 2-6 months post-stimulation. Male Sprague-Dawley rats were implanted with bipolar electrodes in the basolateral nucleus and stimulated once per day until 3-5 stage 5 seizures were observed. Coronal sections containing the amygdala were processed for GABA-IR using the contralateral side of the brain. Results indicate that, in comparison to controls, fully kindled animals showed a significant decrease in total number of GABA-IR amygdala neurons. Decreases in GABA-positive punctate structures surrounding unlabeled pyramidal cells were also observed, but not quantified. The present data suggest that epileptogenesis of the amygdala is associated with a significant reduction of GABA-IR in the lateral and basolateral areas throughout the contralateral amygdaloid nucleus.     

Cholecystokinin immunoreactive neurons in the basolateral amygdala of the rhesus monkey (Macaca mulatta)

Several distinct subpopulations of interneurons (INs) in the amygdalar basolateral nuclear complex (BNC) of the rat can be recognized on the basis of their expression of calcium-binding proteins and neuropeptides, including parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK). In the rat BNC CCK is expressed in two separate IN subpopulations, termed large (CCK_L) and small (CCK_S). These subpopulations exhibit distinct connections indicative of discrete functional roles in the circuitry of the BNC. Although there have been several studies of PV+, SOM+, and CR+ INs in the primate BNC, there is almost no information regarding CCK+ INs in these species. Therefore, in the present study the distribution and morphology of CCK+ INs and their axon terminals in the BNC of the monkey was investigated. CCK immunoreactivity in the BNC was observed in somata and proximal dendrites of nonpyramidal neurons, as well as in axon terminals. A moderate density of CCK+ INs was found in all nuclei of the BNC. CCK+ INs in the BNC were morphologically heterogeneous, with both small and large varieties observed. All CCK+ somata gave rise to 2–4 dendrites that branched sparingly and were aspiny. CCK+ axon terminals in the BNC were found both in the neuropil and forming pericellular baskets contacting somata of pyramidal cells. In addition, many CCK+ neurons were contacted by multiple CCK+ terminals, indicative of the existence of a CCK interneuronal network. These data indicate that the morphology of CCK+ INs in the monkey is very similar to that of the rat.     

Development of immunoreactive lhrh neurons in the fetal rat hypothalamus

The ontogenesis of immunoreactive (ir) LHRH neurons was investigated in rats applying the double-bridge PAP method of Vacca et al. (J. Histochem. Cytochem. 28, 297–307, 1980). Ir LHRH was first evident in the cell bodies confined in the ventromedial surface of the anterior part of the forebrain vesicles on day 16.5 of gestation. Only one or two cells, if any, were found in the brains examined; the cells are oval showing ir brown granules within the perikarya and also within the processes extended from both poles. On day 17.5 of gestation, a few ir cells appeared in the olfactory cortex, medial septum, medial preoptic area, diagonal band of Broca, and ventro-lateral surface of the anterior hypothalamus, and, with development, their numbers increased gradually. During the prenatal period, no ir cell exists in the arcuate nucleus or in the medial-basal hypothalamus. Beaded ir fibers appeared in the organum vasculosum of the lamina terminalis (OVLT) and in the external layer of the median eminence on 18.5 and 19.5 days of gestation, respectively. The present findings ascertain that the hypothalamic regulation of hypophysial gonadotrophic function exists during fetal period in rats.     

Calretinin-immunoreactive neurons in the human striatum

The human striatum contains two types of neurons displaying immunoreactivity for the calcium-binding protein calretinin (CR): (1) large (22–44 μm), multipolar neurons with 5–7 long, aspiny and highly branched dendrites, and (2) medium-sized (9–18 μm), round-to-oval neurons with 2–3 long, varicose and poorly branched dendrites. These CR neurons represent only a small proportion of the total neuronal population and they are heterogeneously distributed in the striatum. The large CR neurons are more numerous in the putamen than in the caudate nucleus, whereas the inverse is true for the medium-sized CR neurons. The ratio of large- to medium-sized CR neurons is 1:4 in the putamen compared to 1:16 in the caudate nucleus. The existence of these two distinct subsets of chemospecific striatal neurons suggest that CR may play an important role in the intrinsic organization of the human striatum.     

Immunohistochemical localization of neurocalcin in the rat inner ear

Localization in the rat inner ear of neurocalcin, a three EF-hand calcium-binding protein, was examined immunohistochemically. Neurocalcin-like immunoreactivity was restricted to neurons in neuroepithelial receptor organs, while hair cells and supporting cells showed no such immunoreactivity. In the organ of Corti, both afferent and efferent nerve terminals, which formed synapses on both inner and outer hair cells, showed distinct immunoreactions. Spiral ganglion neurons and cochlear nerves were immunopositive. In the cristae ampullaris, macula utriculi and macula sacculi, afferent nerve terminals forming nerve calices or terminal boutons were strongly immunopositive. Efferent nerve terminals making synapses either on nerve calices or on hair cells showed an intense immunoreactivity. Vestibular ganglion neurons were strongly immunopositive. In electron microscopy, immunoreaction products were diffuse in the cytoplasm of ganglion neurons and nerve terminals. Neurocalcin-like immunoreactivity occurred in association with microtubules, outer mitochondrial membranes, synaptic vesicles and synaptic membranes. It is thus likely that neurocalcin is involved in neural functions in each type of afferent and efferent transmission in the inner ear.     

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.     

Calretinin immunoreactivity in the anterior olfactory nucleus of the rat

Calretinin-immunoreactive structures in the anterior olfactory nucleus of the rat were studied using a polyclonal antibody, which does not cross-react with the highly homologous calcium-binding protein calbindin-D28k, and the avidin–biotin–peroxidase technique. Calretinin-immunopositive neurons were found in all regions of the anterior olfactory nucleus, with the highest number in the medial subdivision and dorsal transition area. The immunostained neurons, although morphologically heterogeneous, demonstrated typically small size. In addition to neuronal somata, calretinin-immunopositive fibres and terminals, some of them forming basket-like arrangements surrounding immunonegative neurons, were observed. Although calretinin and calbindin-D28k colocalize in several brain regions, and both proteins showed an extensive overlap in the anterior olfactory nucleus, immunostained semithin sections demonstrated that calretinin does not co-localize with calbindin-D28k in this nucleus.     

Calretinin, calbindin-D28k and parvalbumin-like immunoreactivity in mouse chemoreceptor neurons

Calretinin immunoreactivity was demonstrated in adult mouse olfactory receptor neurons and in the vomeronasal and septal chemoreceptor neurons, whereas parvalbumin expression was restricted to the vomeronasal receptor neurons. Calbindin-D28k-like immunoreactivity was primarily localized in the vomeronasal and septal chemoreceptor neurons although an occassional neuronal staining with calbindin-D28k was also found in restricted areas of the main olfactory epithelium.     

The effect of satiety on responses of gustatory neurons in the amygdala of alert cynomolgus macaques

An alert cynomolgus macaque was fed a sweet solution to satiety as the activity of a gustatory neuron in the amygdala was recorded to that solution and to four other taste stimuli. This experiment was conducted a total of 14 times in two monkeys. The responses of individual neurons to the satiety stimuli were suppressed by as little as 1%, and as much as 100% by the induction of satiety (mean suppression = 58%). Nine of the 14 cells responded to the satiety solution with excitation, and their responses were suppressed by a mean of 62% by satiety. Five neurons responded with inhibition, and their responses were suppressed by a mean of 50%. Responses to other taste stimuli, not associated with satiety, were affected to a lesser extent. The amygdala is a taste relay between the primary gustatory cortex, where satiety has no influence on responses to taste stimuli, and the lateral hypothalamic area where the effect of satiety is total. The data presented here indicate that the amygdala is a functional as well as anatomical intermediary between these two areas, and serves as a stage in the process through which sensory stimuli are imbued with motivational significance.     

Bombesin microinjection into the basolateral amygdala influences feeding behavior in the rat

It has been demonstrated that the basolateral amygdala (ABL) represents a satiety mechanism. Experimental data indicate that peripheral or central applications of neuropeptide bombesin (BN) and BN-like peptides inhibit feeding. Since the amygdala (AMY) is rich in BN-like immunoreactive elements, the present study was performed to determine whether 10 or 40 ng doses of BN microinjected bilaterally into the ABL could modify solid food intake. Twenty nanograms of BN (10 ng per injection site) in 24-h deprived rats caused transient inhibition of food intake and 80 ng resulted in a significant reduction of food consumption for 1 h. This inhibitory effect of BN on feeding was eliminated by prior BN antagonist treatment. Results of behavioral tests showed that BN microinjections into the ABL specifically reduced food intake without altering behavioral patterns or influencing the body temperature. Present results suggest that BN-like peptides may act as a complex satiety signal in the ABL.     

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, atM_r = 29k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.     

A subset of calretinin-positive neurons are abnormal in Alzheimer's disease

The distribution of the calcium-binding protein calretinin was investigated by immunohistochemistry in the hippocampus, the subicular areas, and the entorhinal cortex in patients with Alzheimer's disease and in control subjects. By double immunolabelling, the calretinin immunoreactivity was compared to the immunoreactivity for /A4 amyloid or for tau proteins. Calretinin-positive neurons were mainly observed in the molecular layer of the gyrus dentatus, the stratum radiatum of the Ammon's horn, and in layers II and III of the entorhinal cortex. The general pattern of calretinin immunoreactivity was conserved in Alzheimer's disease. Calretinin-positive neurons appeared normal in the hippocampus but had a reduced dendritic tree in the entorhinal cortex. Dystrophic calretinin immunoreactive fibres were often observed in the outer molecular layer of the gyrus dentatus and in the CA4 sector in Alzheimer's disease. Most neurons containing neurofibrillary tangles were not calretinin immunoreactive and most senile plaques were not associated with calretinin positive fibres. These results show that entorhinal calretinin-positive neurons are affected in Alzheimer's disease in spite of an absence of systematic association with neurofibrillary tangles and senile plaques.Supported by grants from the Belgian FRSM (3.4504.91, 3.4517.92), the Fonds de Recherche Divry, the Fondation M.T. de Lava and Alzheimer Belgique     

Subtypes of substance P receptor immunoreactive interneurons in the rat basolateral amygdala

Local injections of the neurotoxin SP-saporin into the basolateral amygdala (BLA) are reported to specifically lesion substance P receptor immunoreactive (SPR-IR) interneurons, and to reduce anxiety related behavior. Hence, this technique might provide a means to study how defined interneuron populations regulate neuronal activity in the BLA. However, what interneuron subgroups in the BLA might be targeted by SP-saporin lesions has not been established. This study has used dual-labeling immunofluorescence in the rat BLA to examine SPR-IR neurons for their colocalization with the calcium-binding proteins; calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR); and the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY). We found that all NPY-IR neurons and 45% of SOM-IR interneurons expressed SPR-IR, and that 50% and 51% of the SPR-IR interneuron population expressed NPY- and SOM-IR, respectively. Previous studies have reported that approximately a third of SOM-IR interneurons also express NPY, which suggests a large degree of overlap between the NPY, SOM and SPR expressing neurons in the BLA. We also found that the majority of SPR-IR cells were CB-IR (62%), but that these interneurons represented only 2.8% of the total CB-IR population. Moreover, SPR-IR interneurons did not express either PV-or CR- IR. Hence, SP-saporin lesions would ablate all interneurons in the BLA that contain NPY, but leave the majority of the CB-IR cells intact, and have no effect on the CR- and PV-IR populations. Consequently, these results support the use of SP-saporin lesions as a useful technique to study the role of NPY-IR interneurons in the BLA.     

Neurocalcin immunoreactivity in the rat accessory olfactory bulb

The distribution and morphology of neurocalcin-immunopositive neurons have been studied in the rat accessory olfactory bulb. Different subsets of neurons displaying neurocalcin immunoreactivity were found in the glomerular layer, the external plexiform layer and the internal plexiform layer. The most abundant staining was detected in the glomerular layer where neurocalcin-immunoreactive periglomerular cells and external tufted cells were observed in the lateral glomeruli, whereas the central region of this layer was practically devoid of immunopositive neurons. In the external plexiform layer, medial tufted cells and Van Gehuchten cells displayed neurocalcin immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunostained. The staining pattern for neurocalcin in the accessory olfactory bulb showed similarities with the immunostaining described in this brain region for another EF-hand calcium binding protein, calbindin D-28k. However, after double immunohistochemical labeling, colocalization of both proteins in the same neuron was not observed, reflecting a biochemical heterogeneity within morphologically homogeneous neuronal groups.     

Differential synaptic distribution of the AMPA-GluR2 subunit on GABAergic and non-GABAergic neurons in the basolateral amygdala

The cellular and ultrastructural distribution patterns of the AMPA glutamate receptor subunit, GluR2, were determined in the rat basolateral amygdala. GluR2 immunoreactivity was widely and uniformly distributed in the basolateral nucleus, with both pyramidal and non-pyramidal neurons labelled. In fact, double label immunohistochemical analyses demonstrated that over 90% of the GABAergic interneurons were labelled for GluR2. Electron microscopic analyses further confirmed the presence of GluR2 in the soma and dendrites of GABAergic interneurons as well as in the soma, spines and dendritic shafts of pyramidal cells. As in our parallel study in the rat hippocampus, immunogold analyses revealed that GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts, as well as the dendritic processes and soma of GABAergic interneurons. However, the number of immunogold particles per labelled synapse on GABAergic neurons was significantly lower than at similar labelled asymmetric synapses on spines of presumed pyramidal cells. Given that the presence of GluR2 within the AMPA receptor complex decreases calcium flux, these data indicate that GABAergic local circuit neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells, as has been suggested for hippocampus. Such cell class-specific differences in the subunit representation and resultant channel properties of AMPA receptors have implications for response properties as well as selective vulnerability of neurons within the basolateral nucleus of the amygdala.