Immunoreactive Neuronal Profiles Research Articles

Localization and phenotypic characterization of brainstem neurons activated by rimonabant and WIN55,212-2

The mechanisms by which the CB1 receptor antagonist rimonabant exerts its appetite-suppressing and energy-dissipating effects are still incompletely resolved. To shed further light on the central pathways influenced by CB1 receptor modulation we examined the expression of the immediate early gene c- fos in male Sprague–Dawley rats at 60, 120 and 240 min after intraperitoneal administration of the CB1R antagonist rimonabant (10 mg/kg) and the CB1R agonist WIN55,212-2 (3 mg/kg). Perfusion-fixed brains were processed for immunohistochemistry and the localization of c-Fos immunoreactive neuronal profiles was assessed qualitatively throughout the brain. Nine areas, including specific hypothalamic and brainstem nuclei known to be involved in appetite regulation, were selected for quantitative analyses. Whereas WIN55,212-2 induced c-Fos immunoreactivity in a time-specific manner in the striatum, the central nucleus of amygdala, the hypothalamic paraventricular nucleus and the arcuate nucleus, no significant increases in c-Fos positive nuclei were found in any forebrain areas following rimonabant administration. In contrast, rimonabant and WIN55,212-2 were both found to significantly increase c-Fos immunoreactivity in the brainstem lateral parabrachial nucleus, the nucleus of the solitary tract and the area postrema. To characterize the phenotype of activated neurons in the nucleus of the solitary tract, a triple immunohistochemical staining technique was used to simultaneously label c-Fos protein and tyrosine hydroxylase (TH), GLP-1 or CART. Interestingly, rimonabant was found to significantly increase c-Fos protein expression in TH-positive neurons. Collectively, these results suggest that brainstem areas including ascending catetholaminergic A2/C2 neurons could play a role in rimonabant-induced inhibition of food intake.

Area-specific increased density of μ-opioid receptor immunoreactive neurons in the cerebral cortex of drug-related fatalities

In animal experiments and in cell culture, chronic morphine treatment has been followed by “up-regulation” as well as “down-regulation” of the μ-opioid receptor (OR) number. The present postmortem morphometric study of morphine-related fatalities of drug-addicts ( n=13, 20–35 years old, with blood unconjugated morphine levels from 27.1 ng/ml to 458 ng/ml, m.v. 198.5 ng/ml) versus a non-addicted control group ( n=13, 10–44 years old) was intended to examine, whether chronic opiate exposure affects the numerical density of μ-OR expressing neurons in the human neocortex (areas 11, 24 and 25 according to Brodmann). For the immunohistochemical procedure, vibratome sections (100 μm) were incubated with a monoclonal antibody against the μ-OR, diluted 1:100, and immunolabelled sites were visualized using an immunoperoxidase protocol. The numerical densities of OR immunoreactive neuronal profiles and Nissl-stained central profiles were assessed morphometrically (camera lucida-drawings). In both groups, the anti-μ-OR-immunoreactivity was mainly localized in pyramidal neurons of layers (L) II/III and V and in multiform neurons of L VI. In the areas 24 and 25, the density of the immunoreactive neuronal profiles did not display a significant difference between the two examined groups. In the area 11, however, the number of immunolabelled neuronal profiles amounted to 2777±206 mm 3 in the drug-related fatalities and to 2320±124 mm 3 in the control group and thus was significantly increased.

Tyrosine kinase B and C receptors in the neostriatum and nucleus accumbens are co-localized in enkephalin-positive and enkephalin-negative neuronal profiles and their expression is influenced by cocaine

Single- and double-label immunohistochemistry were used to determine the extent to which the tyrosine kinase B and C receptors, are expressed in enkephalin-immunopositive or enkephalin-immunonegative neuronal profiles in the rat neostriatum and nucleus accumbens. Results indicate that tyrosine kinase B and C receptors are co-localized in both enkephalin-positive and enkephalin-negative neurons in both of these nuclei, which suggests that these receptors influence both the striatal–pallidal (enkephalin) and striatal–ventral mesencephalic (substance P/dynorphin) pathways. We also examined the influence of acute or repeated injections of cocaine on the number of tyrosine kinase B and C receptors immunoreactive neuronal profiles in the rat neostriatum and nucleus accumbens. Following an acute injection of cocaine (15 mg/kg, i.p.), there were significant decreases in the number of tyrosine kinase B and C receptors immunoreactive profiles in specific regions of the neostriatum and nucleus accumbens relative to saline-pretreated rats. One or 14 days following the last of seven daily injections of 15 mg/kg cocaine or saline there were no differences in the numbers of tyrosine kinase B or C receptors immunoreactive neuronal profiles between these treatment groups. Collectively, the present results indicate that tyrosine kinase B and C receptors in the neostriatum and nucleus accumbens are co-localized in enkephalin-positive and enkephalin-negative neuronal profiles, which suggests that the striatal medium spiny neurons expressing tyrosine kinase B and C receptors include those that project to the pallidum or the ventral mesencephalon. The current results also show that an acute injection of cocaine results in a decrease in the number of tyrosine kinase B and C receptors immunoreactive neuronal profiles in specific regions of the nucleus accumbens and neostriatum, indicating that cocaine-induced increases in extracellular dopamine in the striatal complex result in compensatory decreases in the expression of tyrosine kinase B and C receptors.

Quantitative Distribution of Parvalbumin, Calretinin, and Calbindin D-28k Immunoreactive Neurons in the Visual Cortex of Normal and Alzheimer Cases

The distribution of parvalbumin (PV), calretinin (CR), and calbindin (CB) immunoreactive neurons was studied with the help of an image analysis system (Vidas/Zeiss) in the primary visual area 17 and associative area 18 (Brodmann) of Alzheimer and control brains. In neither of these areas was there a significant difference between Alzheimer and control groups in the mean number of PV, CR, or CB immunoreactive neuronal profiles, counted in a cortical column going from pia to white matter. Significant differences in the mean densities (numbers per square millimeter of cortex) of PV, CR, and CB immunoreactive neuronal profiles were not observed either between groups or areas, but only between superficial, middle, and deep layers within areas 17 and 18. The optical density of the immunoreactive neuropil was also similar in Alzheimer and controls, correlating with the numerical density of immunoreactive profiles in superficial, middle, and deep layers. The frequency distribution of neuronal areas indicated significant differences between PV, CR, and CB immunoreactive neuronal profiles in both areas 17 and 18, with more large PV than CR and CB positive profiles. There were also significantly more small and less large PV and CR immunoreactive neuronal profiles in Alzheimer than in controls. Our data show that, although the brain pathology is moderate to severe, there is no prominent decrease of PV, CR and CB positive neurons in the visual cortex of Alzheimer brains, but only selective changes in neuronal perikarya.

Synaptic Relations of GABAergic Neurons in the Area Postrema

gamma-Aminobutyric acid (GABA)-like immunoreactive neuronal profiles and their synaptic relations in the area postrema of the rat were studied by ultrastructural immunocytochemistry using an antibody directed against GABA. GABA-like immunoreactive (GABA-LI) perikarya and dendrites usually received synaptic inputs from nonimmunoreactive axon terminals. These synaptic contact zones were mostly asymmetrical. Conversely, GABA-LI axon terminals were found to make synapses with nonimmunoreactive perikarya and dendrites, usually of the symmetrical type. Furthermore, GABA-LI axon terminals in synaptic contact with GABA-LI perikarya and dendrites were sometimes found. Although some GABA-LI axon terminals were in close vicinity of other nonimmunoreactive axon terminals, no clear synaptic contacts could be found. On the other hand, GABA-LI profiles could be found close to the capillaries, although they were always separated by a thin layer of glial processes. The above results suggest that the GABAergic neuron receives information from both the circulating blood and other neurons and then performs its depressor function through other neurons.

Serotoninergic innervation of the thalamus in the primate: An immunohistochemical study

Little is known of the serotoninergic innervation of the thalamus in primates; therefore, we undertook a detailed study of the distribution of 5-hydroxytryptamine (5-HT)-immunoreactive neuronal profiles in the thalamus of the squirrel monkey (Saimiri sciureus) with a specific antibody directly raised against 5-HT. All thalamic nuclei in the squirrel monkey displayed 5-HT-immunoreactive fibers, but none contained immunopositive cell bodies. The 5-HT innervation of the thalamus derived from extrinsic fibers arising mostly from the midbrain raphe nuclei and forming the transtegmental system. Most of the fibers destined to the thalamus collected into a major bundle that swept dorsoventrally within the midbrain tegmentum and coursed beneath the thalamus along its entire caudorostral extent. Several fiber fascicles broke off from this main bundle at different levels and ascended dorsally to innervate the various thalamic nuclei. Overall, the 5-HT innervation of the thalamus in the squirrel monkey was more massive than would have been expected from earlier studies in nonprimate species. Marked differences in the regional density of innervation were noted both between the various nuclei and within single nuclei. The most densely innervated nuclei were those delineating the principal subdivisions of the thalamic mass, that is, the midline, rostral intralaminar, limitans, and reticular nuclei, where very dense fields of isolated axonal varicosities occurred. In contrast to the rostral intralaminar nuclei, which were rather uniformly innervated, the centre médian/parafascicular complex contained immunoreactive fibers and isolated varicosities distributed according to a mediolateral gradient. The habenula and the ventral anterior nucleus were among the most weakly innervated nuclei. In the latter nucleus, as well as in more densely innervated nuclei, thin varicose fibers formed numerous pericellular contacts on cell bodies and proximal dendrites of thalamic neurons. The 5-HT innervation of the lateral nuclear group as well as that of the medial and lateral geniculate nuclei ranged from very weak to dense. The mediodorsal nucleus displayed a highly heterogeneous 5-HT innervation that varied from weak in its central portion to moderate or dense in its medial and lateral borders. A moderate 5-HT innervation was observed in the anterior nuclear group. The surprisingly dense and heterogeneous 5-HT innervation of the thalamus noted in the present study suggests that serotonin may be involved in several specific functions of the thalamus in primates.

Dopaminergic innervation of the basal ganglia in the squirrel monkey as revealed by tyrosine hydroxylase immunohistochemistry

The organization of the dopaminergic mesostriatal fibers and their patterns of innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus) were studied immunohistochemically with an antiserum raised against tyrosine hydroxylase (TH). Numerous fibers arose from midbrain TH-positive cell bodies of the substantia nigra pars compacta (group A9), the retrorubral area (group A8), and the lateral portion of the ventral tegmental area (group A10). These fibers accumulated dorsomedially to the rostral pole of the substantia nigra where they formed a massive bundle that coursed through the prerubral field and ascended along the laterodorsal aspect of the medial fore-brain bundle in the lateral hypothalamus. Some ventrally located fibers ran throughout the rostrocaudal extent of the lateral preopticohypothalamic area and could be followed up to the olfactory tubercle, whereas other fibers turned laterodorsally to invade the head of the caudate nucleus. At more dorsal levels in the lateral hypothalamus, many fiber fascicles detached themselves from the main bundle and swept laterally to reach the globus pallidus, the putamen, and the amygdala. Several TH-positive fibers coursed along the dorsal surface of the subthalamic nucleus, and some invaded the dorsomedial third of this structure. The remaining portion of the subthalamic nucleus contained relatively few TH-positive elements. In contrast, the globus pallidus received a dense dopaminergic innervation deriving mostly from two fascicles that coursed backward along the two major output pathways of the pallidum: the lenticular fasciculus caudodorsally and the ansa lenticularis rostroventrally. At the pallidal level, the labeled fibres merged within the medullary laminae and arborized profusely in the internal pallidal segment and less abundantly in the external pallidal segment. However, the caudoventral portion of the external pallidum displayed a dense field of TH-positive axonal varicosities. Other fibers ran through the dorsal two-thirds of the external pallidum en route to the putamen. The striatum contained a multitude of thin axonal varicosities among which a few long and varicosed fibers were scattered. These immunoreactive neuronal profiles were rather uniformly distributed along the rostrocaudal extent of the striatum but appeared slightly more numerous in the ventral striatum than in the dorsal striatum. The pattern of distribution of the TH-positive axonal varicosities in the dorsal striatum was markedly heterogeneous: it consisted of typical zones of poor TH immunoreactivity lying within a matrix of dense terminal labeling.(ABSTRACT TRUNCATED AT 400 WORDS)

Co-localization of fixative-modified glutamate and glutaminase in neurons of the spinal trigeminal nucleus of the rat: an immunohistochemical and immunoradiochemical analysis.

The spinal trigeminal nucleus (STN) is involved in processing orofacial sensory information, including tactile, thermal and nociceptive input, and relaying this information to higher brain centers, such as the thalamus. Very little information is available regarding the major excitatory neurotransmitters of this nucleus. The amino acid glutamate has been proposed as a major excitatory neurotransmitter in the central nervous system. In the present study, a novel monoclonal antibody, specific for fixative-modified glutamate, was utilized in conjunction with polyclonal antisera against glutaminase and aspartate aminotransferase (AATase) in an attempt to identify and map the locations of possible glutamatergic neurons in the STN. Co-localization experiments were performed by radiolabeling our monoclonal antibody and using this antibody in conjunction with the polyclonal antisera against glutaminase and AATase to evaluate the possible coexistence of glutamate with glutaminase or AATase in STN neurons. In all three subnuclei of the STN, immunohistochemically labeled neuronal profiles were observed with both of the polyclonal antisera and with the monoclonal antibody. Subnucleus caudalis contained the greatest number of labeled profiles per coronal section followed by subnucleus interpolaris and subnucleus oralis. The number and the distribution of immunoreactive profiles observed after the use of the glutaminase antiserum was comparable to that obtained with the monoclonal antibody. Co-localization experiments demonstrated that all glutaminase-like immunoreactive neurons also contained fixative-modified glutamate-like immunoradioactivity. These results suggest that glutamatergic neurons are present in the spinal trigeminal nucleus. The AATase antiserum labeled more neuronal profiles in each of the three subnuclei than did the glutaminase antiserum or the monoclonal antibody. In addition, co-localization experiments indicated that glutamate-like immunoreactivity was present in only two-thirds of AATase-like immunoreactive neuronal profiles. These findings suggest that glutaminase may be a more reliable marker of glutamatergic function than AATase.