An immunohistochemical study of the nerve growth factor receptor in developing rats

Nerve growth factor (NGF) receptor expression was studied in rats between embryonic day 11 (E11) to postnatal day 10 (PND10) by using a monoclonal antibody, 192-IgG, that specifically recognizes rat NGF receptor. Sympathetic ganglia were lightly stained by 192-IgG for NGF receptor immunoreactivity (NGFRI) (E11-PND10). Neural crest-derived sensory ganglia were moderately to densely stained (E11-PND10). Areas in CNS innervated by the central processes of these ganglia were also stained. Parasympathetic ciliary ganglion showed some detectable staining (E16-PND6). Placode-derived sensory ganglia were stained more densely than that of neural crest-derived sensory ganglia. The most densely stained tissue for NGFRI was found in all peripheral nerves. Basal forebrain cholinergic neurons were NGFRI positive from E15 throughout the period examined. Motoneurons in both spinal cord and brain stem were positive for NGFRI between E15 and PND10. NGFRI staining was seen in a variety of sensory pathways and related structures, such as olfactory tract and glomerular layer of olfactory bulb; retina, optic nerve and tract, lateral geniculate nucleus, medial terminal nucleus of the accessory optic tract, and olivary pretectal nucleus; ventral cochlear nucleus and to a lesser degree in dorsal cochlear nucleus, superior olive, and nucleus of lateral lemniscus; solitary tract; cuneate nucleus, gracile nucleus, and ventroposterior thalamic nucleus. The specific staining was also found in some other CNS structures, including brain-stem reticular formation; amygdala; medial nucleus of inferior olive but not the rest of inferior olive, external granule cell layer and Purkinje's cells of cerebellum, and deep cerebellar nuclei. Some non-neuronal tissues such as meninges and dental tissue showed very distinctive staining. Limb buds and somites were NGFRI positive starting at E11, and the staining on muscle tissue became very dense at E15-E18 and largely disappeared around PND10. Embryonic thymus was positive for NGFRI. The adventitia surrounding blood vessels was very densely stained. The changes in NGFRI staining seen in this study suggest that NGF may have broader effects during development than previously thought.     

Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) has been used to map the distribution of NGF receptor-containing profiles within the human basal forebrain of four male and three female elderly patients without neurologic or psychiatric illness. Immunohistochemically processed tissue reveals a continuum of NGF receptor-positive neurons located within the medial septum, vertical and horizontal limb nuclei of the diagonal band, and nucleus basalis. NGF receptor-containing neurons are also found within the bed nucleus of the stria terminalis, the anterior commissure, the internal capsule, and the internal and external medullary laminae of the globus pallidus. Virtually all (greater than 95%) NGF receptor-containing neurons colocalize with the specific cholinergic marker choline acetyltransferase (ChAT) or the nonspecific marker acetylcholinesterase (AChE). Conversely, a few cholinergic perikarya are found which are not NGF receptor positive (and vice versa). These findings demonstrate that human basal forebrain neurons on which NGF receptor immunoreactivity is detected are primarily cholinergic and analogous to the nonhuman primate Ch1-Ch4 subgroups of Mesulam et al. (J. Comp. Neurol., 214:170-197, '83). NGF receptor-containing fiber tracts are observed emanating from the medial septum and vertical limb nucleus of the diagonal band coursing medially within the fornix. Another fascicle originating mainly from the nucleus basalis and travelling within the external capsule enroute to the cortex is observed innervating all cortical layers. Comparison of NGF receptor- and ChAT-containing neurons reveals cholinergic perikarya within the striatal complex, whereas virtually no NGF receptor-containing neurons are found in these structures. An occasional displaced NGF receptor-containing neurons is seen in the ventrolateral portion of the putamen and the white matter underlying the nucleus accumbens. These data are discussed in terms of the relationship of NGF receptor- and ChAT-containing neurons within the basal forebrain and in terms of the possible functional significance of NGF in normal and diseased brain.     

Nerve growth factor-like immunoreactive profiles in the primate basal forebrain and hippocampal formation

The distribution of nerve growth factor (NGF), the prototypic neurotrophin, within the basal forebrain and hippocampal formation of young adult monkeys and aged humans was characterized with and affinity purified polyclonal β-NGF antibody raised against mouse β-NGF. In the basal forebrain of both primates, a granular NGF-like immunoreactive (ir) reaction product was observed within neurons of the medial septum, nucleus of the diagonal band, and nucleus basalis of Meynert. NGF-like immunoreactivity exclusively colocalized within p75 NGF receptor (NGFR) containing basal forebrain neurons. The intensity of NGF immunolabeling varied between cell bodies. Many NGF-ir perikarya were highly immunoreactive. In other basal forebrain neurons, NGF-like immunoreactivity was either undetectable or minimally expressed. In the hippocampus of both species, NGF-like immunoreactivity was mainly localized within the hilus of the dentate gyrus and within CA3 and CA2 hippocampal subfields. A marked diminution in NGF-like staining was seen in CA1. Within the hippocampal formation, NGF-like immunoreactivity was heaviest within the neuropil of stratum radiatum, intermediate in stratum oriens, and lightest in stratum pyramidal. NGF-like immunoreactivity was not found within the granule or pyramidal cells of the dentate gyrus and hippocampal formation, respectively. These findings demonstratre the presence of an NGF-like antigen in association with monkey and human magnocellular basal forebrain neurons and within their hippocampal target sites. This lends support to the hypothesis that NGF is internalized from sources located within target regions of the primate cholinergic basal forebrain neurons and is retrogradely transported to these cell bodies where the NGF trophic effect likely occurs.     

Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry

The expression of nerve growth factor (NGF) receptor mRNA and NGF receptor protein was examined in the adult rat basal forebrain using in situ hybridization and immunohistochemical techniques. NGF receptor mRNA and protein were detected within cells in the medial septum, diagonal band of Broca, and nucleus basalis of Meynert. Controls showed that the hybridization signal was not due to nonspecific binding of the probe to heterologous RNAs or other molecules. As expected, the distribution of NGF receptor mRNA-containing cells correlated nicely with the distribution of NGF receptor immunoreactive cells in each of these areas. These data extend previous work which suggests that neurons in these areas express the NGF receptor mRNA and manufacture functional NGF receptors. NGF receptor immunoreactivity was also detected in the arcuate nucleus of the hypothalamus, in the leptomeninges at the base of the brain and overlying the tectum, and within ependymal regions along the lateral walls of the cerebral ventricles. A few weakly stained neurons in the lateral hypothalamus and ventrolateral striatum were also consistently observed. In contrast, NGF receptor mRNA was not detected within any meningial, ependymal, or hypothalamic tissues using in situ hybridization. A cross-linking/immunoprecipitation assay demonstrated normal, membrane-bound NGF receptors within extracts of dorsal superior colliculus, ventromedial hypothalamic, and overlying meningial tissues, proving that the staining observed in these areas was not a non-specific artifact associated with the immunohistochemistry. The lack of hybridization in these areas may reflect levels of NGF receptor mRNA which are too low to be detected by the in situ hybridization methods being used. Alternatively, the staining may represent innervation of these areas by afferents whose cell bodies are located elsewhere, and whose terminals contain the NGF receptor protein.     

Nerve growth factor induces Fos-like immunoreactivity within identified cholinergic neurons in the adult rat basal forebrain

Immunocytochemical techniques were used to examine and compare the effects of intracerebroventricular administration of nerve growth factor (NGF) on Fos expression within identified cholinergic and non-cholinergic neurons located in different regions of the adult rat basal forebrain. Animals were killed 1, 3, 6, and 12 h after receiving NGF (0.5 or 5.0 μg) or vehicle into the left lateral ventricle and sections through the medial septum, diagonal band of Broca, nucleus basalis magnocellularis, and striatum were processed for the combined immunocytochemical detection of Fos and choline acetyltransferase (a marker for cholinergic neurons), or Fos and parvalbumin (a marker for gamma aminobutyric acid (GABA)-containing neurons). NGF produced a significant increase in the percentage of cholinergic neurons containing Fos-like immunoreactivity within all four regions examined. The largest increases were detected in the medial septum (47.8%) and the horizontal limb of the diagonal band of Broca (67.7%). In these areas, NGF-mediated induction of Fos-like immunoreactivity was detected as early as 3 h, peaked at 6 h, and was reduced by 12 h, postinfusion. Small but significant increases in the percentage of cholinergic neurons containing Fos-like immunoreactivity were also detected in the striatum (4.2%) and in the nucleus basalis magnocellularis (19.2%) 3–12 h following administration of the higher dose of NGF. No evidence for an NGF-mediated induction of Fos within parvalbumin-containing neurons was detected in any of the four regions at any of the time-points examined; however, evidence for an NGF-mediated induction of Fos within epithelial cells lining the lateral ventricle was observed. These data demonstrate that NGF induces Fos expression within cholinergic, and not parvalbumin-containing (GABAergic), neurons in the basal forebrain, and furthermore that intracerebroventricular administration of NGF influences the different subgroups of basal forebrain cholinergic neurons to different degrees. ©1977 Elsevier Science B.V. All rights reserved.     

Distribution of galaninergic immunoreactivity in the brain of the mouse

The distribution of galaninergic immunoreactive (-ir) profiles was studied in the brain of colchicine-pretreated and non-pretreated mice. Galanin (GAL)-ir neurons and fibers were observed throughout all encephalic vesicles. Telencephalic GAL-ir neurons were found in the olfactory bulb, cerebral cortex, lateral and medial septum, diagonal band of Broca, nucleus basalis of Meynert, bed nucleus of stria terminalis, amygdala, and hippocampus. The thalamus displayed GAL-ir neurons within the anterodorsal, paraventricular, central lateral, paracentral, and central medial nuclei. GAL-ir neurons were found in several regions of the hypothalamus. In the midbrain, GAL-ir neurons appeared in the pretectal olivary nucleus, oculomotor nucleus, the medial and lateral lemniscus, periaqueductal gray, and the interpeduncular nucleus. The pons contained GAL-ir neurons within the dorsal subcoeruleus, locus coeruleus, and dorsal raphe. In the medulla oblongata, GAL-ir neurons appear in the anterodorsal and dorsal cochlear nuclei, salivatory nucleus, A5 noradrenergic cells, gigantocellular nucleus, inferior olive, solitary tract nucleus, dorsal vagal motor and hypoglossal nuclei. Only GAL-ir fibers were seen in the lateral habenula nucleus, substantia nigra, parabrachial complex, cerebellum, spinal trigeminal tract, as well as the motor root of the trigeminal and facial nerves. GAL-ir was also observed in several circumventricular organs. The widespread distribution of galanin in the mouse brain suggests that this neuropeptide plays a role in the regulation of cognitive and homeostatic functions.     

Plasticity of Galaninergic Fibers Following Neurotoxic Damage within the Rat Basal Forebrain: Initial Observations

Galanin immunoreactive fibers hypertrophy and hyperinnervate remaining cholinergic basal forebrain neurons within the septum–diagonal band complex in Alzheimer's disease. The present investigation determined whether a similar hyperinnervation of galanin immunoreactive fibers occurs following intraparenchymal injections of ibotenic acid within the cholinergic medial septum or diagonal band nucleus in young adult rats. Sections through the medial septum and the diagonal band were either concurrently immunostained for galanin and the low-affinity p75 neurotrophin receptor (an excellent marker of cholinergic basal forebrain neurons) or single stained for choline acetyltransferase. Following chemical lesion, an increase in the density of galanin immunoreactivity was seen within the medial septum on the lesion, as opposed to the contralateral control side. In contrast, within diagonal band-lesioned animals, the increase in galanin immunoreactivity was low to moderate. In either lesion paradigm we did not observe hyperinnervation of remaining cholinergic basal forebrain neurons. In fact, there was no correlation between the galanin hypertrophy and the amount of cholinergic cell loss. We hypothesize that galanin hyperinnervation within the cholinergic basal forebrain may provide a protective effect by down-regulating acetylcholine release following brain insult.     

Distribution of orexin neurons in the adult rat brain

Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.     

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

NGF receptor immunoreactivity in aged rat brain

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.     

Recovery of ChAT lmmunoreactivity in Axotomized Rat Cholinergic Septal Neurons Despite Reduced NGF Receptor Expression

Previous studies have suggested that target-derived nerve growth factor (NGF) is essential for the survival of cholinergic basal forebrain neurons. Thus, axotomy of septohippocampal neurons in adult rats resulting in the withdrawal of target-derived NGF caused a dramatic loss of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum-diagonal band complex. We have recently shown that this loss of immunolabelled neurons does not indicate cell death, since many septohippocampal cholinergic neurons recover their immunoreactivity for ChAT after a long survival time despite disconnection from target-derived neurotrophins. One possibility would be that these surviving ChAT-immunoreactive neurons have gained access to other, probably local, NGF sources. Here we provide evidence that the recovery of ChAT immunoreactivity after axotomy is not accompanied by a similar recovery of NGF receptor expression in these neurons. In situ hybridization for p75^NTR mRNA and trkA mRNA 6 months after bilateral fimbria-fornix transection revealed a substantial loss of labelled cells. In addition, there was a persisting loss of p75^NTR-immunoreactive and NGF-immunoreactive medial septal neurons. Cholinergic neurons in controls did not express NGF mRNA, but were heavily immunostained for NGF protein due to receptor-mediated uptake. These data suggest that at least some cholinergic septohippocampal neurons re-express ChAT either independently of NGF or with a reduced need for NGF.     

The Distribution of Neurons Coexpressing Immunoreactivity to AMPA-sensitive Glutamate Receptor Subtypes (GluR1-4) and Nerve Growth Factor Receptor in the Rat Basal Forebrain

The regional distribution of neurons containing a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR1-4) subunit immunoreactivity, relative to the distribution of cholinergic neurons within the basal forebrain of rats, was assessed using single- and dual-antigen immunocytochemistry. Analysis of serial sections stained with antibodies to nerve growth factor receptor (NGFr) and antibodies against each of the AMPA receptor subunits, GluR1-4, revealed a regional codistribution between NGFr- and GluR1- and GluR4-immunoreactive neurons in the medial septum, diagonal band nuclei and nucleus basalis magnocellularis. Quantitative dual-labelling immunocytochemistry using NGFr in combination with each of the GluR antibodies revealed >65% colocalization between NGFr and GluR4 in each of the major cholinergic nuclei in the basal forebrain and 10–15% colocalization between NGFr, GluR1 and GluR2-3. The reticular nucleus of the thalamus, a structure known to be highly susceptible to AMPA-induced neurotoxicity, expressed GluR4 immunoreactivity exclusively. The observation that cholinergic neurons of the basal forebrain are also highly sensitive to AMPA and express the GluR4 subunit suggests that GluR4 may be important in AMPA receptor-mediated excitotoxicity.     

Medial-to-lateral gradient of neostriatal NGF receptors: relationship to cholinergic neurons and NGF-like immunoreactivity.

High-affinity binding sites for recombinant human NGF (rhNGF) were studied in the caudate-putamen of the adult rat and rabbit. Displaceable 125I-rhNGF binding sites were densely distributed throughout the caudate-putamen and were 2-3-fold more prevalant in the ventrolateral and lateral than in the medial caudate-putamen. The amount of nondisplaceable binding did not vary throughout the caudate-putamen. The medial-to-lateral receptor gradient was correlated (r = +0.99) with a 2-3-fold medial-to-lateral increase in ChAT activity. In contrast, NGF-like immunoreactivity (NGF-LI) was prevalent but uniformly distributed in the caudate-putamen. Lesions of intrinsic cholinergic neurons by quinolinic acid produced extensive gliosis in the medial, central, and lateral caudate-putamen, yet 125I-rhNGF binding was decreased in each of these regions. The activity of ChAT and 125I-rhNGF binding throughout the caudate-putamen were each decreased by 40% following quinolinic acid. Binding was not changed after 70-77% dopamine nerve terminal depletions induced by 6-hydroxydopamine, demonstrating a nonglial, nondopaminergic locus for striatal NGF binding sites. The cholinergiclike topography of NGF binding sites throughout the intact caudate-putamen, the parallel decreases of cholinergic neurons and NGF binding sites following intrinsic neuronal loss, and the uniform neostriatal gradient of NGF-LI are consistent with the trophic role of endogenous NGF for cholinergic interneurons of the caudate-putamen.     

Penetration of fluorescein into the brain: a sex difference

Fluorescein was found to penetrate into the brain via the circumventricular organs. Fluorescein penetrates beyond the borders of the circumventricular organs into the surrounding neuropile. This relationship was found for the area postrema and the nucleus tractus solitarius, the organum vasculosum lamina terminalis and the rostral suprachiasmatic area, the median eminence and the arcuate nucleus. The choroid plexus appears to take up fluorescin, but fluoroscence does not appear in adjacent structures such as the corpus striatum and septum, but rather along the ependymal and plial surfaces. Fluorescein was found to accumulate to a greater extent in the brains of female as compared to male rats. This sex difference is not associated with the blood-brain barrier as cortex and cerebellum did not show increased fluorescence, but only midline structures containing circumventricular organs. Gonadectomy did not alter fluoroscein accumulation.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.     

Dynamic regulation of BDNF and NT-3 expression during visual system development

Bradykinin has long been known to exist in the central nervous system and has been hypothesized to mediate specific functions. Despite an increasing understanding of the functions of bradykinin, little is known about the cell types expressing the bradykinin receptor within the brain. The present investigation employed a monoclonal antibody directed against the 15-amino-acid portion of the C-terminal of the human bradykinin B2 receptor to establish the cellular distribution of bradykinin B2 receptor immunoreactivity in the rat brain. Bradykinin B2 receptor immunoreactivity was ubiquitously and selectively observed in neurons, including those within the olfactory bulb, cerebral cortex, hippocampus, basal forebrain, basal ganglia, thalamus, hypothalamus, cerebellum, and brainstem nuclei. Bradykinin B2 receptor immunoreactivity was also present in the circumventricular organs including choroid plexus, subfornical organ, median eminence, and area postrema. Double-labeling experiments colocalizing the bradykinin B2 receptor with the neuronal marker NeuN or the astrocytic marker glial fibrillary acidic protein revealed that virtually 100% of the bradykinin B2 receptor-immunoreactive positive cells were neurons. The widespread distribution of bradykinin B2 receptor immunoreactivity in neuronal compartments suggests a greater than previously appreciated role for this peptide in neuronal function.     

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of intraneuronal transferrin receptor immunoreactivity in the adult mouse central nervous system

Iron is essential for a variety of intracellular functions. Accordingly, the transfer of iron from blood to brain is vital for normal brain function. In the CNS, the receptor for iron-transferrin is generally accepted to be located in endothelial cells, whereas its occurrence in other cell types is less well established. I have investigated the distribution of the transferrin receptor in the adult mouse central nervous system by immunohistochemistry by using a monoclonal antibody raised against the transferrin receptor protein. Immunoreactive cell types comprised brain capillary endothelial cells, excluding those of circumventricular organs, and choroid plexus epithelial cells. Moreover, transferrin receptor immunoreactivity was detected intraneuronally in several brain regions without access to peripheral blood. The immunoreactive cell bodies were mainly confined to the cerebral cortex, hippocampus, habenular nucleus, red nucleus, substantia nigra, pontine nuclei, reticular formation, several cranial nerve nuclei, deep cerebellar nuclei, and cerebellar cortex. Transferrin receptor immunoreactivity was not detected in astrocytes, oligodendrocytes, or microglial cells. The occurrence of transferrin receptors at brain-barrier sites, i.e., the brain endothelium and choroid plexus epithelium, and the presence of the receptors intraneuronally are in accordance with the generally held belief that iron is released from liver transferrin and transported through capillaries and the choroid plexus into the brain interstitium. Subsequently, iron may be linked to brain transferrin synthesized within oligodendrocytes and choroid plexus epithelial cells followed by a concomitant uptake of iron-transferrin in neurons expressing transferrin receptors. The clinical importance of the intraneuronal transferrin receptor expression is discussed. © 1996 Wiley-Liss, Inc.     

Dose-dependent responses to nerve growth factor by adult rat cholinergic medial septum and neostriatum neurons.

This study describes the relationship between the concentration of intraventricularly infused nerve growth factor (NGF) and several responses by axotomized cholinergic medial septum neurons and normal cholinergic neostriatal neurons of the adult rat. NGF infused for 14 days starting either immediately after a unilateral fimbria-fornix transection or after a 2-week delay period elicited similar dose-response relationships for the maintenance or restoration of ChAT and NGF receptor positivity and cell body size and for intraseptal 'sprouting' of the axotomized medial septum neurons. Thus, in the medial septum it appears that the expression of 'marker' molecules, cell body size and the induction of 'sprouting' are regulated by virtually the same concentrations of NGF in the two treatment strategies. This suggests that NGF has a general regulatory role and injured but untreated neurons remain fully susceptible to NGF at least up to 2 weeks after the lesion. A 14-day infusion with NGF also induced an above-normal cell body size (hypertrophy) both in axotomized medial septum and in intact striatal cholinergic neurons. The hypertrophic response of normal striatal neurons required less NGF than did that of medial septum neurons. Since the striatal response began to be detectable at a similar concentration as that required for the full maintenance or restoration of ChAT and NGF receptor positivity it could be seen as an unwanted side-effect. The definition of a sub-optimal dose with which a significant, but not maximal response can be elicited will allow future evaluations of potentially additive or synergistic actions by other agents.