Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain

The expression of nerve growth factor (NGF) receptor in adult rat brain was studied by immunohistochemistry with a specific anti-rat NGF receptor monoclonal antibody, 192-IgG. Intense NGF receptor immunoreactivity (NGFRI) was found in structures known to be NGF responsive, including forebrain cholinergic neurons in medial septum, diagonal band of Broca, and basal nucleus of Meynert; central processes of neural-crest-derived sensory ganglion neurons and their innervated nucleus also contained such immunoreactivity. Distinct NGFRI staining was also found in many brain areas and cell types not known to be NGF responsive, including some hypothalamic regions, circumventricular organs, some areas related to the optic system, olfactory glomeruli, ependymal and subependymal cells in some locations, mesencephalic nucleus of the trigeminal nerve, cerebellar molecular layer, central linear nucleus, solitary tract and its nucleus, and inferior olive. The NGFRI in the circumventricular organs was further studied by in vivo labeling of 125I-ligands. Intravenously injected 125I-NGF, but not 125I-cytochrome c, was specifically accumulated in the area postrema. Biochemical study of the NGF receptor showed a major band of molecular weight of approximately 90 KDa in the area postrema, choroid plexus, median eminence, and medial septum with the relative content consistent with that seen by immunohistochemistry. No evidence of a truncated NGF receptor was observed. The results of this study suggest that NGF and its receptor have broader roles in adult mammalian brain than previously thought.     

Differential regulation of the low-affinity nerve growth factor receptor during postnatal development of the rat brain.

We studied the temporal and spatial localization of the low-affinity nerve growth factor receptor (LNGF-R) during the early postnatal period in rat brain in order to understand better the relationship between nerve growth factor (NGF)-like responsiveness and the development of specific central neuronal populations. Four different developmental patterns of LNGF-R mRNA hybridization were found in this study. First, some neurons contain high levels of LNGF-R mRNA from postnatal time points into adulthood, as exemplified by neurons of the cholinergic basal forebrain and mesencephalic trigeminal nucleus. Second, several cell groups exhibit robust hybridization during the early postnatal period but contain much reduced levels of LNGF-R mRNA in the adult brain. These include striatal neurons, Purkinje cells of the cerebellum, and several medullary nuclei. A third group of cells produces the LNGF-R transiently during development, including cranial nerve nuclei of the brainstem, the periolivary nuclei complex, the reticular formation, and the deep cerebellar nuclei. Finally, cell populations which may exist only transiently during central nervous system (CNS) development, such as subplate neurons of the cerebral cortex, appear to express the LNGF-R during only a brief period. These results show that the LNGF-R gene is differentially regulated in a cell type-specific manner during development, and suggests that diverse neuronal populations require only transient growth factor sensitivity, while others exhibit NGF-like responsitivity into maturity.     

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.     

A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat.

Ascending projections from the dorsal raphe nucleus (DR) were examined in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). The majority of labeled fibers from the DR ascended through the forebrain within the medial forebrain bundle. DR fibers were found to terminate heavily in several subcortical as well as cortical sites. The following subcortical nuclei receive dense projections from the DR: ventral regions of the midbrain central gray including the 'supraoculomotor central gray' region, the ventral tegmental area, the substantia nigra-pars compacta, midline and intralaminar nuclei of the thalamus including the posterior paraventricular, the parafascicular, reuniens, rhomboid, intermediodorsal/mediodorsal, and central medial thalamic nuclei, the central, lateral and basolateral nuclei of the amygdala, posteromedial regions of the striatum, the bed nucleus of the stria terminalis, the lateral septal nucleus, the lateral preoptic area, the substantia innominata, the magnocellular preoptic nucleus, the endopiriform nucleus, and the ventral pallidum. The following subcortical nuclei receive moderately dense projections from the DR: the median raphe nucleus, the midbrain reticular formation, the cuneiform/pedunculopontine tegmental area, the retrorubral nucleus, the supramammillary nucleus, the lateral hypothalamus, the paracentral and central lateral intralaminar nuclei of the thalamus, the globus pallidus, the medial preoptic area, the vertical and horizontal limbs of the diagonal band nuclei, the claustrum, the nucleus accumbens, and the olfactory tubercle. The piriform, insular and frontal cortices receive dense projections from the DR; the occipital, entorhinal, perirhinal, frontal orbital, anterior cingulate, and infralimbic cortices, as well as the hippocampal formation, receive moderately dense projections from the DR. Some notable differences were observed in projections from the caudal DR and the rostral DR. For example, the hippocampal formation receives moderately dense projections from the caudal DR and essentially none from the rostral DR. On the other hand, virtually all neocortical regions receive significantly denser projections from the rostral than from the caudal DR. The present results demonstrate that dorsal raphe fibers project significantly throughout widespread regions of the midbrain and forebrain.     

Topographical projection of cholinergic neurons in the basal forebrain to the cingulate cortex in the rat

The cholinergic innervation of the rat's posterior cingulate cortex (Brodmann's area 29) was studied using acetylcholinesterase (AChE) histochemistry. Electrolytic lesion of the ipsilateral medial septum and diagonal band region (MS-DB) reduced the diffuse AChE staining in layers I, II, III and V of the cingulate cortex. Kainic acid lesion of the ipsilateral globus pallidus and substantia innominata area (GP-SI) abolished the dense band of AChE stain in layer IV, with small reductions of AChE stain in other layers. The results indicate that the medial cholinergic pathway from MS-DB terminates diffusely in layers I, II, III and V while the lateral cholinergic pathway from the GP-SI predominantly ends in layer IV of the posterior cingulate cortex.     

Connections of the parahippocampal cortex in the cat. II. Subcortical afferents

The organization of subcortical inputs to the parahippocampal cortex, which in the present study in the cat is considered to comprise the entorhinal and perirhinal cortices, was studied by using retrograde and anterograde tracing techniques. The results of the retrograde tracer horseradish peroxidase (HRP), HRP conjugated with wheat germ agglutinine (WGA-HRP), Fast Blue (FB) or Nuclear Yellow (NY] injections indicate that the entorhinal and perirhinal cortices receive inputs from the magnocellular basal forebrain and from distinct portions of the amygdaloid complex, the claustrum, and the thalamus. The two cortices are further projected upon by fibers from the supramamillary region of the hypothalamus, the ventral tegmental area of the mesencephalon, the dorsal raphe nucleus, the nucleus centralis superior, and the locus coeruleus. The entorhinal cortex, in addition, receives projections from the medial septum. As regards the projections from the amygdaloid complex, it was observed that the entorhinal cortex receives its heaviest input from the basolateral amygdaloid nucleus, whereas the perirhinal cortex receives a strong projection from the lateral nucleus and a weaker projection from the basomedial nucleus of the amygdala. Of the thalamic nuclei that project to the parahippocampal cortex, the nucleus reuniens is only connected with the entorhinal cortex, while fibers from the medial geniculate nucleus and the lateral posterior nucleus terminate in the perirhinal cortex. Injections of tritiated amino acid (3H-leucine) were placed in the medial septum, the dorsal and ventral claustrum, the basolateral and basomedial amygdaloid nuclei, and the nucleus reuniens of the thalamus. The results of these experiments demonstrate that, with the exception of the claustrum, these subcortical areas project mainly to the superficial layers I-III and the lamina dissecans of the parahippocampal cortex, and to a lesser degree to the deep layers V and VI.     

A parvalbumin-containing, axosomatic synaptic network in the rat medial septum: relevance to rhythmogenesis

The medial septal diagonal band complex (MS/DB), made up of cholinergic and GABAergic neurons, plays an important role in the generation of the hippocampal theta rhythm. A GABAergic neuron type in the MS/DB that has fast spiking properties was shown previously to contain parvalbumin immunoreactivity and to form axosomatic connections with unidentified somata. The aim in the current study was to determine the neurochemical identities of these target neurons. In slices and in perfused-fixed brain, staining for parvalbumin immunoreactivity first of all revealed the presence of two types of parvalbumin-positive somata in the MS/DB: medially located neurons with parvalbumin-positive basket-like terminals on them, and more laterally located neurons with fewer parvalbumin-positive contacts on them. In MS/DB slices, the terminals of fast spiking neurons filled with biocytin correspondingly made either numerous contacts that surrounded the parvalbumin-positive cell body in basket-like formation, or 1-5 contacts on a localized patch of the soma. These contacts were shown by electron microscopy to form synaptic junctions. No terminals of biocytin-filled fast spiking neurons were observed on cholinergic neurons, and dual staining in perfused-fixed brain did not reveal the presence of parvalbumin-containing terminals on cholinergic somata. Our results suggest therefore that there are two subtypes of parvalbumin-containing neuron in the MS/DB, and that these are interconnected via axosomatic synapses. The contrasting topographical organization of the two types of parvalbumin-containing neuron suggests that they may receive different types of afferent input, but this will require substantiation in future studies. We propose that generation of rhythmic activity in the MS/DB is controlled by contrasting contributions from two types of parvalbumin-positive neuron, and that the role of the cholinergic neuron is modulatory.     

The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat

Following injections of [3H]leucine into the formatio reticularis gigantocellularis (Rgc), reticularis pontis caudalis (Rpc), reticularis pontis oralis (Rpo), reticularis mesencephali (Rmes), or the locus coeruleus (LC) of the rat, autoradiographic study revealed prominent reticuloreticular projections from all areas and secondary projections onto cranial nerve motor nuclei from most areas within the brain stem. Common long descending projections extended the full length of the spinal cord terminating in the ventromedial ventral horn and intermediate zone and more sparsely in the base of the dorsal horn and (particularly from Rgc) the region of the motoneurons. Common long ascending projections extended into the forebrain via Forel's tegmental fascicles. A dorsal branch of fibers innervated the intralaminar and midline nuclei of the thalamus. The major fiber system continued forward through Forel's fields and ascended into the pallidum from Rpo, Rmes, and LC and into the neostriatum from Rmes and LC. Fascicles from all areas also ascended in the medial forebrain bundle through the lateral hypothalamus to the lateral preoptic area, substantia innominata, and nuclei of the diagonal band. From Rpo, Rmes, and LC, fibers continued forward to reach the cerebral cortex, where the innervation was sparse and discrete from Rpo and Rmes but moderate and ubiquitous from LC. Retrograde transport of true blue and/or nuclear yellow revealed inverse gradients along the brain stem longitudinal axis of interdigitated cells respectively projecting caudally into the spinal cord (with the greatest number of cells in Rgc, Rpc, and Rpo) and rostrally into the diencephalon (with the greatest number of cells in Rmes and LC), with very few cells projecting both to the spinal cord and the diencephalon. From the basal forebrain, a large number of reticular and LC cells were retrogradely labelled, whereas from the frontal cortex, a much smaller number of reticular cells was labelled. These results document the widespread efferent projections from the reticular formation and overlapping, yet more extensive, projections from the LC.     

Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and ³⁵S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain. © 1994 Wiley-Liss, Inc.     

Fos induction by nerve growth factor in the adult rat brain

The distribution of Fos, the protein product of the immediate early gene c-fos, was studied with immunocytochemistry in the adult male rat brain after nerve growth factor (NGF) administration. NGF was injected in the lateral cerebral ventricle through a previously implanted cannula. The total number of Fos-immunoreactive (ir) neurons in the brain was 2–3 times higher after NGF administration than in control animals (untreated or injected with cytochrome c). With respect to control rats, in the NGF-treated cases Fos-ir cells were more numerous in the anterior olfactory nucleus, in the medial prefrontal and anterior cingulate cortices, in the basal forebrain, in the preoptic and ventromedial nuclei of the hypothalamus, as well as interior hypothalamic area, in the thalamic midline nuclei, and in some brainstem structures, such as the parabrachial nucleus. The relative quantitative increase of Fos-ir neurons varied in the different structures. In addition, Fos-ir neurons were evident after NGF administration in areas devoid of immunopositive cells in control animals. These included: frontoparietal and occipital cortical fields, the hypothalamic arcuate nucleus, and many brainstem structures, such as the dorsal nucleus of the lateral lemniscus, posterodorsal tegmental, medial and lateral vestibular, ventral cochlear, and prepositus hypoglossal nuclei. These findings demonstrate that the intracerebroventricular administration of NGF can induce c-fos expression in neurons in vivo. The distribution of Fos-ir neurons indicates that NGF can induce activation of functionally and chemically hetergeneous neuronal subsets in the brain.     

Novel Disabled‐1‐expressing neurons identified in adult brain and spinal cord

Components of the Reelin‐signaling pathway are highly expressed in embryos and regulate neuronal positioning, whereas these molecules are expressed at low levels in adults and modulate synaptic plasticity. Reelin binds to Apolipoprotein E receptor 2 and Very‐low‐density lipoprotein receptors, triggers the phosphorylation of Disabled‐1 (Dab1), and initiates downstream signaling. The expression of Dab1 marks neurons that potentially respond to Reelin, yet phosphorylated Dab1 is difficult to detect due to its rapid ubiquitination and degradation. Here we used adult mice with a lacZ gene inserted into the dab1 locus to first verify the coexpression of β‐galactosidase (β‐gal) in established Dab1‐immunoreactive neurons and then identify novel Dab1‐expressing neurons. Both cerebellar Purkinje cells and spinal sympathetic preganglionic neurons have coincident Dab1 protein and β‐gal expression in dab1^lacZ/+ mice. Adult pyramidal neurons in cortical layers II–III and V are labeled with Dab1 and/or β‐gal and are inverted in the dab1^lacZ/lacZ neocortex, but not in the somatosensory barrel fields. Novel Dab1 expression was identified in GABAergic medial septum/diagonal band projection neurons, cerebellar Golgi interneurons, and small neurons in the deep cerebellar nuclei. Adult somatic motor neurons also express Dab1 and show ventromedial positioning errors in dab1‐null mice. These findings suggest that: (i) Reelin regulates the somatosensory barrel cortex differently than other neocortical areas, (ii) most Dab1 medial septum/diagonal band neurons are probably GABAergic projection neurons, and (iii) positioning errors in adult mutant Dab1‐labeled neurons vary from subtle to extensive.     

Reelin-immunoreactive neurons,axons, and neuropil in the adult ferret brain: evidence for axonal secretion of reelin in long axonal pathways

Reelin is a large secretable protein which, when developmentally defective, causes the reeler brain malformation in mice and a recessive form of lissencephaly with cerebellar hypoplasia in humans. In addition, Reelin is heavily expressed throughout the adult brain, although its function/s there are still poorly understood. To gain insight into which adult neuronal circuits may be under the influence of Reelin, we systematically mapped Reelin-immunoreactive neuronal somata, axons, and neuropil in the brain and brainstem of ferrets. Results show that Reelin immunoreactivity is found in widespread but specific sets of neuronal bodies, axonal tracts, and gray matter neuropil regions. Depending on the region, the immunoreactive neuronal somata correspond to interneurons, projection neurons, or both. Some well-defined axonal projection systems are immunoreactive, whereas most other white matter tracts are unlabeled. The labeled pathways include, among others, the lateral olfactory tract, the entorhinohippocampal (perforant) pathway, the retroflex bundle, and the stria terminalis. Labeled axons in these tracts contain large numbers of discrete, very small, immunoreactive particles, suggestive of secretory vesicles under the light microscope. The neuropil in the terminal arborization fields of these axons is also heavily immunoreactive. Taken together, our observations are consistent with the notion that some neurons may anterogradely transport Reelin along their axons in large membrane-bound secretory vesicles (Derer et al. [2001] J. Comp. Neurol. 440:136-143) and secrete it into their terminal arborization fields, which may be quite distant from the somata synthesizing the protein. These findings have implications for identifying where Reelin acts in adult brain circuits.     

A population of nicotinic receptors is associated with thalamocortical afferents in the adult rat: Laminal and areal analysis

In the adult rat brain, a prominent population of nicotinic cholinoceptors binds ³H-nicotine with nanomolar affinity. These receptors are abundant in most thalamic nuclei and in neocortical layers 3/4, which receive a major thalamic input. To test whether cortical nicotinic receptors are associated with thalamocortical afferents, unilateral excitotoxic (N-methyl-D-aspartate) lesions were made in one of four thalamic nuclear groups (anterior, ventral, medial geniculate, or dorsal lateral geniculate) or in temporal cortex. After 1 or 4 weeks of survival, cortical ³H-nicotine binding was quantified via autoradiography. Thalamic lesions resulted in a partial loss of ³H-nicotine binding in ipsilateral cerebral cortex. In each thalamic lesion group, the greatest decrease (35–45%) occurred within the cortical layers and area (i.e., cingulate, parietal, temporal, or occipital cortex) receiving the densest thalamocortical innervation. Binding of ³H-nicotine was also reduced within the thalamus local to the lesion, particularly at the longer survival time. Saturation analysis, performed in frontoparietal cortical tissue homogenates following ventral thalamic lesions, revealed a significant (34%) reduction in receptor density but not affinity. Direct excitotoxic lesions of the neocortex (temporal cortex) tended to preserve ³H-nicotine binding in layers 3/4, despite local neuronal loss. These results, taken with other published findings, suggest that some nicotinic cholinoceptors in adult rat cerebral cortex are located on thalamocortical terminals. This organizing principle appears to apply not only to sensory and motor relay projections but also to association nuclei that project to allocortical areas. These receptors may provide a local mechanism for nicotinic cholinergic modulation of thalamocortical input. J. Comp. Neurol. 380:175–190, 1997. © 1997 Wiley-Liss, Inc.     

Apoptosis in the rat basal forebrain during development and following lesions of connections

Evidence suggests that neurotrophins are essential for the survival and phenotypic maintenance of cholinergic basal forebrain (BF) neurons. We evaluated the pattern of programmed cell death in the BF of the rat during development and after ablations of the cerebral cortex, a major target area and source of neurotrophins for BF neurons. We identified dying cells using the TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling) method and confirmed their apoptotic morphology with electron microscopy. Moreover, we demonstrated the expression of the apoptotic marker active caspase-3 in cells with features of apoptosis. TUNEL(+) cells were present in the developing BF during the first two postnatal weeks. Their frequency peaked at postnatal day (P)1 and at P5. TUNEL used in conjunction with immunofluorescence for neuronal nuclear protein (NeuN) showed that, at both peak stages, the majority of apoptotic cells were neurons. Extensive lesions of the cerebral cortex at different ages (P0, P7 and P14) did not induce significant changes in the frequency of apoptotic BF neurons. However, they resulted in alterations in the morphological phenotype of choline acetyltransferase (ChAT)-immunoreactive neurons in the BF, and a reduction in their number which was inversely proportional to the age at which the lesions were performed. We suggest that: (i) apoptosis is temporally coordinated with the morphological and neurochemical differentiation of BF neurons and the establishment of connections with their target areas; and (ii) cortical ablations do not affect the survival of BF neurons, but they influence the phenotype of cholinergic BF neurons.     

Cholinergic regulation of fear learning and extinction

Cholinergic activation regulates cognitive function, particularly long‐term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and pharmacological evidence supporting the cholinergic regulation of Pavlovian contextual and cue‐conditioned fear learning and extinction. Basal forebrain cholinergic neurons provide inputs to neocortical regions and subcortical limbic structures such as the hippocampus and amygdala. Pharmacological manipulations of muscarinic and nicotinic receptors support the role of cholinergic processes in the amygdala, hippocampus, and prefrontal cortex in modulating the learning and extinction of contexts or cues associated with threat. Additional evidence from lesion studies and analysis of in vivo acetylcholine release with microdialysis similarly support a critical role of cholinergic neurotransmission in corticoamygdalar or corticohippocampal circuits during acquisition of fear extinction. Although a few studies have suggested a complex role of cholinergic neurotransmission in the cellular plasticity essential for extinction learning, more work is required to elucidate the exact cholinergic mechanisms and physiological role of muscarinic and nicotinic receptors in these fear circuits. Such studies are important for elucidating the role of cholinergic neurotransmission in disorders such as posttraumatic stress disorder that involve deficits in extinction learning as well as for developing novel therapeutic approaches for such disorders. © 2016 Wiley Periodicals, Inc.     

Nerve growth factor induces galanin gene expression in the rat basal forebrain: Implications for the treatment of cholinergic dysfunction

Nerve growth factor (NGF) is a potential treatment for cholinergic dysfunction associated with Alzheimer's disease (AD). In rats, NGF activates gene expression of the acetylcholine synthetic enzyme choline acetyltransferase (ChAT) and prevents age- and lesion-induced degeneration of basal forebrain (BF) cholinergic neurons. Cholinergic neurons in the BF coexpress galanin (GAL), a neuropeptide that has been shown to impair performance on memory tasks possibly through the inhibition of cholinergic memory pathways. NGF up-regulates both ChAT and GAL gene expression in cultured pheochromocytoma cells; however, the effect of chronic in vivo NGF administration on GAL gene expression within the BF has not been studied. We used in situ hybridization and quantitative autoradiography to assess GAL and ChAT gene expression within the BF of adult male rats following chronic intracerebroventricular infusion of NGF or cytochrome c. We now report that, in addition to stimulating ChAT gene expression, NGF strongly up-regulated the GAL gene in the rat cholinergic BF. NGF had no effect on GAL gene expression in other noncholinergic forebrain regions. NGF induction of GAL gene expression in the BF was specific, because gene expression for another neuropeptide, neurotensin, present within noncholinergic BF neurons was unchanged. Our data provide the first evidence that in vivo NGF administration up-regulates GAL gene expression in the cholinergic BF. These results suggest that the concurrent induction of GAL in the BF could limit the ameliorating actions of NGF on cholinergic dysfunction. J. Comp. Neurol. 379;563–570, 1997. © 1997 Wiley-Liss, Inc.     

Dopamine D5 receptor localization on cholinergic neurons of the rat forebrain and diencephalon: a potential neuroanatomical substrate involved in mediating dopaminergic influences on acetylcholine release

The study of dopaminergic influences on acetylcholine release is especially useful for the understanding of a wide range of brain functions and neurological disorders, including schizophrenia, Parkinson's disease, Alzheimer's disease, and drug addiction. These disorders are characterized by a neurochemical imbalance of a variety of neurotransmitter systems, including the dopamine and acetylcholine systems. Dopamine modulates acetylcholine levels in the brain by binding to dopamine receptors located directly on cholinergic cells. The dopamine D5 receptor, a D1-class receptor subtype, potentiates acetylcholine release and has been investigated as a possible substrate underlying a variety of brain functions and clinical disorders. This receptor subtype, therefore, may prove to be a putative target for pharmacotherapeutic strategies and cognitive-behavioral treatments aimed at treating a variety of neurological disorders. The present study investigated whether cholinergic cells in the dopamine targeted areas of the cerebral cortex, striatum, basal forebrain, and diencephalon express the dopamine D5 receptor. These receptors were localized on cholinergic neurons with dual labeling immunoperoxidase or immunofluorescence procedures using antibodies directed against choline acetyltransferase (ChAT) and the dopamine D5 receptor. Results from this study support previous findings indicating that striatal cholinergic interneurons express the dopamine D5 receptor. In addition, cholinergic neurons in other critical brain areas also show dopamine D5 receptor expression. Dopamine D5 receptors were localized on the somata, dendrites, and axons of cholinergic cells in each of the brain areas examined. These findings support the functional importance of the dopamine D5 receptor in the modulation of acetylcholine release throughout the brain.     

Lesion‐induced transneuronal plasticity of the cholinergic innervation in the adult rat entorhinal cortex

The present experiments were designed to determine the effect that lesions of the basal forebrain cholinergic system exert on cholinergic interneurons within the entorhinal cortex (EC) in the rat. Unilateral infusion of 192 IgG-saporin into the nucleus of the horizontal diagonal band of Broca (HDB) decreased the number of ipsilateral choline acetyltransferase immunoreactive (ChAT-ir) neurons by 54%. Two–four weeks after the lesion, the ipsilateral EC exhibited a moderate but significant loss of ChAT-ir fibres and interneurons. Adjacent sections revealed a parallel loss of vasoactive intestinal polypeptide (VIP) immunoreactivity. Cell counts in the cingulate cortex were unaffected, suggesting that this effect was indeed specific to the main target area for HDB neurons. Ibotenic acid lesions also induced a significant 36% decrease in the number of cholinergic neurons in the ipsilateral HDB, and disappearance of ChAT terminals in the EC, whereas the number of ChAT-ir neurons in the EC was unchanged. Since ibotenic acid affects all cells and not only cholinergic ones, our results suggest that the specific degeneration of cholinergic neurons in the HDB after 192 IgG-saporin treatment could be inducing transsynaptic effects on their targets. Injections of 192 IgG-saporin directly into the EC also lesioned the cholinergic projection from the HDB, but had no effect on the intrinsic population. Eight weeks after immunolesion, the number of interneurons immunoreactive for ChAT and VIP in the EC had returned to normal values, and persisted for as long as 6 months after the lesion. By contrast, ChAT-ir neurons in the HDB were permanently lost. Our results suggest that the transient down-regulation of the cholinergic phenotype in entorhinal cortex interneurons could be a manifestation of activity-dependent plasticity, and that the loss of cholinergic innervation from the basal forebrain could be responsible for these effects through an imbalance of inputs. We hypothesize that the recovery of the phenotypic expression of entorhinal interneurons could be due to a recovery in their innervation, perhaps from sprouting axons in the same fields, belonging to surviving cholinergic neurons in the basal forebrain.     

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.