Decreased level of nerve growth factor (NGF) and its messenger RNA in the aged rat brain

Trophic factors such as nerve growth factor (NGF) are thought to support survival, differentiation and maintenance of neurons. Recent results indicate that NGF produced in cortical and hippocampal areas is required for the function of cholinergic neurons in the basal forebrain. With the use of enzyme immunoassay and RNA blot hybridization we studied the NGF protein and NGF mRNA, respectively, in regions of the brain innervated by basal forebrain cholinergic neurons in adult and aged rats. Levels of NGF protein were decreased by 40% in hippocampus of aged (28 months) Fischer 344 rats compared with adults (6 months), whereas no alterations were observed in cerebral cortex. Moreover, a reduction by 50% in the NGF mRNA was found in samples of the aged forebrain (cerebral cortex, hippocampus, basal forebrain and hypothalamus) compared to the adult. NGF deficiencies may thus account for the loss of cholinergic neurons in the basal forebrain generally found to accompany normal aging and resulting in altered cognitive functions.     

Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in areas of the brain, including the septal nucleus. Certain growth factors may promote the long-term survival of this subpopulation of neurons at risk. This study was undertaken to characterize growth factors' long-term effects on survival and development of neurons expressing the calcium-binding protein calbindin. In order to accomplish this, embryonic day 16 rat septal neurons were grown in bilaminar culture with astrocytes and in the absence of serum. These cultures were chronically treated with estrogen (Es), insulin-like growth factors I/II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Insulin-like growth factor II significantly increased the number of neurons immunoreactive for calbindin by 155%, suggesting either an increase in the survival of this subpopulation or an increase in the percentage of cells expressing calbindin. Chronic treatment with NGF, IGF-II, and Es significantly increased the number of primary neuritic processes on calbindin-positive neurons, whereas NGF and Es caused significant increases in the number of secondary processes and in the total lengths of the neuritic processes. Thus, effects of IGF-II, estrogen, and NGF on survival and maintenance of this neuronal subpopulation may be dependent on alterations in neurons which are immunopositive for calbindin.     

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF. © 1995 Wiley-Liss, Inc.     

Changes of NGF level in mouse hypothalamus following intermale aggressive behaviour: biological and immunohistochemical evidence

Nerve growth factor (NGF) immunoreactivity was detected in the hypothalamus of adult male mice. NGF-immunoreactive cell bodies were examined through consecutive brain sections, and it was found that most of the NGF-positive cells were located in the dorsomedial and mediolateral portions of the hypothalamus. Tissue culture bioassays showed that hypothalamic extract elicits neurite outgrowth from both chick sensory ganglia and rat superior cervical ganglia neurons, and that these effects are inhibited by addition of NGF antibodies. Our results also showed that intermale aggressive behaviour induced by 6-8 weeks of social isolation induces an NGF increase in the hypothalamic area, which is not abolished by sialoadenectomy, suggesting that the increased brain NGF is locally synthesized, and does not come from salivary sources. Likewise, the level of NGF in the hypothalamus of adrenalectomized fighting mice increased, although to a much lesser extent, when compared to hypothalamic levels of sham-operated fighting mice. The present results and a recent report showing that aggressive behaviour causes an increase of mRNANGF in hypothalamic areas are discussed in relation to a possible functional role of NGF in these brain structures.     

Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo

We have previously reported the developmental profiles of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) bio- and immunocytochemically, assessing GABAergic and cholinergic neuronal phenotypes respectively, in neuroblast-enriched cultures from 3-day-old chick embryo, plated on poly-L-lysine. We have also reported that collagen as culture substrate inhibits neuronal aggregation and neuritic fasciculation in this culture system. In this study we assessed the same parameters for cultures on collagen. In addition, we evaluated the effects of nerve growth factors (NGF) on cholinergic and GABAergic expression on neurons plated either on polylysine or collagen. We found that non-neuronal cells and NGF prolonged the survival of cholinergic and GABAergic neuronal populations and that both markedly stimulated GABAergic expression. In contrast, cholinergic expression was only enhanced by NGF. Immunostaining for GABA and ChAT reflected the biochemical findings. Glutamine synthetase and cyclic nucleotide phosphohydrolase, used as markers for astrocytes and oligodendrocytes respectively, showed very low activity in both substrata and were not related to GAD or ChAT peak activities. Our findings suggest that humoral factors and cell-cell contacts markedly influence neuronal phenotypic expression in culture. Moreover, it appears that during early neuronal differentiation GABAergic neurons are more responsive to microenvironmental regulation compared to cholinergic neurons.     

Cellular phenotype of androgen receptor-immunoreactive nuclei in the developing and adult rat brain

Androgen exposure during development and adulthood promotes cell-to-cell communication, modulates the size of specific brain nuclei, and influences hormone-dependent behavioral and neuroendocrine functions. Androgen action involves the activation of androgen receptors (AR). To elucidate the mechanisms involved in AR-mediated effects on forebrain development, double-label fluorescent immunohistochemistry and confocal microscopy were employed to identify the cellular phenotype of AR-immunoreactive (AR(+)) cells in the developing (embryonic day 20, postnatal days 0, 4, 10) and adult male rat forebrain. Sections were doubly labeled with antibodies directed against AR and one of the following: neurons (immature, nestin; mature, NeuN) or astrocytes [immature, vimentin; mature, glial fibrillary acidic protein (GFAP)] or mature oligodendrocytes (mGalC). In all brain regions examined, by far the majority of AR(+) cells were neurons. In addition, small subsets of AR(+) cells were identified as mature astrocytes (GFAP(+)) but only in specific brain regions at specific ages. AR(+)/GFAP(+) cells were observed in the cerebral cortex but only in postnatal day 10 rats and in the arcuate nucleus of the hypothalamus but only in adult rats. Immature neurons, immature astrocytes, and oligodendrocytes were not AR(+) at any age, in any region. Thus, both neurons and astrocytes in the male rat forebrain contain ARs, suggesting that androgens, via ARs, may exert effects on both cell types in an age- and region-dependent manner.     

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.     

S-100 immunoreactivity in rat brain glial cultures is associated with both astrocytes and oligodendrocytes

S-100 protein, a Ca²⁺-binding protein of the EFhand type, is most abundant in the brain, and is involved in cell differentiation and the molecular mechanisms underlying cytoskeletal organization. We have investigated the immunocytochemical localization of S-100 protein in rat brain glial cultures prepared from the cerebral hemispheres of newborn rats. In mixed glial cultures, containing astrocytes type I and II and oligodendrocytes at various stages of differentiation, S-100 immunoreactivity was detected in all three cell types. Double immunofluorescence analysis revealed that in astrocytes, S-100 immunoreactivity was mainly colocalized with glial fibrillary acidic protein (GFAP), while in oligodendrocytes a close association with microtubular structures was observed. For immunoblot analysis, highly enriched oligodendrocytes and astrocytes were separately cultured for another week, and their extracts were analyzed by immunoblotting. The immunoblots of the cell extracts of both cell types showed a single S-100-immunoreactive polypeptide with an apparent molecular weight of approximately 12,000 daltons. Thus, the data presented here demonstrate that S-100 protein is not confined to astrocytes but occurs also in oligodendrocytes of rat brain. The close association with the oligodendroglial cytoskeleton suggests that this protein could also play a regulatory role in the organization of microtubules in oligodendrocytes and hence may be involved in the formation and maintenance of the myelin-containing membrane sheets. © 1995 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.     

Organization of ascending hypothalamic projections to the rostral forebrain with special reference to the innervation of cholinergic projection neurons

Axonal projections from hypothalamic nuclei to the basal forebrain, and their relation to cholinergic projection neurons in particular, were studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with choline acetyltransferase (ChAT) immunocytochemistry. Discrete iontophoretic PHA-L injections were delivered to different portions of the caudal lateral hypothalamus, as well as to various medial hypothalamic areas, including the ventromedial, dorsomedial, and paraventricular nuclei, and anterior hypothalamic and medial preoptic areas. The simultaneous detection of PHA-L-labeled fibers/terminals and ChAT-positive neurons was performed by using nickel-enhanced diaminobenzidine (DAB) and nonenhanced DAB as chromogens. Selected cases were investigated at the electron microscopic level. Ascending hypothalamic projections maintained an orderly lateromedial arrangement within the different components of the medial forebrain bundle, as well as with respect to their terminal projection fields (e.g., within the bed nucleus of the stria terminalis and lateral septal nucleus). The distribution pattern of hypothalamic inputs to cholinergic projection neurons corresponded to the topography of ascending hypothalamic axons. Axons originating from neurons in the far-lateral hypothalamus reached cholinergic neurons in a zone that extended from the dorsal part of the sublenticular substantia innominata (SI) caudolaterally, to the lateral portion of the bed nucleus of the stria terminalis rostromedially, encompassing a narrow band along the ventral part of the globus pallidus and medial portion of the internal capsule. Axons originating from cells in the medial portion of the lateral hypothalamus reached cholinergic cells primarily in more medial and ventral parts of the SI, and in the magnocellular preoptic nucleus and horizontal limb of the diagonal band nucleus (HDB). Axons from medial hypothalamic cells appeared to contact cholinergic neurons primarily in the medial part of the HDB, and in the medial septum/vertical limb of the diagonal band complex. Electron microscopic double-labeling experiments confirmed contacts between labeled terminals and cholinergic cells in the HDB and SI. Individual hypothalamic axons established synapses with both cholinergic and noncholinergic neuronal elements in the same regions. These findings have important implications for our understanding of the organization of afferents to the basal forebrain cholinergic projection system.     

Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulin-like growth factors I and II

To study the selectivity of neurotrophic actions in the brain, we analyzed the actions of several known growth factors on septal cholinergic, pontine cholinergic, and mesencephalic dopaminergic neurons in culture. Similar to nerve growth factor (NGF), basic fibroblast growth factor (bFGF) stimulated choline acetyltransferase activity in septal cultures. In contrast to NGF, bFGF also enhanced dopamine uptake in mesencephalic cultures and stimulated cell proliferation in all 3 culture types. Insulin and the insulin-like growth factors I and II stimulated transmitter-specific differentiation and cell proliferation in all culture types. Epidermal growth factor (EGF) produced a small increase in dopamine uptake by mesencephalic cells and stimulated cell proliferation in all culture types. In septal cultures, bFGF was most effective when given at early culture times, NGF at later times. The stimulatory actions of bFGF and insulin did not require the presence of glial cells and were not mediated by NGF. In mesencephalic cultures, the stimulation of dopamine uptake by bFGF and EGF was dependent on glial proliferation. The results suggest different degrees of selectivity of the neurotrophic molecules. NGF and, very similarly, bFGF seem to influence septal cholinergic neurons directly and rather selectively, whereas the neurotrophic actions of insulin and the insulin-like growth factors appear to be more general.     

Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons

Nerve growth factor (NGF) has recently been implicated as a trophic agent in the survival and maintenance of basal forebrain cholinergic neurons. To test the hypothesis that NGF may play a role in the age-related degeneration of basal forebrain neurons and decline of cerebral cholinergic function, we have used a monoclonal antibody to the NGF receptor, 192 IgG, to immunocytochemically visualize and compare rat basal forebrain neurons responsive to NGF in aged (30 months) and young adult (10 months) rats. In a subpopulation of aged rats, NGF receptor-immunoreactive cells in the basal forebrain appear vacoulated and shrunken, and the neuropil staining is markedly reduced. While no substantial decline in cell density is apparent in Nissl-stained sections, the number of NGF receptor-positive cell profiles within the vertical limb of diagonal band nuclei is reduced by an average of 32% in aged rats. Marked reduction in the expression of NGF receptors in aged rats may signify loss of capacity of the basal forebrain neurons to bind and transport NGF from their terminals in the hippocampus and cortex, subsequent decrease in NGF delivered to the cell bodies, and eventual cellular dysfunction and death of neurons in aging.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the brain of the African lungfish Protopterus annectens

The localization of the enzymes responsible for the biosynthesis of neurosteroids in the brain of dipnoans has not yet been determined. In the present study, we investigated the immunohistochemical distribution of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 5 alpha-reductase (5 alpha-R) in the brain and pituitary of the African lungfish Protopterus annectens by using antibodies raised against type I human 3 beta-HSD and type I human 5 alpha-R. The 3 beta-HSD and 5 alpha-R immunoreactivities were detected in cell bodies and fibers located in the same areas of the lungfish brain, namely, in the pallium, thalamus, hypothalamus, tectum, and periaqueductal gray. Identification of astrocytes, oligodendrocytes, and neurons with antisera against glial fibrillary acidic protein, galactocerebroside and neurofilaments revealed that, in the lungfish brain, 3 beta-HSD immunolabeling is expressed exclusively by neurons, whereas the 5 alpha-R-immunoreactive material is contained in both neurons and glial cells. In the pituitary gland, 3 beta-HSD- and 5 alpha-R-like immunoreactivity was localized in both the pars distalis and the pars intermedia. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the brain and pituitary of a lungfish. These data strongly suggest that neurosteroid biosynthesis occurs in the brain of fishes, as previously shown for amphibians, birds, and mammals.     

Oligodendrocytes use lactate as a source of energy and as a precursor of lipids.

Lactate is an important metabolic substrate for the brain during the postnatal period and also plays a crucial role in the traffic of metabolites between astrocytes and neurons. However, to date there are no clues with regard to lactate utilization by oligodendrocytes, the myelin-forming cells in the brain. In the present work, lactate utilization by oligodendrocytes in culture was investigated and compared with its utilization by cultured neurons, type 1 and type 2 astrocytes. Our results clearly indicate that oligodendrocytes readily use lactate both as a metabolic fuel and as a precursor to build carbon skeletons. Oligodendrocytes oxidize lactate at a higher rate than that observed for neurons and astrocytes, and their rate of lipid synthesis from lactate was at least 6-fold higher than that found in astrocytes or neurons. The rate of glucose utilization through different pathways was also investigated. The flux of glucose through the pentose phosphate pathway and the rate of lipid synthesis were at least 2-fold higher in oligodendrocytes than in astrocytes or neurons. These findings indicate that oligodendrocyte metabolism is designed specifically for the synthesis of lipids, presumably those of myelin.     

The immunohistochemical localization of choline acetyltransferase in the cat brain

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.     

Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture

We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.     

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.