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Basal forebrain cholinergic (BFC) neurons are an important substrate of cognitive function and are hypothesized to require the presence of nerve growth factor (NGF) for survival and target innervation. NGF-deficient mice develop BFC neurons that extend projections into telencephalic targets, but the mice perish before innervation is fully established. Rescue of NGF-deficient mice by transgenic expression of NGF under the keratin promoter yields viable mice with disrupted CNS expression of NGF. In the current study, rescued NGF-deficient mice contain normal numbers of septal cholinergic neurons yet reveal severe compromise of cholinergic innervation of both cortex and hippocampus. Surprisingly, intracerebroventricular infusion of NGF into juvenile mice can induce an essentially normal pattern of cholinergic innervation of the hippocampus. These results indicate that NGF is required for induction of proper innervation by BFC neurons, but that the cellular pattern of expression of this factor is not critical for specifying the distribution of axon terminals.

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.

Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.

The Eph-related tyrosine kinase receptor, REK7/EphA5, mediates the effects of AL-1/Ephrin-A5 and related ligands and is involved in the guidance of retinal, cortical, and hippocampal axons during development. The continued expression of REK7/EphA5 in the adult brain, in particular in areas associated with a high degree of synaptic plasticity such as the hippocampus, raises the question of its function in the mature nervous system. In this report we examined the role of REK7/EphA5 in synaptic remodeling by asking if agents that either block or activate REK7/EphA5 affect synaptic strength in hippocampal slices from adult mouse brain. We show that a REK7/EphA5 antagonist, soluble REK7/EphA5-IgG, impairs the induction of long-term potentiation (LTP) without affecting other synaptic parameters such as normal synaptic transmission or paired-pulse facilitation. In contrast, perfusion with AL-1/Ephrin-A5-IgG, an activator of REK7/EphA5, induces a sustained increase in normal synaptic transmission that partially mimics LTP. The sustained elevation of normal synaptic transmission could be attributable to a long-lasting binding of the AL-1/Ephrin-A5-IgG to the endogenous REK7/EphA5 receptor, as revealed by immunohistochemistry. Furthermore, maximal electrical induction of LTP occludes the potentiating effects of subsequent treatment with AL-1/Ephrin-A5-IgG. Taken together these results implicate REK7/EphA5 in the regulation of synaptic plasticity in the mature hippocampus and suggest that REK7/EphA5 activation is recruited in the LTP induced by tetanization., (Copyright 1998 Academic Press.)

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.

BatK is a second member of the Csk family of regulatory kinases that phosphorylate a key inhibitory tyrosine on Src family kinases, leading to down-regulation. To investigate the roles of BatK and Csk, both of which are expressed in the brain, we compared their temporal expression patterns during development of the central nervous system (CNS) in rats. BatK mRNA is undetectable at embryonic day 12 (E12), appears in the developing nervous system at approximately E15, and its expression progressively increases up to the time of birth, thereafter remaining high throughout the adult brain. In striking contrast, Csk is highly expressed throughout embryonic development and remains high in the CNS until birth. It is then dramatically down-regulated in the adult brain except in the olfactory bulb. BatK and Csk thus exhibit complementary temporal expression patterns. Since BatK expression correlates with late-stage development and terminal differentiation, we speculated that it might be involved in regulating neuronal differentiation. Using PC12 cells as a model system, we show that overexpression of BatK is sufficient to induce neurite outgrowth in the absence of nerve growth factor. Further, overexpression of BatK activates the mitogen-activated protein kinase cascade. We propose a model suggesting that, despite overlapping in vitro activities, BatK and Csk regulate different targets in vivo and have different functions during and after neuronal development, BatK being the dominant regulator of Src kinases in the fully differentiated adult brain.

Administration of nerve growth factor (NGF) to aged or lesioned animals has been shown to reverse the atrophy of basal forebrain cholinergic neurons and ameliorate behavioral deficits. To examine the importance of endogenous NGF in the survival of basal forebrain cholinergic cells and in spatial memory, mice bearing a disruption mutation in one allele of the NGF gene were studied. Heterozygous mutant mice (ngf+/-) have reduced levels of NGF mRNA and protein within the hippocampus and were found to display significant deficits in memory acquisition and retention in the Morris water maze. The behavioral deficits observed in NGF-deficient mice were accompanied by both shrinkage and loss of septal cells expressing cholinergic markers and by a decrease in cholinergic innervation of the hippocampus. Infusions of NGF into the lateral ventricle of adult ngf+/- mice abolished the deficits on the water maze task. Prolonged exposure to NGF may be required to induce cognitive effects, because reversal of the acquisition deficit was seen after long (5 weeks) but not short (3 d) infusion. Although NGF administration did not result in any improvement in the number of septal cells labeled for choline acetyltransferase, this treatment did effectively correct the deficits in both size of cholinergic neurons and density of cholinergic innervation of the hippocampus. These findings demonstrate the importance of endogenous NGF for survival and function of basal forebrain cholinergic neurons and reveal that partial depletion of this trophic factor is associated with measurable deficits in learning and memory.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.

Expression of trk receptors is a major determinant of neurotrophin responsiveness of sensory neurons. Although it has been apparent for some time that subpopulations of dorsal root and trigeminal ganglion neurons respond in vitro to each of the members of the neurotrophin family, the extent to which functionally distinct subclasses of sensory neurons are dependent on the actions of different neurotrophins for their development and function remains an active area of investigation. One step towards elucidating the role of various neurotrophins in development and function of sensory neurons has been to examine the distribution of trk receptors on sensory neurons. These studies have clearly revealed that members of the trk family are differentially expressed in functionally distinct populations of both developing and mature sensory neurons and, further, have provided evidence consistent with a shift in neurotrophin responsiveness during the development of sensory neurons.

Localization of mRNA encoding trkB indicates that two truncated isoforms of trkB, T1trkB and T2trkB, are differentially distributed in the rodent nervous system, and that each of these transcripts is co-expressed with catalytic trkB (TK+trkB) in adult motor neurons. In contrast to the prominent expression of T1trkB by non-neuronal cells, T2trkB expression appeared to be restricted to neurons and demonstrated significant overlap with the pattern of TK+trkB distribution. In developing spinal cord ventral horn, an age-related increase in hybridization was observed for truncated isoforms. These findings suggest that truncated trkB may modulate neuronal responses to neurotrophins which act via trkB.

Using molecular cloning techniques, human homologs of the known members of the trk family of neurotrophin receptors have been cloned and sequenced. Overall, there is a high degree of similarity between the human sequences and those from other mammals; however, there are differences in splicing patterns. There are two spliced forms of the extracellular domain of trkC in the human, a finding that has not been described in other species. In contrast, fewer spliced forms were detected of the intracellular domains of human trkB and trkC than has been described in other mammals. Northern analysis and in situ hybridization experiments indicate that the human trks are expressed in a similar pattern to that described in other mammals. Expression of the trk extracellular domains as fusion proteins with IgG heavy chain yields soluble molecules that mimic intact trks in their binding specificity and affinity. These soluble chimeras block the biological activity of their cognate neurotrophin(s) in vitro.

The vertebrate ventral midbrain contains 3-4 x 10(4) dopaminergic neurons that influence motor activity, emotional behavior, and cognition. Recently, glial cell line-derived neurotrophic factor (GDNF) was shown to be a potent survival factor for these dopaminergic neurons in culture. However, many midbrain dopaminergic neurons project to targets that do not express GDNF. We report here that transforming growth factors (TGFs) TGF beta 2 and TGF beta 3, which are distantly related to GDNF, also prevent the death of cultured rat embryonic midbrain dopaminergic neurons at picomolar concentrations. Furthermore, we find that TGF beta 2, TGF beta 3, and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of dopaminergic neurons projecting to distinct targets have access to only one of these factors. These findings are consistent with the idea that GDNF, TGF beta 2, and TGF beta 3 are physiological survival factors for developing midbrain dopaminergic neurons and may have applications as therapeutics for Parkinson's disease, a neurodegenerative disorder of dopaminergic neurons.

To determine whether neurotrophins act on functionally distinct populations of adult sensory neurons, the distributions of mRNAs for TrkA and tyrosine kinase-containing isoforms of TrkB and TrkC were determined in rat DRG neurons projecting to different peripheral targets. Whereas trkA was expressed by a very high percentage of visceral afferents, trkC was expressed frequently only in muscle afferents. Among cutaneous afferents, the size distributions for trkA- and trkC-positive cells showed little overlap. The percentages and size distributions of cells labeled for the trks argue strongly that almost all trkB-expressing cells must also express trkA or trkC. These results indicate that NGF and NT-3 act on functionally distinct populations of adult sensory neurons and suggest that a sizeable number of small DRG neurons may not respond to neurotrophins via a known Trk in the adult rat.

Homologous recombination was utilized to generate mice with a deletion in the coding sequence of the nerve growth factor (NGF) gene. Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia. Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. Examination of the central nervous system revealed that, in marked contrast with neurons of sensory and sympathetic ganglia, basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the null mutant mice. Thus, differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.

Embryonic spinal motor neurons are thought to depend for survival on unidentified factors secreted both by their peripheral targets and by cells within the central nervous system. The neurotrophins are a family of polypeptides required for survival of discrete central and peripheral neuronal populations in vivo and in vitro. In spite of their ability to reduce motor neuron death in vivo, the known neurotrophins have been thought to be without direct effect on motor neurons. Here we show that picomolar concentrations of three of them, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-5, can prevent the death of cultured embryonic rat spinal motor neurons. Furthermore, messenger RNA coding for neurotrophins is present at appropriate stages in spinal cord and limb bud, and mRNA for their receptors is found in motor neurons. These neurotrophins may therefore be physiological motor neuron growth factors.

A growing literature suggests that the hippocampus can be damaged by glucocorticoids, the adrenal steroids secreted during stress. Thus, considerable interest was generated by recent reports that prolonged elimination of glucocorticoids by adrenalectomy (ADX) damages hippocampal dentate gyrus neurons. To date, this phenomenon has only been observed in rats of peripubertal age or younger; moreover, reports differ considerably as to the magnitude of the damage induced. Therefore, we examined this issue in rats ADXd at 5 months of age. Three months later, there was a significant 26% loss of dentate neurons in a subset of rats. In agreement with these previous reports, this subset had attenuated weight gain and electrolyte imbalances, suggestive of complete removal of the adrenals and accessory adrenal tissue. As a novel observation, we also observed significant (19%) loss of CA4 pyramidal neurons. Thus, both severe under- or overexposure to glucocorticoids can be deleterious to a number of hippocampal neuron types.

The adrenal stress hormones glucocorticoids (GCs) impair the ability of hippocampal neurons to survive neurological insults, including hypoxia-ischemia and seizure. These insults are thought to be toxic via a cascade of excessive synaptic concentrations of excitatory neurotransmitters (e.g. glutamate), activation of the NMDA receptor, and pathologic mobilization of cytosolic calcium post-synaptically. We tested whether GCs exacerbate these insults by exacerbating this 'NMDA cascade'. We sought a toxin which damaged independently of the NMDA cascade, and whose toxicity was enhanced by GCs. After testing a number of neurotoxins, we found that the antimetabolite 3-acetylpyridine (3AP) fit this requirement. We then tested if blockade of the NMDA receptor blocks the ability of GCs to enhance 3AP toxicity. Hippocampi were microinfused with 160 micrograms of 3AP. Elevating circulating GC concentrations to the range seen during major stressors for a week before and after microinfusion caused a significant increase in 3AP-induced damage (when compared to adrenalectomized rats kept GC-free for the same period). Infusing the NMDA receptor blocker APV with 3AP did not alter the toxicity in adrenalectomized rats. However, APV reduced 3AP-induced damage in GC-treated rats to levels seen in adrenalectomized rats. This suggests that GCs endanger hippocampal neurons by enhancing glutamatergic signals and/or enhancing vulnerability to such signals. As a possible explanation for this observation, GCs inhibit glucose uptake into hippocampal neurons, and numerous steps in the NMDA cascade are exacerbated when neuronal energy stores are diminished.

Feedback inhibition of the adrenocortical axis by circulating glucocorticoids occurs at the pituitary and CNS sites. In the CNS, both hypothalamic and suprahypothalamic sites have been implicated as mediators of glucocorticoid feedback activity. In the present experiments, we have attempted to identify specific CNS regions mediating the feedback and to characterize which hypothalamic adrenocorticotropic hormone secretagogues are under glucocorticoid inhibitory control. Adrenalectomized rats were presented with a delayed feedback signal in the form of systemic infusion with corticosterone or dexamethasone. Hypophysialportal concentrations of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), and oxytocin (OT) were determined before and during a hypotensive stressor in the face of varying levels of feedback. The rats were then killed, and the extent of total, type I, and type II corticosteroid receptor occupancy in hippocampus, hypothalamus, and amygdala was determined. The following observations were made: (1) increased hippocampal corticosteroid receptor occupancy was associated with suppressed adrenocorticotropic hormone secretagogue concentrations; (2) the major, significant predictor of initial (prehypotensive) concentrations of CRF, AVP, and OT was the extent of occupancy of hippocampal type II receptors, often in combination with occupancy of hippocampal type I or hypothalamic receptors; (3) secretion of CRF induced by hypotension was best predicted by hippocampal type I and type II receptor occupancy (stress-induced OT secretion was best predicted by hippocampal type II and hypothalamic receptor occupancy), and (4) the 'shape' of the hippocampal type II receptor occupancy versus initial AVP concentration curve suggested a nonlinear, threshold type of relationship, implying tight hippocampal regulation of AVP secretion.

Glucocorticoid feedback inhibition at the level of the brain is extremely complex, involving feedback at both hypothalamic and suprahypothalamic levels. The hippocampus has been implicated as a suprahypothalamic mediator of such feedback, based on numerous lesion, stimulation, and steroid implantation studies. These reports, however, predated the isolation and characterization of CRF and recognition of the multifactorial control of ACTH release. Thus, it is not clear which hypothalamic ACTH secretagogues are under inhibitory control of the hippocampus. To answer this, we measured hypophysialportal concentrations of CRF, arginine vasopressin, and oxytocin in rats with fornix transections, which disrupt hippocampal communication with the hypothalamus. Hypophysial-portal blood was collected in rats exposed to either low or high circulating corticosterone concentrations in the presence or absence of the coincident stressor of hypotension. We observed that fornix transection produced hypersecretion of all three secretagogues. However, the pattern of hypersecretion differed for each as follows: 1) fornix transection did not affect either initial CRF secretion or the magnitude of the stress response, but made rats resistant to a high feedback signal during stress; 2) fornix transection led to initial arginine vasopressin hypersecretion, which remained sensitive to a high feedback signal; and 3) fornix transection led to initial oxytocin hypersecretion as well as resistance to a high corticosterone feedback signal during hypotension.