The entorhinal cortex is a major relay between the hippocampus and other cortical and subcortical regions. Glutamatergic axons from layer II neurons form the entorhinal cortical projection to the hippocampus via the perforant pathway. We have demonstrated previously that lesion of the perforant pathway causes the death of approximately 30% of entorhinal layer II (ECL2) neurons. To elucidate mechanisms contributing to neuronal death and to investigate strategies preventing it, we identified the phenotype of the vulnerable neuronal population. Sections were immunolabeled with antibodies to the neuronal markers NeuN, glutamate, and calbindin-D28k, and to receptors for fibroblast growth factor-2 (FGFR1) and NMDA (NMDAR1) and were examined using confocal microscopy. Calbindin immunoreactivity was strikingly lamina-specific to ECL2, where one-third of all ECL2 neurons were calbindin-positive. Localization of glutamate revealed that half of the glutamatergic ECL2 neurons coexpressed calbindin. Quantification using unbiased stereology at 9 weeks after lesion of the perforant pathway revealed that the only ECL2 neuronal population that experienced a significant (70%) loss (20% of the total) was the population of glutamatergic ECL2 neurons that did not coexpress calbindin. All ECL2 neurons expressed FGFR1; therefore, we tested the role of FGF-2 in the survival of glutamatergic ECL2 neurons. We grafted fibroblasts genetically engineered to express nerve growth factor or FGF-2 and found that only FGF-2 grafts prevented loss of the vulnerable glutamatergic/calbindin-negative neurons. We present a hypothesis for the selective vulnerability of these glutamatergic/calbindin-negative ECL2 neurons and address the role of FGF-2 in neuronal rescue.

To investigate the molecular mechanisms of cholinergic sprouting in the hippocampus after removal of entorhinal cortical inputs, we evaluated trophic factor gene expression in the denervated hippocampus. Despite the proposed role for nerve growth factor (NGF) in this sprouting, we observed no change in NGF mRNA or protein at several postlesion time points. In contrast, FGF-2 mRNA was increased within 16 hr. FGF-2 immunoreactivity was localized within GFAP-positive hypertrophic astrocytes distributed specifically within the denervated outer molecular layer after the lesion. To address the functional significance of this increase in FGF-2, we assessed the magnitude of cholinergic sprouting in animals receiving chronic intracerebroventricular infusions of neutralizing antibodies specific for FGF-2 and compared it with that observed in lesioned animals receiving infusate controls. Animals given FGF-2 antibodies displayed a marked reduction in cholinergic sprouting as compared with controls. In fact, many of these animals exhibited virtually no sprouting at all despite histological verification of complete lesions. These results suggest that endogenous FGF-2 promotes cholinergic axonal sprouting in the injured adult brain. Furthermore, immunocytochemical localization of receptors for FGF-2 (i.e., FGFR1) on projecting basal forebrain cholinergic neurons suggests that FGF-2 acts directly on these neurons to induce the lesion-induced sprouting response.

Local delivery of brain-derived neurotrophic factor (BDNF) by genetically modified cells provides the unique opportunity to examine the effects of BDNF on adult dopaminergic and cholinergic neurons in vivo. Primary rat fibroblasts were genetically engineered to produce BDNF. Conditioned media from BDNF-transduced fibroblasts supported embryonic chick dorsal root ganglion neurons as well as rat fetal mesencephalic neurons. BDNF-transduced fibroblasts grafted to the rat brain survived and showed continued mRNA production for at least 2 weeks. The effects of BDNFtransduced fibroblast grafts on the dopaminergic and cholinergic systems were then assessed. BDNFtransduced fibroblasts grafted into the normal intact substantia nigra induced sprouting of tyrosine hydroxylase-and neurofilament-immunoreactive fibers into the graft. Fibroblast grafts implanted into the normal intact striatum and midbrain as well as the 6-hydroxydopamine-lesioned brain did not induce sprouting of dopaminergic fibers; neither did they affect drug-induced rotational behavior. BDNF-transduced fibroblasts did, however, significantly increase the homovanillic acid/dopamine ratio when grafted into the normal midbrain. Following transection of the fimbria-fornix, BDNFtransduced fibroblasts grafted into the septum were unable to rescue the septal cholinergic population, as did nerve growth factor-producing fibroblast grafts. Genetically modified fibroblast grafts may provide an effective, localized method of BDNF delivery in vivo to test biological effects of this factor on the central nervous system. Indexing termscholinergic; dopaminergic; neurotrophic factor; NGF; transplantation Neurotrophic factors are present in the central nervous system (CNS) and play important roles in neural development, differentiation, and survival (for reviews, see Barde, 1989; Thoenen, 1991). A family of related neurotrophic molecules, called neurotrophins, whose members affect overlapping as well as distinct populations of neurons, has been recently isolated. Members of the neurotrophin family include nerve growth factor (NGF), brain-derived NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and neurotrophic factors-4/-5 (NT-4/5). Extensive conservation between species and sequence similarity between members (over 50% homology in amino acid identity) suggest important roles for neurotrophins in the CNS (Leibrock et al., 1989;Maisonpierre et al., 1990;Berkemeier et al., 1991).Neurotrophins exert their effects through specific binding to cell surface receptors that exist in both low (Kd ~ 10 −9 )-and high (Kd ~ 10 −11 )-affinity forms (Meakin and Shooter, 1991). The low-affinity NGF receptor (p75 NGFR ) comprises a 75 kD protein (Chao et al., 1986) and binds all known members in the neurotrophin family. Specificity in binding is afforded by high-affinity binding sites on trk protooncogenes: trkA., trkB, and trkC. Neurotrophic factors appear to mediate their effects by inducing phos...

In vivo model systems that can evaluate neuronal death, survival, and regeneration are critical to revealing basic mechanisms of neuronal response and developing strategies for CNS repair. We propose a distinct experimental model of CNS degeneration following lesions to the perforant path connecting the hippocampus and the entorhinal cortex. Within 2 weeks of a unilateral aspirative perforant path lesion, 30% of the ipsilateral entorhinal cortex layer II (ECL II) projection neurons had died with no change in the contralateral ECL II population. Although there was no loss of ECL II neurons with normal aging, animals that survived for 15 months postlesion experienced an almost 50% loss of ipsilateral neurons compared to unlesioned controls. This progressive neuronal death was bilateral, with the contralateral ECL II experiencing a 30% decline in neuronal number relative to unlesioned controls. The use of unbiased stereology ensured that estimates of total number were not distorted by changes in the reference volume. The documented progressive neuronal death resulted in delayed behavioral impairment in spatial learning and performance (latency nearly 200% of controls). We propose, therefore, that the perforant path model is suitable for experimental investigation of neuronal survival and regeneration following CNS trauma.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.