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Lack of axon regeneration in the adult CNS has been attributed partly to myelin inhibitors and the properties of astrocytes. After spinal cord injury, proliferating astrocytes not only represent a physical barrier to regenerating axons but also express and secrete molecules that inhibit nerve growth, including chondroitin sulfate proteoglycans (CSPGs). Epidermal growth factor receptor (EGFR) activation triggers astrocytes into becoming reactive astrocytes, and EGFR ligands stimulate the secretion of CSPGs as well as the formation of cribriform astrocyte arrangements that contribute to the formation of glial scars. Recently, it was shown that EGFR inhibitors promote nerve regeneration in vitro and in vivo. Blocking a novel Nogo receptor interacting mechanism and/or effects of EGFR inhibition on astrocytes may underlie these effects. Here we show that rats subjected to weight-drop spinal cord injury can be effectively treated by direct delivery of a potent EGFR inhibitor to the injured area, leading to significantly better functional and structural outcome. Motor and sensory functions are improved and bladder function is restored. The robust effects and the fact that other EGFR inhibitors are in clinical use in cancer treatments make these drugs particularly attractive candidates for clinical trials in spinal cord injury.

DeltaFosB is a transcription factor that accumulates in a region-specific manner in the brain after chronic perturbations. For example, repeated administration of drugs of abuse increases levels of DeltaFosB in the striatum. In the present study, we analyzed the effect of spontaneous wheel running, as a model for a natural rewarding behavior, on levels of DeltaFosB in striatal regions. Moreover, mice that inducibly overexpress DeltaFosB in specific subpopulations of striatal neurons were used to study the possible role of DeltaFosB on running behavior. Lewis rats given ad libitum access to running wheels for 30 d covered what would correspond to approximately 10 km/d and showed increased levels of DeltaFosB in the nucleus accumbens compared with rats exposed to locked running wheels. Mice that overexpress DeltaFosB selectively in striatal dynorphin-containing neurons increased their daily running compared with control littermates, whereas mice that overexpress DeltaFosB predominantly in striatal enkephalin-containing neurons ran considerably less than controls. Data from the present study demonstrate that like drugs of abuse, voluntary running increases levels of DeltaFosB in brain reward pathways. Furthermore, overexpression of DeltaFosB in a distinct striatal output neuronal population increases running behavior. Because previous work has shown that DeltaFosB overexpression within this same neuronal population increases the rewarding properties of drugs of abuse, results of the present study suggest that DeltaFosB may play a key role in controlling both natural and drug-induced reward.

Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta) related to GDNFR-alpha. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-beta mRNA in developing and adult mice, and comparisons with GDNFR-alpha and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-beta mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-alpha in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-beta was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-beta was expressed in surrounding tissue. Neurturin and GDNFR-beta were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-beta was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-beta in the developing and adult organism. Although NTN and GDNFR-beta expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-alpha and GDNFR-beta.

The nicotinic cholinergic antagonist alpha-bungarotoxin (alpha-BT) binds throughout the rat hippocampal formation. The binding is displaceable by d-tubocurarine. The most heavily labeled cells are GABA- containing interneurons in the dentate and in Ammon's horn. These neurons have several different morphologies and contain several neuropeptides. alpha-BT-labeled interneurons in the dentate are small cells between the granular and molecular layers that often contain neuropeptide Y. alpha-BT-labeled interneurons in CA1 are medium-sized interneurons, occasionally found in stratum pyramidale, but more often found in stratum radiatum and stratum lacunosum moleculare. These neurons often contain cholecystokinin. The largest alpha-BT-labeled interneurons are found in CA3, in both stratum radiatum and stratum lucidum. These neurons are multipolar and frequently are autofluorescent. They often contain somatostatin or cholecystokinin. These large interneurons have been found to receive medial septal innervation and may also have projections that provide inhibitory feedback directly to the medial septal nucleus. The cholinergic innervation of the hippocampus from the medial septal nucleus is under the trophic regulation of NGF and brain-derived neurotrophic factor, even in adult life. Expression of mRNA for both these factors is increased in CA3 and the dentate after intraventricular administration of alpha-BT, but not after administration of the muscarinic antagonist atropine. alpha-BT-sensitive cholinergic receptors on inhibitory interneurons may be critical to medial septal regulation of the hippocampal activity, including the habituation of response to sensory input.

Blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) was used to test at which levels of the neuroaxis signals are elicited when different modalities of sensory information from the limbs ascend to cortex cerebri. We applied graded electric stimuli to the rat hindlimbs and used echo-planar imaging to monitor activity changes in the lumbar spinal cord and medulla oblongata, where primary afferents of painful and nonpainful sensation synapse, respectively. BOLD signals were detected in ipsilateral lumbar spinal cord gray matter using sufficiently strong stimuli. Using stimuli well below the threshold needed for signals to be elicited in the spinal cord, we found BOLD responses in dorsal medulla oblongata. The distribution of these signals is compatible with the neuroanatomy of the respective synaptic relay stations of the corresponding sensory pathways. Hence, the sensory pathways conducting painful and nonpainful information were successfully distinguished. The fMRI signals in the spinal cord were markedly decreased by morphine, and these effects were counteracted by naloxone. We conclude that fMRI can be used as a reliable and valid method to monitor neuronal activity in the rat spinal cord and medulla oblongata in response to sensory stimuli. Previously, we also documented BOLD signals from thalamus and cortex. Thus, BOLD responses can be elicited at all principal synaptic relay stations along the neuroaxis from lumbar spinal cord to sensory cortex. Rat spinal cord fMRI should become a useful tool in experimental spinal cord injury and pain research.

We have examined the influence of glutamate on cortical brain-derived neurotrophic factor (BDNF) expression using in situ hybridization and immunohistochemistry. Kainic acid (KA) produced an upregulation of hippocampal and neocortical BDNF mRNA as well as BDNF protein that was blocked by a non-NMDA antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but was not affected by the NMDA antagonist 2-amino-7- phosphonoheptanoic acid (AP7). Basal levels of BDNF mRNA were not affected by NMDA, DNQX, or AP7 treatment. BDNF protein was also increased after kainate exposure with a spatial and temporal course distinct from that seen for the expression of BDNF mRNA. A dramatic shift in BDNF immunoreactivity (-IR) was observed from intracellular compartments to the neuropil surrounding CA3 pyramidal cells 2–3 hr after KA exposure. This shift in localization of BDNF-IR suggests a constitutive release of BDNF at the level of the cell body and dendrites. Moreover, we have localized mRNAs for full-length and truncated trkB, to a co-incident population of neurons and glia. These data suggest the neurons that produce BDNF also express components necessary for a biological response to the same neurotrophic factor. The present study also demonstrates increased BDNF-IR in the mossy fiber terminal zone of hippocampus after exposure to KA, as well as an increase in trkB mRNA, and provides evidence of local release of this neurotrophin into the surrounding neuropil where it would be available for local utilization. The synthesis and putative release of BDNF from somatic and/or dendritic sites within the hippocampus provide evidence of a potential autocrine or paracrine role for BDNF, and establish a local source of trophic support for the maintenance of synaptic plasticity and anatomic reorganization in the mature nervous system.

Human fetal mesencephalic tissue obtained from elective first-trimester abortions was grafted to 6-hydroxydopamine-denervated striatum of athymic (nude) rats. After 3–6 months, the transplants were evaluated by light and electron microscopy using antibodies against tryosine hydroxylase (TH), human specific Thy-1 (Thy-1), 5-hydroxytryptamine (5- HT), and laminin. In vivo chronoamperometric studies of K+-induced release of electroactive species were done prior to the histochemical evaluations. At the light microscopical level, Thy-1-immunoreactivity was evenly distributed throughout the entire transplants. Thy-1- immunoreactive nerve fibers were observed radiating from the graft into the host striatum. In sections that were double-stained with antibodies against Thy-1 and TH, such nerve fibers contained both markers. Also 5- HT-immunoreactive cells were found in the grafts with processes both in the grafts and radiating into host neuropil. Laminin immunohistochemistry showed an even distribution of capillaries in the graft with less density than in host brain, suggesting immaturity of graft tissue. At the ultrastructural level, TH-immunoreactive axons made symmetric contacts with unlabeled dendritic shafts and dendritic spines within the host brain. A few asymmetric contacts with TH- immunoreactive axons were seen. 5-HT-immunoreactive terminals made both symmetric and asymmetric contacts with unlabeled dendritic shafts and spines. In vivo chronoamperometry using local application of K+ revealed average signals that were lower on the transplanted side than in control striatum. However, close to the grafts significant amounts of the K+-evoked signal amplitudes were as large as 1.3 microM, and the ratio of the reduction to oxidation currents suggested release of a mixture of dopamine and 5-HT. Taken together, this study shows that human fetal mesencephalic tissue pieces survive grafting into nude rats, develop normal vascularization, and express coexistence of TH- and Thy-1-immunoreactivity. Human TH- and 5-HT-immunoreactive nerve fibers form synapses in host striatum and release monoamine neurotransmitters.

Using immunohistochemistry with antisera raised against the glial fibrillary acidic protein (GFA), we have studied the appearance and distribution of GFA-like immunoreactivity in whole mounts of rodent iris and in sectioned cat and cow iris. In the adult rat iris, a dense plexus of GFA-positive fibers was seen in both the dilator plate and the sphincter. The fluorescent fibers formed large meandering bundles and a dense irregular network of thinner fibers. In the sphincter, mainly thinner fibers were seen. Thin fibers were also seen winding around blood vessels in the dilator plate. In adult mouse iris, the GFA- positive fibers had a quite different distribution with a few radially oriented fiber bundles superimposed on a more regular network of thinner fibers. Adult guinea pig irides showed still another pattern of GFA-positive fibers with a low number of bundles and thinner fibers forming a sparse irregular network. In thicker fiber bundles of all three rodent species, as well as at branching sites of the thinner fibers, negative or weakly fluorescent swellings surrounded by GFA-like immunoreactivity were present. These structures probably represent the cellular origin of the GFA-positive fibers. Thick, strongly fluorescent fiber bundles, as well as numerous thinner fibers, were seen in sections of cat and cow iris. Prenatally, fibers were visualized at embryonic day 18 in the rat. In these irides as well as in irides from 21-day-old embryos and 1-day-old pups, most fibers were organized in a gradually increasing system of thin meandering fiber bundles that showed limited branching. At postnatal day 6, a more mature network of thinner fibers had developed between the now more numerous fiber bundles. No obvious increase or decrease in the amount of GFA-positive fibers was seen in irides grafted to the anterior eye chamber of adult rat recipients examined 1 and 6 days after grafting. However, in these irides, as well as in the host irides, strongly fluorescent spider-like cells with short branching processes and a negative nucleus were seen. These cells were more numerous and more strongly fluorescent in grafted irides as compared to recipient irides and in the 6-day iris grafts as compared to the 2-day grafts. In all probability, the GFA-positive fibers and cells forming a network in adult irides from different species and in embryonic and grafted rat irides represent Schwann cells and their processes. The cellular origin of the spider-like cells in the iris grafts is less clear.

Selective autonomic denervations of the iris have been used to study the possible redistribution of adrenergic markers within adult nerve fiber systems and to reveal the cellular origin of a nonsympathetic fiber plexus induced to express such markers. The presence and distribution of fibers showing neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-like immunoreactivity was studied in the rat iris using stretch-prepared whole mounts. Normal irides contained a dense regular network of NPY-positive varicose fibers. Such fibers were regularly seen innervating blood vessels. The choroid membrane had a high number of fluorescent fibers. A similar, although slightly denser TH-positive fiber system was visualized in the iris. One or 2 days after surgical removal of the superior cervical ganglion, almost all NPY- and TH-positive fibers had disappeared, suggesting that most, if not all, NPY-positive fibers in the iris originate in the superior cervical ganglion. In irides from long-term sympathectomized animals, a high number of TH- and NPY-immunoreactive fibers had reappeared, while such irides were devoid of catecholamine-containing fibers, as evidenced by Falck-Hillarp histochemistry. The appearance of TH- and NPY-positive fibers in sympathetically denervated irides was clearly time dependent. The distribution of fluorescent fibers in irides from intact and sympathectomized animals showed obvious dissimilarities such as a lower fluorescence intensity and fewer varicose fibers in denervated irides. Furthermore, in irides from sympathectomized rats, TH- and NPY-positive fibers were not associated with blood vessels. Unilateral removal of the parasympathetic ciliary ganglion, which supplies the iris with cholinergic fibers, 3 days prior to sacrifice in animals bilaterally sympathectomized 1 month earlier, led to a drastic reduction in numbers of TH- and NPY-positive iris fibers on the ciliarectomized/sympathectomized side as compared to the sympathectomized-alone side. The present experiments thus suggest that adult cholinergic neurons in vivo are capable of expressing adrenergic characteristics under experimental conditions.