Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2

Prostaglandin D2 (PGD2) is an extensively studied sleep-promoting substance, but the neuroanatomical basis of PGD2-induced sleep is only partially understood. To determine potential regions involved in this response, we used Fos immunohistochemistry to identify neurons activated by infusion of PGD2 into the subarachnoid space below the rostral basal forebrain. PGD2 increased nonrapid eye movement sleep and induced striking expression of Fos in the ventrolateral preoptic area (VLPO), a cluster of neurons that may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD2 also increased Fos immunoreactivity in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO.

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse

Although it has been known for some time that estrogen exerts a profound influence on brain development a definitive demonstration of the role of the classical estrogen receptor (ERα) in sexual differentiation has remained elusive. In the present study we used a sexually dimorphic population of dopaminergic neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) to test the dependence of sexual differentiation on a functional ERα by comparing the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the AVPV of wild-type (WT) mice with that of mice in which the ERα had been disrupted by homologous recombination (ERKOα). Only a few ERα-immunoreactive neurons were detected in the AVPV of ERKOα mice, and the number of TH-immunoreactive neurons was three times that of WT mice, suggesting that disruption of the ERα gene feminized the number of TH-immunoreactive neurons. In contrast, the AVPV contains the same number of TH-immunoreactive neurons in testicular feminized male mice as in WT males, indicating that sexual differentiation of this population of neurons is not dependent on an intact androgen receptor. The number of TH-immunoreactive neurons in the AVPV of female ERKOα mice remained higher than that of WT males, but TH staining appeared to be lower than that of WT females. Thus, the sexual differentiation of dopamine neurons in the AVPV appears to be receptor specific and dependent on the perinatal steroid environment.

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale.

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxy-dopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

A cGMP-signaling pathway in a subset of olfactory sensory neurons

It is well established that signal transduction in sensory neurons of the rat olfactory epithelium involves a cAMP-signaling pathway. However, a small number of olfactory neurons specifically express cGMP-signaling components, namely a guanylyl cyclase (GC-D) and a cGMP-stimulated phosphodiesterase (PDE2). Here, we show that this subset of olfactory neurons expressing GC-D and PDE2 does also express the subunit of a cGMP-selective cyclic nucleotide-gated (CNG) channel that has been previously identified in cone photoreceptors. Further, components of the prototypical cAMP-signaling pathway could not be detected in this subpopulation of cells. These results imply that these neurons use an alternative signaling pathway, with cGMP as the intracellular messenger, and that, in these cells, the receptor current is initiated by the opening of cGMP-gated channels.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

F-spondin is a contact-repellent molecule for embryonic motor neurons

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural–functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP+ cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP+ cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.