Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system

The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ–TTC gene fusion retains the biological functions of both proteins in vivo—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.

Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis

The lipid bilayer of the myelin membrane of the central nervous system (CNS) and the peripheral nervous system (PNS) contains the oligodendrocyte- and Schwann cell-specific glycosphingolipids galactocerebrosides (GalC) and GalC-derived sulfatides (sGalC). We have generated a UDP-galactose ceramide galactosyltransferase (CGT) null mutant mouse (cgt−/−) with CNS and PNS myelin completely depleted of GalC and derived sGalC. Oligodendrocytes and Schwann cells are unable to restore the structure and function of these galactosphingolipids to maintain the insulator function of the membrane bilayer. The velocity of nerve conduction of homozygous cgt−/− mice is reduced to that of unmyelinated axons. This indicates a severely altered ion permeability of the lipid bilayer. GalC and sGalC are essential for the unperturbed lipid bilayer of the myelin membrane of CNS and PNS. The severe dysmyelinosis leads to death of the cgt−/− mouse at the end of the myelination period.

Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691–10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Carbon monoxide and nitric oxide as coneurotransmitters in the enteric nervous system: Evidence from genomic deletion of biosynthetic enzymes

Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 (HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes (nNOSΔ/Δ and HO2Δ/Δ) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOSΔ/Δ and HO2Δ/Δ mice are depolarized, with apparent additive effects in the double knockouts (HO2Δ/Δ/nNOSΔ/Δ). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2Δ/Δ preparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.

Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system

Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology.

Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features.

T cells, B cells, and antibody are found in the white matter of the central nervous system in multiple sclerosis. The epitope center for the antibody response to human myelin basic protein (MBP) fits precisely the minimal epitope Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96 for that reported for HLA DR2b (DRB1*1501)-restricted T cells that recognize MBP [Wucherpfenning, K.W., Sette, A., Southwood, S., Oseroff, C., Matsui, M., Strominger, J. & Hafler, D. (1994) J. Exp. Med. 179, 279-290], and overlaps with the reported DR2a-restricted epitope for T cells reactive to MBP [Martin, R., Howell, M. D., Jaraquemada, D., Furlage, M., Richert, J., Brostoff, S., Long, E. O., McFarlin, D. E. & McFarland, H. F. (1991) J. Exp. Med. 173, 19-24]. We describe a molecular model of this epitope.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

Local nitric oxide production in viral and autoimmune diseases of the central nervous system.

Because of the short half-life of NO, previous studies implicating NO in central nervous system pathology during infection had to rely on the demonstration of elevated levels of NO synthase mRNA or enzyme expression or NO metabolites such as nitrate and nitrite in the infected brain. To more definitively investigate the potential causative role of NO in lesions of the central nervous system in animals infected with neurotropic viruses or suffering from experimental allergic encephalitis, we have determined directly the levels of NO present in the central nervous system of such animals. Using spin trapping of NO and electron paramagnetic resonance spectroscopy, we confirm here that copious amounts of NO (up to 30-fold more than control) are elaborated in the brains of rats infected with rabies virus or borna disease virus, as well as in the spinal cords of rats that had received myelin basic protein-specific T cells.

Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin

Mice deficient for plasminogen exhibit a variety of pathologies, all of which examined to date are reversed when the animals are also made fibrin(ogen) deficient. These results suggested that the predominant, and perhaps exclusive, physiological role of plasminogen is clearance of fibrin. Plasminogen-deficient mice also display resistance to excitotoxin-induced neurodegeneration, in contrast with wild-type mice, which are sensitive. Based on the genetic interaction between plasminogen and fibrinogen, we investigated whether resistance to neuronal cell death in the plasminogen-deficient mice is dependent on fibrin(ogen). Unexpectedly, mice lacking both plasminogen and fibrinogen are resistant to neurodegeneration to levels comparable to plasminogen-deficient mice. Therefore, plasmin acts on substrates other than fibrin during experimental neuronal degeneration, and may function similarly in other pathological settings in the central nervous system.

Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the rat

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system

Mice devoid of PrPC (Prnpo/o) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnpo/o brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnpo/o mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnpo/o mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

Intrinsic responses to Borna disease virus infection of the central nervous system

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs for egr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor α, macrophage inflammatory protein 1β, interleukin 6, and mob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.

Induction of myelination in the central nervous system by electrical activity.

The oligodendrocyte is the myelin-forming cell in the central nervous system. Despite the close interaction between axons and oligodendrocytes, there is little evidence that neurons influence myelinogenesis. On the contrary, newly differentiated oligodendrocytes, which mature in culture in the total absence of neurons, synthesize the myelin-specific constituents of oligodendrocytes differentiated in vivo and even form myelin-like figures. Neuronal electrical activity may be required, however, for the appropriate formation of the myelin sheath. To investigate the role of electrical activity on myelin formation, we have used highly specific neurotoxins, which can either block (tetrodotoxin) or increase (alpha-scorpion toxin) the firing of neurons. We show that myelination can be inhibited by blocking the action potential of neighboring axons or enhanced by increasing their electrical activity, clearly linking neuronal electrical activity to myelinogenesis.