Hypothyroidism in transgenic mice expressing IFN-γ in the thyroid

IFN-γ has been implicated with contradictory results in the pathogenetic process of autoimmune (Hashimoto's) thyroiditis, the most common cause of hypothyroidism in adults. To test whether the local production of IFN-γ can lead to thyroid dysfunction, we have generated transgenic mice that express constitutively IFN-γ in the thyroid follicular cells. This expression resulted in severe hypothyroidism, with growth retardation and disruption of the thyroid architecture. The hypothyroidism derived from a profound inhibition of the expression of the sodium iodide symporter gene. Taken together, these results indicate a direct role of IFN-γ in the thyroid dysfunction that occurs in autoimmune thyroiditis.

Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively γ-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in Aplysia sensory neurons

Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa1, that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 μM Zn2+. Indicating particularly high permeability through Ca2+-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury

Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Cholecystokinin-8 protects central cholinergic neurons against fimbria–fornix lesion through the up-regulation of nerve growth factor synthesis

In this study, we demonstrate that cholecystokinin-8 (CCK-8) induces an increase in both nerve growth factor (NGF) protein and NGF mRNA in mouse cortex and hippocampus when i.p. injected at physiological doses. By using fimbria–fornix-lesioned mice, we have also demonstrated that repeated CCK-8 i.p. injections result in recovery of lesion-induced NGF deficit in septum and restore the baseline NGF levels in hippocampus and cortex. Parallel to the effects on NGF, CCK-8 increases choline acetyltransferase (Chat) activity in forebrain when injected in unlesioned mice and counteract the septo-hippocampal Chat alterations in fimbria–fornix-lesioned mice. To assess the NGF involvement in the mechanism by which CCK-8 induces brain Chat, NGF antibody was administrated intracerebrally to saline- and CCK-8-injected mice. We observe that pretreatment with NGF antibody causes a marked reduction of NGF and Chat activity in septum and hippocampus of both saline- and CCK-8-injected mice. This evidence indicates that the CCK-8 effects on cholinergic cells are mediated through the synthesis and release of NGF. Taken together, our results suggest that peripheral administration of CCK-8 may represent a potential experimental model for investigating the effects of endogenous NGF up-regulation on diseases associated with altered brain cholinergic functions.

Reelin controls position of autonomic neurons in the spinal cord

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell–cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157–172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055–1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons

Astrocytes can release glutamate in a calcium-dependent manner and consequently signal to adjacent neurons. Whether this glutamate release pathway is used during physiological signaling or is recruited only under pathophysiological conditions is not well defined. One reason for this lack of understanding is the limited knowledge about the levels of calcium necessary to stimulate glutamate release from astrocytes and about how they compare with the range of physiological calcium levels in these cells. We used flash photolysis to raise internal calcium in astrocytes, while monitoring astrocytic calcium levels and glutamate, which evoked slow inward currents that were recorded electrophysiologically from single neurons grown on microislands of astrocytes. With this approach, we demonstrate that modest changes of astrocytic calcium, from 84 to 140 nM, evoke substantial glutamatergic currents in neighboring neurons (−391 pA), with a Hill coefficient of 2.1 to 2.7. Because the agonists glutamate, norepinephrine, and dopamine all raise calcium in astrocytes to levels exceeding 1.8 μM, these quantitative studies demonstrate that the astrocytic glutamate release pathway is engaged at physiological levels of internal calcium. Consequently, the calcium-dependent release of glutamate from astrocytes functions within an appropriate range of astrocytic calcium levels to be used as a signaling pathway within the functional nervous system.

Modifications of retinal neurons in a mouse model of retinitis pigmentosa

Animal models of retinitis pigmentosa include the rd mouse, in which a mutation of a rod-specific phosphodiesterase leads to the rapid loss of photoreceptors during the early postnatal life. Very little is known about changes occurring in inner retinal neurons after photoreceptor loss. These changes are important in view of the possibility of restoring vision in retinas with photoreceptor degeneration by means of cell transplantation or direct stimulation of inner layers. In this paper, we show that bipolar and horizontal cells of the rd mouse retina undergo dramatic morphological modifications accompanying photoreceptor loss, demonstrating a dependence of second order neurons on these cells. While describing modifications of the rd retina, we also provide quantitative information about neurons of the wild-type mouse retina, useful for future studies on genetically altered animals.

Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Cholesterol depletion inhibits the generation of β-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer’s disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called β-amyloid (Aβ) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Aβ that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-β-cyclodextrin, we show that the formation of Aβ is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Aβ formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Aβ formation to occur and imply a link between cholesterol, Aβ, and Alzheimer’s disease.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-d-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Connexin expression in electrically coupled postnatal rat brain neurons

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription–PCR revealed a differential expression pattern of connexins in these identified neurons.