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1.
Transl Psychiatry ; 7(2): e1025, 2017 02 07.
Article in English | MEDLINE | ID: mdl-28170001

ABSTRACT

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1ß is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.


Subject(s)
Chemokine CCL2/metabolism , Monocyte Chemoattractant Proteins/metabolism , Stress Disorders, Post-Traumatic/metabolism , Adult , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Chemokine CCL17/metabolism , Chemokine CCL4/metabolism , Chronic Disease , Circadian Rhythm , Cytokines/metabolism , Female , Humans , Male , Sex Factors
2.
Brain Res ; 1061(2): 118-23, 2005 Nov 09.
Article in English | MEDLINE | ID: mdl-16242674

ABSTRACT

The co-expression of calretinin with parvalbumin and calbindin D-28k was examined in the rat cranial and spinal sensory ganglia by triple immunofluorescence method. In the trigeminal and nodose ganglia, 9% and 5% of calretinin-immunoreactive neurons, respectively, also contained both parvalbumin- and calbindin D-28k immunoreactivity. These neurons had large cell bodies. In the trigeminal ganglion, they were restricted to the caudal portion. Such neurons were evenly distributed throughout the nodose ganglion. The co-expression could not be detected in the dorsal root, jugular or petrosal ganglia. Nerve fibers which co-expressed all the three calcium-binding proteins were observed in the inferior alveolar nerve but not the infraorbital nerve or palate. In the periodontal ligament, these nerve fibers formed Ruffini-like endings. These findings suggest that (1) the co-expression in trigeminal neurons is intimately related to their peripheral receptive fields; (2) the three calcium-binding proteins (calretinin, parvalbumin, calbindin D-28k) co-expressed in the trigeminal neurons may have mechanoreceptive function in the periodontal ligament.


Subject(s)
Ganglia, Sensory/metabolism , Neurons/physiology , Parvalbumins/biosynthesis , S100 Calcium Binding Protein G/biosynthesis , S100 Calcium Binding Protein G/physiology , Spinal Cord/metabolism , Animals , Calbindin 2 , Calbindins , Fluorescent Antibody Technique, Indirect , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Male , Mechanoreceptors/physiology , Nerve Fibers/metabolism , Nodose Ganglion/cytology , Nodose Ganglion/metabolism , Periodontal Ligament/innervation , Periodontal Ligament/physiology , Rats , Rats, Sprague-Dawley , Trigeminal Ganglion/cytology , Trigeminal Ganglion/metabolism
3.
Neurotox Res ; 6(4): 245-57, 2004.
Article in English | MEDLINE | ID: mdl-15545008

ABSTRACT

The substantia nigra cells of a normal and Parkinson's disease human brain were obtained by the micropunch procedure and total RNA was isolated. Differential display RT-PCR of the total RNA revealed differentially expressed cDNAs that were identified by sequencing. This resulted in the identification of a panel of known and unknown differentially expressed genes. Complex I (NADH ubiquinone oxidoreductase) and Complex IV (cytochrome oxidase) whose expressions are decreased in Parkinson's disease were reduced in the Parkinson brain. Of the various differentially expressed genes, flotillin-1, also known as reggie-2, was of great interest to us. It is a relatively new protein which is an integral membrane component of lipid rafts and has been implicated in signal transduction pathway events. In situ hybridization histochemical studies with human and rat brain sections revealed the presence of this mRNA in discrete neuronal (and possibly glial) cells of the substantia nigra, locus coeruleus, cortex, hippocampus, hypothalamus, thalamus, motor nuclei, nucleus basalis, raphe nucleus, and other brain regions. Immunohistochemical studies revealed that flotillin-1 is not present in all the regions where the message was found. In the rat brain, the most prominent observation was the revelation of all catecholamine cells (dopamine, norepinephrine, epinephrine) by the flotillin-1 antibody (1:100 dilution). At a more concentrated dilution (1:10) other neuronal cells (e.g., cortex, thalamus, hindbrain) were observed. At both dilutions dense dopaminergic fibers were observed in the rat caudate-putamen, nigrostriatal tract, and substantia nigra. It is significant that there is an increased gene expression of flotillin-1 in the Parkinson substantia nigra/ventral tegmental area. The role of flotillin in these cells is unclear although it is interesting that the reggie-2/flotillin-1 gene was upregulated during retinal axon regeneration in the goldfish visual pathway (Schulte et al., Development 124:577-87, 1997) which suggests that flotillin-1/reggie-2 might play a role in axonal growth from the remaining substantia nigra cells of the Parkinson brain.


Subject(s)
Catecholamines/biosynthesis , Membrane Proteins/biosynthesis , Nerve Fibers/metabolism , Parkinson Disease/metabolism , Substantia Nigra/metabolism , Aged , Aged, 80 and over , Animals , Catecholamines/analysis , Catecholamines/genetics , Humans , Male , Membrane Proteins/analysis , Membrane Proteins/genetics , Nerve Fibers/chemistry , Parkinson Disease/genetics , Parkinson Disease/pathology , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Substantia Nigra/chemistry
4.
Neuroscience ; 113(3): 537-46, 2002.
Article in English | MEDLINE | ID: mdl-12150774

ABSTRACT

Immunohistochemistry for parvalbumin, calbindin D-28k, calretinin and calcitonin gene-related peptide (CGRP) was performed on the trigeminal ganglion and oro-facial tissues in Brn-3a wildtype and knockout mice at embryonic day 18.5 and postnatal day 0. In wildtype mice, the trigeminal ganglion contained abundant parvalbumin-, calbindin D-28k- and CGRP-immunoreactive neurons while the ganglion was almost devoid of calretinin-immunoreactive neurons. In Brn-3a knockout mice, a 63% decrease of parvalbumin-immunoreactive neurons was detected. In contrast, the absence of Brn-3a dramatically increased the number of calbindin D-28k-immunoreactive (3.5-fold increase) and calretinin-immunoreactive neurons (91-fold increase). The number of CGRP-immunoreactive neurons, however, was not altered by the Brn-3a deficiency. Cell size analysis indicated that loss of Brn-3a increased the proportions of small (<100 microm (2)) parvalbumin-, calbindin D-28k- and CGRP-immunoreactive neurons while it decreased those of large (>200 microm(2)) immunoreactive cells. Calretinin-immunoreactive neurons were either small or medium (100-200 microm (2)) in mutant mice. The oro-facial tissues contained parvalbumin-, calbindin D-28k- and CGRP-immunoreactive fibers, but not calretinin-immunoreactive ones in wildtype mice. In Brn-3a knockout mice, the number of parvalbumin-immunoreactive fibers markedly decreased in the infraorbital nerve and parvalbumin-immunoreactive endings disappeared in the vibrissa. In contrast, the number of calbindin D-28k-immunoreactive fibers increased significantly in the infraorbital and mental nerves. In addition, calbindin D-28k-immunoreactive endings appeared in the vibrissa. As well, some fibers showed calretinin-immunoreactivity in the infraorbital nerve of the mutant. However, no obvious change of CGRP-immunoreactive fibers was observed in the oro-facial region of knockout mice. Taken together, our data suggest that Brn-3a deficiency has effects on the expression of neurochemical substances in the trigeminal ganglion.


Subject(s)
Calcitonin Gene-Related Peptide/analysis , DNA-Binding Proteins/deficiency , Neurons, Afferent/metabolism , Parvalbumins/analysis , S100 Calcium Binding Protein G/analysis , Transcription Factors/deficiency , Trigeminal Ganglion/metabolism , Animals , Animals, Newborn , Calbindin 2 , Calbindins , Calcitonin Gene-Related Peptide/immunology , DNA-Binding Proteins/genetics , Face/innervation , Immunohistochemistry , Mice , Mice, Knockout , Nerve Fibers/chemistry , Nerve Fibers/metabolism , Neurons, Afferent/chemistry , Parvalbumins/immunology , S100 Calcium Binding Protein G/immunology , Transcription Factor Brn-3 , Transcription Factor Brn-3A , Transcription Factors/genetics , Trigeminal Ganglion/chemistry , Trigeminal Ganglion/embryology , Vibrissae/innervation
5.
Neurology ; 57(5): 780-4, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11552003

ABSTRACT

OBJECTIVE: To characterize the specificity of anti-GAD(65) antibodies in patients with stiff person syndrome (SPS), quantify antibody titers, and examine antibody production within the CNS. METHODS: The authors studied 18 patients with SPS and positive serum immunoreactivity to gamma-aminobutyric acid (GABA)-ergic neurons. The reactivity of serum and CSF to purified GAD antigen was examined by Western blots, and the anti-GAD(65) antibody titers in serum and CSF were quantified by ELISA and compared with 70 disease controls (49 with other autoimmune disorders and 11 with insulin-dependent diabetes mellitus). The intrathecal synthesis of anti-GAD(65) IgG was calculated, and the functional significance of the antibodies was examined by measuring the GABA levels in the CSF. RESULTS: The serum and CSF of all selected patients with SPS had high anti-GAD(65) titers (from 7.0 to 215 microg/mL in serum and from 92 to 2500 ng/mL in CSF) and immunoreacted strongly with recombinant GAD(65) on Western blots and with GABA-ergic neurons on rat cerebellum. Among controls, only the serum of eight patients with insulin-dependent diabetes mellitus had low anti-GAD(65) antibody titers (from 200 to 1760 ng/mL) but no reactivity to recombinant GAD(65). The CSF showed oligoclonal IgG bands in 10 (67%) of 15 patients and an increased anti-GAD(65)-specific IgG index in 11 (85%) of 13. The mean level of GABA in the CSF was lower in patients with SPS than in controls. CONCLUSIONS: In patients with SPS, there is marked intrathecal antibody response against neuronal GAD(65) epitopes, indicating a clonal B cell activation in the CNS. Anti-GAD(65) antibodies at high titers, when confirmed with immunoblots, are highly specific for SPS and appear to impair GABA synthesis.


Subject(s)
Antibody Specificity , Autoantibodies/biosynthesis , Autoantibodies/cerebrospinal fluid , Glutamate Decarboxylase/cerebrospinal fluid , Glutamate Decarboxylase/immunology , Isoenzymes/cerebrospinal fluid , Isoenzymes/immunology , Stiff-Person Syndrome/cerebrospinal fluid , Stiff-Person Syndrome/immunology , Animals , Autoantibodies/blood , Cerebellum/metabolism , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/cerebrospinal fluid , Glutamate Decarboxylase/blood , Humans , Immunoglobulin G/blood , Immunoglobulin G/cerebrospinal fluid , Isoenzymes/blood , Rats
6.
Exp Neurol ; 163(2): 311-23, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10833305

ABSTRACT

Calretinin (CR) is a calcium-binding protein purported to have neuroprotective properties. This study was designed to characterize the types of neurons containing CR in two different primary cultures and to determine which, if any, CR-immunoreactive (CR-ir) neurons are resistant to excitotoxic insults. Calretinin-containing neurons in cortical primary cultures derived from E14 rat embryos were not resistant to either kainic acid or a brief calcium overload induced by the calcium ionophore A23187. Equal proportions of CR-ir and GABAergic cortical neurons were lost after a 24-h exposure to 100 or 500 microM kainic acid. A 3 microM, 3-h exposure to A23187 induced equivalent amounts of cell loss in both the total cell and CR-ir cortical neuron culture populations. Cortical cultures grown for 6-7 days were more vulnerable than 12- to 13-day-old cultures to short-term, low-concentration treatments of A23187. Older cultures, however, were more severely affected when examined 24 h after a 3-h exposure to A23187. Calretinin-immunoreactive neurons derived from the diencephalon were relatively more resistant than cortical neurons to kainic acid at 6-7 days in vitro. In cortical or diencephalic cultures, CR was rarely coexpressed with GABA or calbindin D-28k. No vasoactive intestinal peptide, substance P, or parvalbumin was detected in CR-ir neurons in either culture system. We suggest that the presence of CR alone is not sufficient to spare neurons from a toxic calcium overload. Calretinin may still buffer calcium at low concentrations or be a component in a calcium-based signal transduction system.


Subject(s)
Calcium/metabolism , Neurons/metabolism , S100 Calcium Binding Protein G/metabolism , Animals , Calbindin 2 , Calcimycin/pharmacology , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Diencephalon/drug effects , Diencephalon/metabolism , Embryo, Mammalian , Excitatory Amino Acid Agonists/pharmacology , Ionophores/pharmacology , Kainic Acid/pharmacology , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Receptors, AMPA/drug effects , Receptors, AMPA/metabolism , S100 Calcium Binding Protein G/drug effects
7.
Int J Dev Neurosci ; 17(4): 331-45, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10479068

ABSTRACT

An investigation of the developmental expression of calretinin immunoreactivity and mRNA expression was carried out in the developing mouse diencephalon. Attention was focused on the thalamic eminence, which is a prominent structure previously described in the thalamus of the fetal mammalian diencephalon and adult lower vertebrates. Calretinin-positive staining was first observed in the thalamic eminence beginning at embryonic day 11. In situ hybridization histochemistry confirmed the presence of calretinin mRNA in the thalamic eminence. During subsequent embryonic development calretinin expression was very intense in neurons in the thalamic eminence though embryonic day 17, and thereafter, was increasingly difficult to distinguish. By postnatal day 0 the thalamic eminence was no longer discernable. Additional neurons within the murine diencephalon also expressed calretinin positive cell bodies and, or neuronal processes, including the stria medullaris, the habenular commissure and the paraventricular thalamic nucleus. It is possible that the thalamic eminence may form during development in order to act as an organizing center for the diencephalon.


Subject(s)
S100 Calcium Binding Protein G/metabolism , Thalamus/embryology , Animals , Calbindin 2 , Embryonic and Fetal Development/physiology , Fetus/metabolism , Fetus/physiology , Histocytochemistry , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL
8.
Cell Tissue Res ; 298(1): 11-9, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10555535

ABSTRACT

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.


Subject(s)
Nerve Tissue Proteins/metabolism , S100 Calcium Binding Protein G/metabolism , Supraoptic Nucleus/metabolism , Animals , Calbindin 1 , Calbindin 2 , Calbindins , Calcium/metabolism , Fluorescent Antibody Technique , Male , Microscopy, Confocal , Neurons/metabolism , Oxytocin/metabolism , Rats , Rats, Sprague-Dawley , Supraoptic Nucleus/cytology , Vasopressins/metabolism
9.
Neuroreport ; 10(10): 2089-94, 1999 Jul 13.
Article in English | MEDLINE | ID: mdl-10424680

ABSTRACT

TA20 cDNA was previously cloned as a neurite outgrowth factor from a hybridoma of mouse and rat cells, NG108-15. To clarify the detailed function and tissue distribution of this gene, homologous sequences of rat and mouse were identified. The cloned sequences had no homology with known genes, and was designated as sproutin. A predicted open reading frame of rat sproutin was transfected into human SK-N-SH cells. The over-expressed protein was distributed in cytoplasm and neurites, and caused an increase in the levels of microtuble associated proteins, but not that of phosphorylated neurofilament-H. The percentage of cells with neurites, the length of neurites and the number of neurites per cell were increased by sproutin transfection. Sproutin mRNA was brain specific. These results suggest that an increase in sproutin promotes dendritic extension.


Subject(s)
Nerve Growth Factors/pharmacology , Neurites/drug effects , Animals , Base Sequence , Cloning, Molecular , Humans , Male , Mice , Molecular Sequence Data , Open Reading Frames , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
10.
J Neurosci ; 19(14): 6017-26, 1999 Jul 15.
Article in English | MEDLINE | ID: mdl-10407039

ABSTRACT

Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cell cycle-related kinases. Previous neuropathological analysis of cdk5(-/-) mice showed significant changes in CNS development in regions from cerebral cortex to brainstem. Among the defects in these animals, a disruption of the normal pattern of cell migrations in cerebellum was particularly apparent, including a pronounced abnormality in the location of cerebellar Purkinje cells. Complete analysis of this brain region is hampered in the mutant because most of cerebellar morphogenesis occurs after birth and the cdk5(-/-) mice die in the perinatal period. To overcome this disadvantage, we have generated chimeric mice by injection of cdk5(-/-) embryonic stem cells into host blastocysts. Analysis of the cerebellum from the resulting cdk5(-/-) left arrow over right arrow cdk5(+/+) chimeric mice shows that the abnormal location of the mutant Purkinje cells is a cell-autonomous defect. In addition, significant numbers of granule cells remain located in the molecular layer, suggesting a failure to complete migration from the external to the internal granule cell layer. In contrast to the Purkinje and granule cell populations, all three of the deep cerebellar nuclear cell groupings form correctly and are composed of cells of both mutant and wild-type genotypes. Despite similarities of the cdk5(-/-) phenotype to that reported in reeler and mdab-1(-/-) (scrambler/yotari) mutant brains, reelin and disabled-1 mRNA were found to be normal in cdk5(-/-) brain. Together, the data further support the hypothesis that Cdk5 activity is required for specific components of neuronal migration that are differentially required by different neuronal cell types and by even a single neuronal cell type at different developmental stages.


Subject(s)
Cerebellum/abnormalities , Cyclin-Dependent Kinases/metabolism , Purkinje Cells/physiology , Stem Cells/physiology , Aging/physiology , Animals , Blastocyst , Cell Adhesion Molecules, Neuronal/genetics , Cerebellum/growth & development , Cerebellum/pathology , Chimera , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/deficiency , Cyclin-Dependent Kinases/genetics , Extracellular Matrix Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Morphogenesis , Nerve Tissue Proteins/genetics , Purkinje Cells/pathology , RNA, Messenger/genetics , Reelin Protein , Serine Endopeptidases , Stem Cells/cytology , Transcription, Genetic
11.
Brain Res ; 818(2): 275-84, 1999 Feb 13.
Article in English | MEDLINE | ID: mdl-10082813

ABSTRACT

ATP-sensitive inwardly rectifying potassium channels (KATPs) couple cell metabolism with its membrane potential. The best characterized KATP is the pancreatic KATP which is an heteromultimer of Kir6.2 and SUR1 protein subunits. KATPs are found in a variety of excitable cells, including neurons of the central nervous system. Basal ganglia (BG), especially in the substantia nigra (SN) reticulata and the globus pallidus (GP), have a high density of KATPs. Pharmacological modulation of the KATPs within the BG alters GABAergic activity and produces behavioural changes. However, the relatively high concentrations of drugs used might not have been entirely selective for the KATPs and may have acted at presynaptic nerve terminals as well as on the post-synaptic neurons. As an alternative means of examining the role of KATPs in regulating motor behavior, we used oligoantisense technology to diminish selectively Kir6.2 formation in the GP neurons. We then examined the effect of reduction in Kir6.2 expression on apomorphine-induced turning behavior in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the SN. Two weeks after injection of 6-OHDA, contralateral circling in response to apomorphine (0.25 mg/kg sc) was recorded. Kir6.2 antisense oligodeoxyribonucleotide (ODN) was then administered daily for 6 days into the GP ipsilateral to the 6-OHDA injection. Responses to apomorphine were then tested again and the animals killed to determine the effect of the antisense ODN on Kir6. 2 mRNA. Administration of Kir6.2 antisense ODN significantly attenuated apomorphine-induced contralateral turning and specifically reduced Kir6.2 mRNA in the injected GP. These results are consistent with pharmacological experiments which suggest that KATP channels in the GP are involved in motor responses to apomorphine in 6-OHDA lesioned rats, localizing the effects to the GP neurons, probably through modulation of the GABAergic system.


Subject(s)
Apomorphine/antagonists & inhibitors , Globus Pallidus/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Oxidopamine/toxicity , Parkinson Disease, Secondary/drug therapy , Peptide Fragments/pharmacology , Animals , Male , Motor Activity/drug effects , Neurons/drug effects , Parkinson Disease, Secondary/pathology , Rats , Rats, Sprague-Dawley , Rotation
12.
Int J Dev Neurosci ; 16(2): 135-45, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9762586

ABSTRACT

A calretinin enriched cell culture system which comprised approximately 40% of the total neuronal population of the E14 rat embryo was established from the region of the thalamic eminence (TE), and the effects of several neurotrophins on the neurite growth of calretinin-immunoreactive (CR-IR) neurons was investigated. A 4-day treatment of BDNF significantly increased the ratio of CR-IR to microtubule-associated protein 2-immunoreactive neurons at concentrations between 50 and 250 ng/ml. IGF-I at 100 ng/ml and TGF-alpha at 250 ng/ml also increased this ratio. None of the neurotrophins examined increased the number of primary neurites. BDNF did, however, increase the number of secondary neurites. BDNF-treated primary and secondary neurites were also significantly longer than neurites from neurons in control cultures. IGF-I elicited an increase in primary neurite length, but did not affect either number or length of secondary neurites. TGF-alpha had no effect on either number or length of the primary and secondary neurites. These results indicate that the maturation and development of CR-IR neurites is specifically affected by BDNF. It is suggested that BDNF increases the CR concentration above the threshold of detection by immunohistochemistry in cells and stimulates the sprouting of secondary CR-IR neurites.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Nerve Tissue Proteins/physiology , Neurites/physiology , S100 Calcium Binding Protein G/physiology , Animals , Brain-Derived Neurotrophic Factor/metabolism , Calbindin 2 , Cell Count , Cells, Cultured , Immunohistochemistry , Microtubule-Associated Proteins/metabolism , Nerve Growth Factors/metabolism , Neurites/ultrastructure , Rats , Rats, Sprague-Dawley , Stimulation, Chemical , Thalamus/cytology , Thalamus/embryology , Thalamus/metabolism
13.
Neurology ; 51(1): 85-93, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9674784

ABSTRACT

OBJECTIVE: To test inhibitory spinal circuits in patients with stiff-person syndrome (SPS). BACKGROUND: Patients with SPS have fluctuating muscle stiffness and spasms, and most have antibodies against GABAergic neurons. We predicted they would also have abnormalities of spinal GABAergic circuits. DESIGN/METHODS: Physiologic methods using H-reflexes were used to test reciprocal inhibition in the forearm and thigh, vibration-induced inhibition of flexor carpi radialis and soleus H-reflexes, recurrent inhibition, and nonreciprocal (1b) inhibition of soleus H-reflexes. RESULTS: Vibration-induced inhibition of H-reflexes was diminished in eight of nine patients tested, but the presynaptic period of reciprocal inhibition was normal in most patients. Both circuits are presumed to involve presynaptic inhibition and GABAergic interneurons. Presumed glycinergic circuits, including the first period of reciprocal inhibition and nonreciprocal (1b) inhibition, showed occasional abnormalities. Recurrent inhibition was normal in all five patients tested. CONCLUSION: Differences between the two presumptive GABAergic circuits may indicate that not all populations of GABAergic neurons are uniformly affected in SPS. The involvement of presumptive glycinergic circuits in some patients could point to impairment of nonGABAergic neurons, unrecognized involvement of GABAergic neurons in these inhibitory circuits, or, more likely, alterations of supraspinal systems that exert descending control over spinal circuits.


Subject(s)
H-Reflex/physiology , Neural Inhibition/physiology , Spinal Cord/physiopathology , Stiff-Person Syndrome/physiopathology , Adult , Aged , Electromyography , Femoral Nerve/cytology , Femoral Nerve/physiology , Glycine/physiology , Humans , Interneurons/chemistry , Interneurons/physiology , Median Nerve/cytology , Median Nerve/physiology , Middle Aged , Motor Neurons/chemistry , Motor Neurons/physiology , Muscle, Skeletal/innervation , Physical Stimulation , Spinal Cord/chemistry , Spinal Cord/cytology , Tendons/physiology , Vibration , gamma-Aminobutyric Acid/physiology
14.
Cell Tissue Res ; 292(2): 345-54, 1998 May.
Article in English | MEDLINE | ID: mdl-9560477

ABSTRACT

Prior studies have revealed the presence of chymotrypsinlike protease in peripheral organs, although no definitive evidence for the synthesis of this enzyme in tissue other than the pancreas is available. In an attempt to detect chymotrypsinogen mRNA in peripheral organs, a fragment of the pancreatic chymotrypsin mRNA from rat was amplified using PCR. The sequence was identified as a portion of the rat chymotrypsin B gene overlapping exon 5 through exon 7. It was subcloned into the pGEM-4Z vector and used as a template for the vitro transcription of an antisense riboprobe. Using ribonuclease protection and Northern blot analyses, chymotrypsin mRNA was detected in the rat pancreas, stomach, duodenum, ovary, and spleen. Monoclonal and polyclonal antisera against chymotrypsin detected chymotrypsinlike immunoreactivity in rat and human pancreas, rat stomach, duodenum and jejunum. Electrophoresis and immunoblotting revealed chymotrypsin-chymotrypsinogen bands (25-29 kDa) in the stomach and duodenum. Synthesis of a potent protease such as chymotrypsin in tissue other than pancreas is significant, suggesting a potential physiological and/or pathological role in these tissues.


Subject(s)
Chymotrypsin/genetics , Gastric Mucosa/metabolism , Intestinal Mucosa/metabolism , Pancreas/metabolism , Animals , Chymotrypsin/biosynthesis , Fluorescent Antibody Technique, Indirect , Gene Expression , Immunoblotting , Male , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Tissue Distribution
15.
Exp Neurol ; 149(2): 464-7, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9500959

ABSTRACT

This article provides basic guidelines for a rapid analysis of subpopulation proportions and neurite morphology in primary cultures. We describe, in E14 mesencephalic primary cultures, an immunohistochemical method for the simultaneous identification of multiple neuronal phenotypes and an estimation of the ratio of subpopulations. In addition, we describe the use of the Renaissance TSA-Direct kit (NEN, DuPont) to enhance the visualization of neurites when the antigen is in low abundance. Finally, a modified sholl analysis is used to rapidly and reliably estimate neurite number and length.


Subject(s)
Mesencephalon/cytology , Neurites/ultrastructure , Neurons/cytology , Animals , Calbindin 2 , Cell Culture Techniques/methods , Cells, Cultured , Embryo, Mammalian , Fluorescent Dyes , Guidelines as Topic , Immunohistochemistry/methods , Nerve Tissue Proteins/analysis , Phenotype , Rats , Rats, Sprague-Dawley , Reagent Kits, Diagnostic , Reproducibility of Results , S100 Calcium Binding Protein G/analysis , Tyrosine 3-Monooxygenase/analysis
16.
Nihon Yakurigaku Zasshi ; 112 Suppl 1: 1P-4P, 1998 Oct.
Article in English | MEDLINE | ID: mdl-10190122

ABSTRACT

Neuropeptides have now been localized throughout the nervous system. This talk focuses attention on (1) the interplay of peptides and other neurotransmitter systems in the hypothalamus--median eminence--pituitary gland. The multiplicity of neurochemicals is perceived to be responsible for the integrated control of pituitary hormone releasing factors; (2) the role of neuropeptides in the regulation of cardiovascular function in the hypothalamus-preoptic area. We investigated the effects of discrete intrahypothalamic injections of a variety of peptides on blood pressure and heart rate. We concluded that neuropeptides have a diversity of central cardiovascular actions and that not all areas containing a given peptide respond with cardiovascular change when the peptide is injected. Also, peptide specific actions originating within the same nucleus have been demonstrated, and the same peptide may have different vascular effects in different segments of the same nucleus; (3) the colocalization of neuropeptides with other classical neurotransmitters. We have found modulatory behavioral effects ("boxing") of combinations of transmitters and peptides injected into the postsynaptic site in the brain.


Subject(s)
Brain Chemistry , Neuropeptides/analysis , Neuropeptides/physiology , Animals , Cardiovascular Physiological Phenomena , Humans , Hypothalamo-Hypophyseal System/physiology , Neurotransmitter Agents/physiology
18.
Brain Res Mol Brain Res ; 49(1-2): 175-87, 1997 Oct 03.
Article in English | MEDLINE | ID: mdl-9387877

ABSTRACT

The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.


Subject(s)
Brain/metabolism , Mice/genetics , Neurons/metabolism , Promoter Regions, Genetic , S100 Calcium Binding Protein G/biosynthesis , S100 Calcium Binding Protein G/genetics , 3T3 Cells , Animals , Base Sequence , Binding Sites , Brain/cytology , Brain/embryology , Calbindin 2 , Cells, Cultured , Cloning, Molecular , DNA-Binding Proteins/metabolism , Female , Genes, Reporter , Humans , Luciferases/biosynthesis , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neurons/cytology , PC12 Cells , Rats , Recombinant Fusion Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Alignment , Sequence Deletion , TATA Box , Transfection , Uterus/cytology
19.
Exp Neurol ; 146(1): 25-32, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9225735

ABSTRACT

Levodopa, which is used in the treatment of Parkinson's disease, has known cytotoxic effects on dopaminergic neurons grown in culture. Calretinin (CR) is a cytosolic calcium-binding protein found in specific subpopulations of neurons as well as in some nonneuronal tissue. CR is expressed in 10% of rat embryo dopaminergic neurons grown in vitro. Since it has been postulated that CR provides neuroprotection due to its calcium-binding properties, we investigated whether CR-containing dopaminergic neurons were spared from levodopa toxicity. Incubation of mesencephalic cells with 10(-5) to 10(-7) M levodopa on Days 1-6 in vitro produced no significant effects on the number of dopaminergic neurons containing CR, but resulted in the loss of approximately 65% of the dopaminergic cells which did not contain CR. The remaining CR-negative dopaminergic neurons exhibited dose-dependent reductions in neurite length. The neuronal processes in CR-containing dopaminergic cells retained a smooth bipolar appearance. CR-immunoreactive cells which did not contain dopamine showed slight neurite length decreases at the highest drug concentrations but no changes in neuron number. These results indicate that CR may protect dopaminergic neurons from levodopa-induced toxicity.


Subject(s)
Dopamine/physiology , Levodopa/toxicity , Mesencephalon/cytology , Nerve Tissue Proteins/physiology , Neurons/drug effects , Neurons/metabolism , Neurotoxins , S100 Calcium Binding Protein G/physiology , Animals , Calbindin 2 , Cell Survival/drug effects , Cells, Cultured , Drug Resistance , Embryo, Mammalian , Neurites/drug effects , Neurites/physiology , Neurites/ultrastructure , Neurons/cytology , Rats , Rats, Sprague-Dawley , S100 Calcium Binding Protein G/analysis , S100 Calcium Binding Protein G/biosynthesis , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/physiology , Tyrosine 3-Monooxygenase/analysis
20.
J Comp Neurol ; 383(1): 112-21, 1997 Jun 23.
Article in English | MEDLINE | ID: mdl-9184990

ABSTRACT

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR-IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR-IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR-IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR-IR levels at 3 hours, 1 day, or 3-4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound-evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching.


Subject(s)
Brain Stem/physiology , Cochlear Nerve/physiology , Hearing/physiology , Nerve Tissue Proteins/biosynthesis , S100 Calcium Binding Protein G/biosynthesis , Acoustic Stimulation , Animals , Auditory Pathways/cytology , Auditory Pathways/metabolism , Brain Stem/cytology , Brain Stem/metabolism , Calbindin 2 , Chick Embryo , Deafness/physiopathology , Immunohistochemistry , In Situ Hybridization , Neurons, Afferent/physiology , Oligonucleotide Probes , RNA, Messenger/biosynthesis , Synapses/physiology
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