Xin-Ming Ma

The arginine vasopressin and corticotrophin-releasing hormone gene transcription responses to varied frequencies of repeated stress in rats.

1 Rats habituate to repeated exposure to homotypic stressors. The present studies were designed to define how altered frequency of exposure to a stressor affects the development of habituation and how this habituation is reflected in alterations in basal expression and responsiveness of hypothalamic corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP) messenger and heteronuclear RNA (hnRNA). 2 Rats were exposed to a 60 min period of restraint stress every 7th day, every 3rd day, alternate days or daily for 2 weeks and their response to a final episode of stress on day 15 was compared with that of a control group of unstressed rats. 3 The response of plasma corticosterone to the final stressor on day 15 was diminished in animals which had been stressed on only two previous occasions, 7 days apart, and diminished further with increasing frequency of previous stressors until it failed to respond at all in animals stressed daily. 4 The pattern of CRH hnRNA and mRNA responses were similar, decreasing with increasing frequency of exposure to the stressor, while AVP mRNA responses increased in response to repeated stress. 5 The gradual emergence of increased AVP transcription at a time of diminishing CRH response suggests that repeated stress results in a specific facilitation of AVP gene expression, perhaps by impairment of corticosterone feedback.

Kalirin 7 is required for synaptic structure and function

Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), the predominant adult splice form of the multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain and localizes to the postsynaptic side of excitatory synapses. In vitro studies demonstrated that overexpression of Kal7 increased dendritic spine density, whereas reduced expression of endogenous Kal7 decreased spine density. To evaluate the role of Kal7 in vivo, mice lacking the terminal exon unique to Kal7 were created. Mice lacking both copies of the Kal7 exon (Kal7KO) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7KO mice. Behaviorally, Kal7KO mice showed decreased anxiety-like behavior in the elevated zero maze and impaired acquisition of a passive avoidance task, but normal behavior in open field, object recognition, and radial arm maze tasks. Kal7KO mice were deficient in hippocampal long-term potentiation. Western blot analysis confirmed the absence of Kal7 and revealed compensatory increases in larger Kalirin isoforms. PSDs purified from the cortices of Kal7KO mice showed a deficit in Cdk5, a kinase known to phosphorylate Kal7 and play an essential role in synaptic function. The early stages of excitatory synaptic development proceeded normally in cortical neurons prepared from Kal7KO mice, with decreased excitatory synapses apparent only after 21 d in vitro. Expression of exogenous Kal7 in Kal7KO neurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

Effect of Repeated Lipopolysaccharide Administration on Tissue Cytokine Expression and Hypothalamic‐Pituitary‐Adrenal Axis Activity in Rats

The effects of chronic immune challenge on cytokine expression and hypothalamic‐pituitary‐adrenal axis (HPA) axis responses to stress were studied in Wistar rats after administration of increasing doses of lipopolysaccharide (LPS). Repeated LPS (R‐LPS) decreased body weight and increased adrenal weight and pituitary pro‐opiomelanocortin mRNA levels. LPS injection increased plasma adrenocorticotropic hormone (ACTH) and corticosterone but the effect was attenuated in R‐LPS. Plasma corticosterone but not ACTH responses to restraint were also reduced in R‐LPS. Basal and restraint‐stimulated corticotropin releasing hormone (CRH) mRNA levels were lower in R‐LPS, but responses to a new LPS injection were similar to controls. In contrast, type 1 CRH receptor (CRH‐R1) mRNA responses to both LPS and restraint were blunted in R‐LPS. Vasopressin mRNA levels in parvocellular neurones were higher in R‐LPS, and increased further after restraint but not after a new LPS injection. Glucocorticoid receptor (GR) levels in the paraventricular nucleus (PVN) increased after a single LPS or R‐LPS (24 h after the last injection) but declined after a new injection in R‐LPS. Interleukin (IL)‐1β and IL‐6 mRNAs increased in the pituitary, spleen and circumventricular organs after single or R‐LPS, suggesting that cytokines may contribute to the activation of the HPA axis though pathways from the circumventricular organs as well as paracrine effects in the pituitary. The data show that (i) adaptation of the HPA axis during repeated LPS injection involves increases in vasopressin : CRH expression ratios in parvocellular neurones; (ii) that hypothalamic CRH and vasopressin responses to acute stimulation are independent of CRH‐R1 expression in the PVN; and (iii) there is a dissociation between pituitary and adrenal responses to acute stress suggesting a decrease of adrenal sensitivity to ACTH.

Hypothalamic and amygdaloid corticotropin-releasing hormone (CRH) and CRH receptor-1 mRNA expression in the stress-hyporesponsive late pregnant and early lactating rat

This study investigated the expression of corticotropin releasing hormone (CRH) and its receptor CRHR-1, and arginine vasopressin (AVP) mRNAs during the stress hyporesponsive periods of late pregnancy and lactation (day-3) and in virgin stress-responsive females. In situ hybridization histochemistry showed that basal CRH mRNA in the paraventricular nucleus (PVN) decreased in pregnant and increased in lactating rats (compared with virgin controls), whereas it increased after restraint stress only in virgin rats. Basal PVN CRHR-1 mRNA increased markedly in all groups but reached lower levels in pregnant rats. Basal AVP mRNA in the parvocellular PVN was higher in lactating rats, and in contrast to CRH mRNA, it increased after stress in all groups. In medial preoptic area (MPOA) CRH mRNA levels were higher in lactating females compared with virgin and pregnant rats, and unexpectedly they decreased markedly after stress only in virgin rats. CRH mRNA levels in the central and medial nuclei of the amygdala were higher in lactating rats than in virgin or pregnant ones, and stress had no effect in either group. These data suggest that these stress hyporesponsive periods: (1) do not depend on basal CRH mRNA expression in the PVN; (2) appear to have intact stress-activated afferent pathways to the PVN, as shown by preservation of CRHR-1 and AVP responses to stress, but the information may be differently processed; (3) are associated with an alteration in a CRH mediated pathway from the MPOA.

Kalirin-7 Is an Essential Component of both Shaft and Spine Excitatory Synapses in Hippocampal Interneurons

Kalirin, a multifunctional Rho GDP/GTP exchange factor, plays a vital role in cytoskeletal organization, affecting process initiation and outgrowth in neurons. Through alternative splicing, the Kalirin gene generates multiple functionally distinct proteins. Kalirin-7 (Kal7) is the most prevalent isoform in the adult rat hippocampus; it terminates with a postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 (PDZ) binding motif, is localized to the postsynaptic density, interacts with PSD-95, and causes the formation of dendritic spines when overexpressed in pyramidal neurons. Levels of Kal7 are low in the dendrites of hippocampal aspiny interneurons. In these interneurons, Kal7 is localized to the postsynaptic side of excitatory synapses onto dendritic shafts, overlapping clusters of PSD-95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD-95-positive, bassoon-positive clusters along the dendritic shaft of hippocampal interneurons. Overexpression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to vesicular glutamate transporter 1-positive, bassoon-positive presynaptic endings; GAD-positive, vesicular GABA transporter-positive inhibitory endings are unaffected. Almost every Kal7-positive dendritic cluster contains PSD-95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules with a level of expression at excitatory synapses that titrates the number of dendritic spines.

Transcriptional responses of the vasopressin and corticotropin-releasing hormone genes to acute and repeated intraperitoneal hypertonic saline injection in rats.

The contribution of corticotropin releasing hormone (CRH) and vasopressin (VP) to the adaptation of ACTH responses to chronic stress was studied by analysis of CRH and VP expression in the hypothalamic paraventricular nucleus (PVN) of rats receiving acute or chronic i.p. hypertonic saline injection (ipHS), a stress model in which the HPA axis is not desensitized after repeated stimulation. Repeated ipHS for 14 days had no effect on CRH hnRNA levels but increased CRH mRNA levels by 42.2%. Parallel with preserved plasma corticosterone responses to repeated ipHS, CRH hnRNA responses and CRH mRNA response to the last injection in repeatedly stressed rats were identical to those in naive rats (8.6-fold increase by 15 min, returning to basal level by 1 h). Parvocellular VP hnRNA responses to a single ipHS were slower and more prolonged than for CRH (7.1-, 11.5-, 9.8- and 4.6-fold by 1, 2, 4 and 6 h), and VP mRNA levels increased by 4 h and remained elevated 12 h later. Parvocellular VP hnRNA was at basal levels after 14 days ipHS, but VP mRNA levels remained elevated as during acute stimulation. Despite high basal mRNA levels, VP hnRNA responses to the last repeated ipHS were minor, suggesting increases in mRNA stability. This study shows that conserved pituitary ACTH responsiveness to a homotypical repeated stress is associated with the ability of parvocellular PVN neurons to increase CRH transcription after repeated stimulation.

Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment.

ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal copper injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A-specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopulations from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopting the cell-specific and developmentally-regulated morphology of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neurodevelopmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease.

Hypothalamic-Pituitary-Adrenal Function

Basal hypothalamic-pituitary-adrenal (HPA) function is characterised by pulses of corticosterone secretion followed by a transient refractory period when the axis appears to be inhibited. In females pulses of corticosterone secretion occur approximately once per hour with variation in pulse amplitude underlying a diurnal rhythm. Males show smaller pulses of secretion which become widely spaced during the early light phase nadir. Pulsatility is altered by genetic programming, early life experiences and reproductive status. Activation of the HPA axis during adjuvant induced arthritis results in an increase in the pulse frequency. This is associated with a marked change in hypothalamic gene expression with a diminution of CRH mRNA and a marked increase of AVP mRNA which becomes the predominant HPA secretagogue.

Behavioral and morphological responses to cocaine require Kalirin7

BACKGROUND Long-lasting increases in dendritic spine density and gene expression in the nucleus accumbens and in the ambulatory response to cocaine occur following chronic cocaine treatment. Despite numerous reports of these findings, the molecular mechanisms leading to these morphological, biochemical, and behavioral changes remain unclear. METHODS We used mice genetically lacking Kalirin7 (Kal7(KO)), a Rho guanine nucleotide exchange factor that regulates dendritic spine formation and function. Both wild-type (Wt) and Kal7(KO) mice were given high-dose cocaine (20 mg/kg) for 4 or 8 consecutive days. Locomotor sensitization and conditioned place preference elicited by cocaine were evaluated. The nucleus accumbens core was diolistically labeled and spine density and morphology were quantified using confocal microscopy. RESULTS Cocaine increased Kalirin7 messenger RNA and protein expression in the nucleus accumbens of Wt mice. The Kal7(KO) animals showed greater locomotor sensitization to cocaine than Wt mice. In contrast, Kal7(KO) mice exhibited decreased place preference for cocaine, despite displaying a normal place preference for food. While Wt mice showed a robust increase in dendritic spine density after 4 and 8 days of cocaine treatment, dendritic spine density failed to increase in cocaine-exposed Kal7(KO) mice. Wild-type mice treated with cocaine for 8 days exhibited larger dendritic spines than cocaine-treated Kal7(KO) mice. CONCLUSIONS Kalirin7 is an essential determinant of dendritic spine formation following cocaine treatment. The absence of this single isoform of one of the many Rho guanine nucleotide exchange factors expressed in the nucleus accumbens results in enhanced locomotor sensitization and diminished place preference in response to cocaine.

Altered ATP7A expression and other compensatory responses in a murine model of Menkes disease.

Mutations in the copper-transporter ATP7A lead to severe neurodegeneration in the mottled brindled hemizygous male (MoBr/y) mouse and human patients with Menkes disease. Our earlier studies demonstrated cell-type- and -stage-specific changes in ATP7A protein expression during postnatal neurodevelopment. Here we examined copper and cuproenzyme levels in MoBr/y mice to search for compensatory responses. While all MoBr/y neocortical subcellular fractions had decreased copper levels, the greatest decrease (8-fold) was observed in cytosol. Immunostaining for ATP7A revealed decreased levels in MoBr/y hippocampal pyramidal and cerebellar Purkinje neurons. In contrast, an upregulation of ATP7A protein occurred in MoBr/y endothelial cells, perhaps to compensate for a lack of copper in the neuropil. MoBr/y astrocytes and microglia increased their physical association with the blood-brain barrier. No alterations in ATP7A levels were observed in ependymal cells, arguing for specificity in the alteration observed at the blood-brain barrier. The decreased expression of ATP7A protein in MoBr/y Purkinje cells was associated with impaired synaptogenesis and dramatic cytoskeletal dysfunction. Immunoblotting failed to reveal any compensatory increase in levels of ATP7B. While total levels of several cuproenzymes (peptide-amidating monooxygenase, SOD1, and SOD3) were unaltered in the MoBr/y brain, levels of amidated cholecystokinin (CCK8) and amidated pituitary adenylate cyclase-activating polypeptide (PACAP38) were reduced in a tissue-specific fashion. The compensatory changes observed in the neurovascular unit provide insight into the success of copper injections within a defined neurodevelopmental period.