Anxiolytic reversal of classically conditioned / chronic stress-induced gene expression and learning in the Stress Alternatives Model.

Social decision-making is critically influenced by neurocircuitries that regulate stress responsiveness. Adaptive choices, therefore, are altered by stress-related neuromodulatory peptide systems, such as corticotropin releasing factor (CRF). Experimental designs that take advantage of ecologically salient fear-inducing stimuli allow for revelation of neural mechanisms that regulate the balance between pro- and anti-stress responsiveness. To accomplish this, we developed a social stress and conditioning protocol, the Stress Alternatives Model (SAM), that utilizes a simple dichotomous choice, and produces distinctive behavioral phenotypes (Escape or Stay). The experiments involve repeated social aggression, a potent unconditioned stimulus (US), from a novel larger conspecific (a 3X larger Rainbow trout). Prior to the social interaction, the smaller test fish is presented with an auditory conditioning stimulus (water off = CS). During the social aggression, an escape route is available, but is only large enough for the smaller test animal. Surprisingly, although the new aggressor provides vigorous attacks each day, only 50% of the test fish choose Escape. Stay fish, treated with the CRF1 antagonist antalarmin, a potent anxiolytic drug, on day 4, promotes Escape behavior for the last 4 days of the SAM protocol. The results suggest that the decision to Escape, required a reduction in stress reactivity. The Stay fish that chose Escape following anxiolytic treatment, learned how to use the escape route prior to stress reduction, as the Escape latency in these fish was significantly faster than first time escapers. In Escape fish, the use of the escape route is learned over several days, reducing the Escape latency over time in the SAM. Fear conditioning (water off + aggression) resulted in elevated hippocampal (DL) Bdnf mRNA levels, with coincident reduction in the AMPA receptor subunit Glua1 expression, a result that is reversed following a one-time treatment (during SAM aggression on day 4) with the anxiolytic CRF1 receptor antagonist antalarmin.

Cleavage of proBDNF to BDNF by a tolloid-like metalloproteinase is required for acquisition of in vitro eyeblink classical conditioning.

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Activation of mammalian Tolloid-like 1 expression by hypoxia in human neuroblastoma SH-SY5Y cells.

Mammalian Tolloid-like 1 (mTll-1) is an astacin metalloprotease that is a member of the Tolloid family of proteins. mTll-1 cleaves chordin, an inhibitor of bone morphogenetic proteins (BMPs) and potentiates activity of the BMPs. Prenatal stress and glucocorticoids decrease mTll-1 expression whereas voluntary exercise increase mTll-1 gene expression in the mouse hippocampus. Here, we studied the underlying molecular mechanisms by which hypoxia regulates human mTll-1 gene expression. When cells were subjected to hypoxia, the expression of endogenous mTll-1 was upregulated in SH-SY5Y human neuroblastoma cells. Dual-luciferase assay and site-directed mutagenesis showed the presence of hypoxia responsive elements (HREs) at position 625 that was essential for activation of mTll-1 expression under hypoxic conditions. The binding of hypoxia-inducible factor (HIF-1) protein to the HREs was confirmed by gel shift assay. These results indicate that the HRE motif is directly involved in the activation of the mTll-1 transcription under hypoxic conditions.

Activation of Tolloid-like 1 gene expression by the cardiac specific homeobox gene Nkx2-5.

Mammalian Tolloid-like 1 (Tll-1) is a pleiotropic metalloprotease that is expressed by a small subset of cells within the precardiac mesoderm and is necessary for proper heart development. Following heart tube formation Tll-1 is expressed by the endocardium and regions of myocardium overlying the region of the muscular interventricular septum. Mutations in Tll-1 lead to embryonic lethality due to cardiac defects. We demonstrate that the Tll-1promoter contains Nkx2-5 binding sites and that the Tll-1 promoter is activated by and directly binds Nkx2-5.Tll-1 expression is ablated by a dominant negative Nkx2-5 or by mutation of the Nkx2-5 binding sites within theTll-1 promoter. In vivo, Tll-1 expression is decreased in the hearts of Nkx2-5 knockout embryos when compared with hemizygous and wild-type embryos. These results show that Nkx2-5 is a direct activator of Tll-1 expression and provide insight into the mechanism of the defects found in both the Tll-1 and Nkx2-5 knockout mice.

Seasonal variation of myostatin gene expression in pectoralis muscle of house sparrows (Passer domesticus) is consistent with a role in regulating thermogenic capacity and cold tolerance.

Winter acclimatization in small birds overwintering in cold climates, including house sparrows (Passer domesticus), is associated with improved cold tolerance, elevated summit metabolic rates (M(sum) = maximum cold-induced metabolic rate), and increased pectoralis muscle mass compared to summer birds. Myostatin is a potent autocrine/paracrine inhibitor of skeletal muscle growth in mammals and birds and is a potential candidate for regulation of seasonal phenotypic flexibility in birds. As a first step toward examining such a role for myostatin in small birds, we measured summer and winter gene expression of myostatin and its potential metalloproteinase activators TLL-1 and TLL-2 in house sparrows from southeastern South Dakota. Gene expression of myostatin decreased significantly in winter, with summer values exceeding winter values by 1.52-fold. Moreover, gene expression of TLL-1 was also significantly reduced in winter, with summer values exceeding winter values by 1.55-fold. These data are consistent with the hypothesis that the winter increases in pectoralis muscle mass, M(sum), and cold tolerance in house sparrows are mediated by reduced levels of myostatin and its activator TLL-1, and they suggest the possibility that myostatin may be a common mediator of phenotypic flexibility of muscle mass in birds.

Characterization of a novel reptilian tolloid-like gene in the pond turtle, Pseudemys scripta elegans.

The Tolloid metalloproteases are pleiotropic enzymes that are important for many developmental processes. This study describes the isolation and characterization of a novel Tolloid family member from the pond turtle Pseudemys scripta elegans. The turtle Tolloid, designated tTll, is found in two forms. The first, tTlls, contains a signal sequence which may provide a mechanism for secretion. The second, tTllc, does not contain a signal sequence and is likely cytoplasmic. Sequence analysis of tTll revealed that it is most closely related to chicken Tll-2 although tTll domain structure is different. We examined the expression of tTll mRNA by real-time RT-PCR and found the highest expression in the cerebellum with lower levels in the brain stem and cortex. This expression pattern is similar to the expression of the Tolloid mouse orthologues Tll-1 and Tll-2 with highest levels of expression in the cerebellum and lower levels in the brain stem and cortex.

Tolloid-like 1 is negatively regulated by stress and glucocorticoids.

Glucocorticoids affect a variety of tissues to enable the organism to adapt to the stress. Hippocampal neurons contain glucocorticoid receptors and respond to elevated glucocorticoid levels by down-regulating the HPA axis. Chronically, however, stress is deleterious to hippocampal neurons. Chronically elevated levels of glucocorticoids result in a decrease in the number of dendritic spines, reduced axonal growth and synaptogenesis, and decreased neurogenesis in the hippocampus. Tolloid-like 1 (Tll-1) is a metalloprotease that potentiates the activity of the bone morphogenetic proteins (BMPs). Neurogenesis in the hippocampus of both developing and adult mammals requires BMPs. In this study, we demonstrate that Tll-1 expression is increased in mice that have increased neurogenesis. The Tll-1 promoter contains glucocorticoid response elements which are capable of binding to purified glucocorticoid receptor. Glucocorticoids decrease Tll-1 expression in vitro. Finally, prenatal stress leads to a decrease in Tll-1 mRNA expression in the hippocampus of adult female mice that is not observed in adult male mice indicating that Tll-1 expression is differentially regulated in males and females. The results of this study indicate that Tll-1 is responsive to glucocorticoids and this mechanism might influence neurogenesis in the hippocampus.

Abducens conditioning in in vitro turtle brain stem without cerebellum requires NMDA receptors and involves upregulation of GluR4-containing AMPA receptors.

Previous work showed that in vitro abducens eyeblink classical conditioning of turtle brain stem-cerebellum preparations involved NMDA-mediated mechanisms and redistribution of GluR4-containing AMPA receptors in the abducens motor nuclei. Since conditioning can be obtained in brain stem preparations without the cerebellum, we examined whether similar mechanisms were involved during conditioning of the brain stem alone. The results showed that conditioning could not be induced in the presence of the NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP-5) and that abducens nerve conditioned responses, once initiated in normal saline, were significantly attenuated in the presence of AP-5. The effects of AP-5 did not generally depress physiological responsiveness of preparations because some abducens nerve reflexes were not significantly reduced by the compound. GluR4-containing AMPA receptors in the abducens motor nuclei were significantly upregulated and positively correlated with the levels of conditioning similar to that of preparations having an intact cerebellum. Furthermore, increased GluR4 subunits after brain stem conditioning was confirmed by Western blot analysis. These results suggest that NMDA receptor-mediated mechanisms and GluR4 upregulation may mediate in vitro abducens eyeblink classical conditioning and that these mechanisms reside in the brain stem eyeblink circuitry.

Role for calbindin-D28K in in vitro classical conditioning of abducens nerve responses in turtles.

Intracellular calcium has a pivotal role in synaptic modifications that may underlie learning and memory. The present study examined whether there were changes in immunoreactivity levels of the AMPA receptor subunits GluR2/3 and calcium binding proteins during classical conditioning recorded in the abducens nerve of in vitro brain stem preparations from turtles. The results showed that abducens motor neurons in unconditioned turtle brain stems were immunopositive for GluR2/3, calbindin-D28K, and calmodulin, but were immunonegative for parvalbumin. After classical conditioning, immunoreactivity for calbindin-D28K in the abducens motor nuclei was significantly reduced, whereas there were no significant changes in GluR2/3, calmodulin, or parvalbumin. This reduction in calbindin-D28K immunoreactivity was not observed following conditioning in the NMDA receptor antagonist AP-5, which blocked conditioned responses, suggesting that these changes are NMDA receptor-dependent. Moreover, the degree of the decrease in calbindin-D28K immunoreactivity was negatively correlated with the level of conditioning. Consistent with the immunocytochemical findings, Western blot analysis showed that calbindin-D28K protein levels were reduced after classical conditioning. The results support the hypothesis that in vitro classical conditioning of abducens nerve responses utilizes intracellular calcium-dependent signaling pathways that require NMDA receptor function and suggest a specific role for the calcium binding protein calbindin-D28K.