The Baram Lab had an excellent time at the 2008 Society for Neuroscience Annual Meeting. In addition to the research posters presented by lab members, Dr. Baram presented findings on stress-induced dendritic spine loss at a press conference which included media such as Scientific American and Science News.
Activity-Dependent Increase of NRSF / REST levels suppresses expression of the hyperpolarization activated cyclic-nucleotide gated (HCN) channel.
S McClelland, Q Zha, C Richichi, CM Dubé, TZ Baram
Abstract (click to expand)
Rationale: Altered expression of the HCN channels contributes to activity-dependent changes in Ih, which alter the firing properties and network response of hippocampal neurons. Indeed, seizure-activity in several models leads to depressed mRNA and protein levels of HCN channels type 1 (HCN1). Neuron-restrictive silencing factor (NRSF or REST) is a transcription factor that suppresses gene expression in a variety of cells, and the HCN1 gene contains several possible NRSF binding sites (NRSEs). Here we tested the possibility that NRSF mediates activity-dependent suppression of HCN1 expression.
Methods: Organotypic hippocampal slice cultures were used to study HCN1 and NRSF/REST in vitro. Whereas direct injection of kainic acid into immature and adult rats was used for in vivo studies. Analysis of both sources of tissue included Western blot, in situ hybridization, and chromatin immunoprecipitation (ChIP).
Results: Seizure-like activity reduced HCN1 expression both in vivo ( in rats) and in organotypic hippocampal slice cultures. In contrast to HCN1, NRSF mRNA and protein levels increased in the in vivo and in vitro hippocampus. The seizure-induced reduction of HCN1 expression was abrogated by treatment with decoy NRSE oligomers, designed to bind and ‘trap’ all of the nuclear NRSF, preventing it from binding the NRSE located on the HCN1 gene. Thus, NRSE oligomers applied to KA exposed (‘seizure-experiencing’) organotypic cultures or infused ICV to adult rats exposed to KA, abolished seizure-provoked reduction of HCN1 expression. The specific binding site of NRSF to the HCN1 gene regulatory-region was elucidated using chromatin immunoprecipitation (ChIP) using antibodies against NRSF. Furthermore, ChIP using antibodies against dimethylated H3K9 histone demonstrated that the histones near the HCN1 gene regulatory region are more methylated or ‘closed’ to transcription.
Conclusions: NRSF contributes critically to activity dependent repression of HCN1 expression. This transcription factor may be involved in the regulation of other crucial genes that, together with HCN1, alter neuronal function after seizure-activity.
Dynamic trafficking of HCN1 channels in hippocampal neurons: live-imaging studies
Y Noam, Q Zha, RL Wu, L Phan, DM Chetkovich, WJ Wadman, TZ Baram
Abstract (click to expand)
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are non-selective cation channels that play several important roles in regulation of neuronal excitability. The effect s of these channels on neuronal excitability depend on their sub-cellular location (Santoro & Baram, 2003). The sub-cellular distribution of HCN channels, and particularly of HCN1, is inhomogeneous: a strong dendritic gradient occurs in cortical and hippocampal pyramidal cells (Magee 1998; Lorincz et al. 2002) and axonal expression of HCN1 has been described in interneurons (Aponte et al. 2006) and during development (Bender et al. 2007). Despite the physiological importance of the channels, little is known about the molecular and cellular mechanisms of their localization to different cell compartments. Previously, we have shown that transfection of GFP-tagged HCN1 channels in cultured neurons is feasible, and results in functional channels with similar properties to the wild-type channel (Noam et al. 2007). Here, we applied time-lapse live-imaging techniques to directly observe and characterize several aspects of channel trafficking. HCN1-GFP containing puncta along dendrites possessed variable degrees of motility: approximately 30% of detected puncta were motile, and moved at velocities as high as 0.5 micron/sec. In addition, whereas most of the motile puncta moved bi-directionally in restricted segments of the dendrite, a sub-population (~10% ) of mobile puncta was characterized by high velocity and long-distance travel almost exclusively in a centripetal direction. Finally, increased excitatory input (induced by glutamate application) extensively influenced the trafficking of the puncta already within minutes of the stimulus. Taken together, these data demonstrate for the first time a direct visualization of dendritic trafficking of the HCN1 channel in live neurons. This approach may help elucidate the rapid changes in the HCN-channel mediated current, Ih, that is provoked by normal and pathological neuronal network activity.
Stress induces dendritic spine loss within hours in adult hippocampus: novel mechanisms
Y Chen, CM Dubé, CJ Rice, A Korosi, A Ivy, TZ Baram
Abstract (click to expand)
Rationale: Chronic stress leads to dendritic remodeling in the hippocampus, and this is likely a result of loss of dendritic spines. However, whether acute stress provokes loss of spines in hippocampal pyramidal cells has not been fully investigated. In addition, the nature of the mechanisms underlying spine and dendritic reduction with stress is unclear. Corticotropin-releasing hormone (CRH) is released in hippocampus by stress, and activates the CRH receptor CRFR1 on pyramidal neurons (Chen et al., 2004, 2006). CRH exposure leads to a rapid spine loss in hippocampal organotypic cultures, which is blocked by a selective CRFR1 antagonist (Chen et al., 2008). Here, we tested whether exposure of adult rodents to acute stress induces loss of dendritic spines, as well as the functional consequences.
Methods: Adult Thy1-YFP transgenic mice were exposed to a 5 hrs restraint and noise stress. At the end of this stress experience, spine densities on apical dendrites of CA3 pyramidal cells were compared among the following groups: (1) stressed, (2) stress-free controls, (3) pre-infused (icv) with the CRFR1 antagonist NBI 30775 (15 ?g in 1 ?l) 30 min prior to the 5 hr stress, and (4) stress-free and antagonist-infused.
Results: Stress induced a rapid reduction of spine density in apical dendrites of CA3 neurons, primarily on the 3rd and 4th order dendritic branches, the main postsynaptic target of excitatory commissural/associational fibers. The selective CRFR1 antagonist, abolished the stress-induced decline of spine density. In further support of a role for CRH in rapid spine loss after stress, two-photon live imaging demonstrated an increased spine retraction within minutes after the infusion of the peptide to organotypic cultures of hippocampus.
Conclusions: (1) Acute stress induces a rapid (within hours) spine loss in selective dendritic domains of adult hippocampus; (2) The activation of CRFR1, most likely by its endogenous ligand CRH, is involved in this process; and (3) CRH may promote spine loss via selective acceleration of spine retraction.
Dysfunctional nurturing behavior in rat dams with limited access to nesting material: a clinically relevant model for early-life stress.
AS Ivy, KL Brunson, K Fok, C Sandman, TZ Baram
Abstract (click to expand)
Background: Early-life emotional stress may be associated with affective and cognitive disorders later in life, yet satisfactory animal models for studying the underlying mechanisms are limited. Because maternal presence and behavior critically influence molecular and behavioral stress responses in offspring, we sought to create a model of dysfunctional, fragmented maternal nurturing behavior that would, in turn, provoke chronic early-life stress in the offspring.
Methods: Dams’ nursing and nurturing behaviors were altered by limiting their ability to create satisfactory nests during postpartum days 2-9. Maternal behavior was observed throughout the diurnal cycle, and the frequency and duration of nurturing behaviors were scored. In addition, potential stress and anxiety of the dams were assessed using behavioral, molecular and hormonal measures.
Results: Both the quantity and the quality of dams’ care of their pups were profoundly influenced by restriction of nesting materials in their cages: licking/grooming activities decreased and the frequency of leaving the pups increased, resulting in fragmented interactions between the dams and pups. The abnormal activity patterns of the dams were accompanied by increased anxiety-like behavior in the open field, but not in the elevated plus maze tests. Additionally, dams’ plasma corticosterone levels and adrenal weights were augmented, suggesting chronic stress of these dams. By the end of the limited-nesting, stress-inducing period, hypothalamic corticotropin releasing hormone (CRH) mRNA expression was reduced in the limited-nesting dams, while arginine-vasopressin (AVP) mRNA levels were not significantly affected.
Conclusion: Limiting dams’ ability to construct a nest for their pups leads to an abnormal repertoire of nurturing behaviors, possibly as a result of chronic stress and mild anxiety of the dams. Because the fragmented and aberrant maternal behavior provoked chronic stress in the pups, the limited-nesting paradigm provides a useful tool for studying the mechanisms and consequences of such early-life stress experience in the offspring.
Re-wiring of CRH-expressing neurons in the PVN is involved in the neuroplasticity of the HPA axis after early-life handling
A Korosi, M Shanabrough, ZW Liu, E Borok, H Duenas, L Phan, XB Gao, TL Horvath, TZ Baram
Abstract (click to expand)
Rationale: The ‘tone’ (reactivity) of the HPA system contributes to an individual’s stress responses and vulnerability to depression and premature cognitive decline. In rat models, early-life experience, including bursts of maternal care, induces enduring neuroplasticity in this system, reducing corticotropin releasing factor (CRH) expression in the paraventricular nucleus of the hypothalamus (PVN), increasing hippocampal glucocorticoid receptor (GR) expression and downregulating hormonal stress responses. Increased maternal care provides sensory signals that reduce CRH mRNA expression in PVN of offspring already on P9, as found using the handling paradigm (Fenoglio J Neurosci, 2006). However, the mechanisms by which augmented maternal care is converted to reduced CRH gene expression in PVN neurons remain unclear. Here we tested the hypothesis that modified innervation of CRH-PVN neurons was responsible for reduced CRH expression.
Methods: Pups were handled (15 min separation of pups from the dams) daily from P2-P8, and sacrificed on P9. Levels of vGAT (inhibitory terminal marker) and vGlut2 (excitatory terminals marker) were quantified using Western blots. Symmetric (inhibitory) and asymmetric (excitatory) synapses on CRH-immunoreactive neurons were quantified using electron microscopy (EM). Functional alterations of excitatory and inhibitory input onto CRH neurons were assessed by electrophysiology.
Results: vGlut2 protein levels were significantly reduced (by 42%) in anterior hypothalamus of handled rats compared to undisturbed cohorts. Numbers of asymmetric synapses on PVN-CRH immunolabeled cells, assessed by EM, was reduced by 70% in handled vs undisturbed groups. The number of miniature excitatory postsynaptic currents (mEPSCs) onto parvocellular PVN cells from handled pups was reduced in preliminary studies. Changes in inhibitory synapses and markers (vGAT) were variable.
Conclusion: The reduction of CRH expression in PVN of rats experiencing bursts of maternal care (handled) may derive from reduced excitatory signals to CRH neurons, which may attenuate intracellular signaling cascades governing CRH expression in these neurons. Because neuroplasticity of CRH and GR expression culminates in the ‘handled’ phenotype conferring cognitive and emotional resilience, understanding its underlying mechanisms will provide valuable insights into this important plasticity.
A novel mouse model for acute and long-lasting consequences of early life stress
CJ Rice, CA Sandman,Y Chen, TZ Baram
Abstract (click to expand)
Chronic early life stress (ES) exerts profound acute and long-lasting effects on the hypothalamic pituitary adrenal system, with relevance to cognitive function and affective disorders. Our ability to determine the molecular mechanisms underlying these effects should benefit greatly from appropriate mouse models, because these would enable utilization of powerful transgenic methods. Therefore, we have characterized a mouse model of chronic ES, that was provoked in mouse pups by abnormal, fragmented, interactions with the dam. Dam-pup interaction was disrupted by limiting the nesting and bedding material in the cages, a manipulation that affected this parameter in a dose-dependent manner. At the end of their week-long rearing in the limited-nesting cages, mouse pups were stressed, as apparent from elevated basal plasma corticosterone levels. In addition, steady-state mRNA levels of corticotropin releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) of ES-experiencing pups were reduced, without significant change in mRNA levels of arginine vasopressin.
Rearing mouse pups in this stress-provoking cage environment resulted in enduring effects: Basal plasma corticosterone levels were still increased and CRH mRNA levels in PVN remained reduced in adult ES mice compared with those of controls. In addition, hippocampus-dependent learning and memory functions were impaired in 4-8 month old ES mice. In summary, this novel, robust model of chronic early life stress in the mouse results in acute and enduring neuroendocrine and cognitive abnormalities. This model should facilitate the examination of the specific genes and molecules involved in the generation of this stress, as well as in its consequences.