Analysis of pyramidal neuron morphology in an inducible knockout of brain-derived neurotrophic factor

doi:10.1016/j.biopsych.2005.01.010

Biological Psychiatry

Volume 57, Issue 8, 15 April 2005, Pages 932-934

https://doi.org/10.1016/j.biopsych.2005.01.010 Get rights and content

Background

The messenger ribonucleic acid (mRNA) for brain-derived neurotrophic factor (BDNF), a regulator of pyramidal neuron dendritic spine density during development, is decreased in the prefrontal cortex of subjects with schizophrenia, and the level of BDNF mRNA expression is positively correlated with dendritic spine density in the same subjects.

Methods

To determine whether reduced BDNF mRNA expression might account for decreased spine density in schizophrenia, a knockout of the BDNF gene was induced in mice during embryogenesis or at 12 weeks of age. Quantitative assessments were made of the dendritic arbor of Golgi-impregnated pyramidal neurons in the prelimbic and anterior cingulate cortices in adulthood.

Results

Despite an 80% reduction in BDNF mRNA levels in both knockouts, neither spine density nor other dendritic or somal measures were decreased compared with wild-type animals.

Conclusions

A reduction in BDNF expression alone does not seem to be sufficient to alter pyramidal neuron morphology in mice. This finding suggests that other molecular abnormalities are also required to produce the pyramidal neuron dendritic spine abnormalities observed in schizophrenia.

Section snippets

Methods and materials

Knockout of the bdnf gene was induced in mice with a combination of the tetracycline-inducible system and the bacteriophage P1-derived Cre/loxP recombination system (Monteggia et al 2004). Specifically, recombination was induced in mice at 12 weeks of age (adult knockout) by removing the suppressor doxycycline from their drinking water. To induce the bdnf knockout during embryogenesis, mice were bred in the absence of doxycycline. Brain-derived neurotrophic factor mRNA expression was regionally

Results

The mean (± SD) percentage of neurons sampled from the prelimbic cortex (as opposed to the anterior cingulate cortex) did not differ [F(2,15) = 2.62, p = .11] among the embryonic (50% ± 24%) and adult (38% ± 11%) knockout animals and the wild-type control animals (27% ± 11%). As shown in Table 1, no differences across the three experimental groups were found for spine density or any of the dendritic measures. The one p value that was less than .05 (dendritic length, branch order 3) reflects a

Discussion

In contrast to our hypothesis, a decrease in BDNF expression alone does not seem to be sufficient to alter basilar dendritic morphology in the prelimbic and anterior cingulate cortices of adult mice, regardless of whether the reduction in BDNF mRNA levels was induced prenatally or in early adulthood. This absence of an effect does not seem to be an artifact of the tissue preparation or experimental measures because the mean values for spine density are consistent with previously reported values

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The molecular and cellular mechanisms underlying the pathogenesis of depression remain unclear. Over the past two decades, studies have highlighted a potential role for neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF), in contributing to the pathophysiology and treatment of depression. Studies indicate opposing effects of stress and antidepressant treatment on BDNF expression in cortical and subcortical brain regions. Stress-evoked reduction of BDNF is implicated in dendritic atrophy, decline in hippocampal neurogenesis, and enhanced depressive-like behavior in animal models. In contrast, antidepressant-induction of BDNF expression is implicated in reversing neuronal atrophy, neurogenic decline, and enhanced despair in animal models of depression. In this chapter, we revisit the neurotrophic hypothesis of depression, examining evidence for, as well as against, the notion that reduced trophic support in key limbic brain regions contributes to the pathogenesis of depression, whereas enhanced trophic factor signaling participates in the therapeutic actions of antidepressants.
Glucocorticoid mechanisms of functional connectivity changes in stress-related neuropsychiatric disorders
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And chronic glucocorticoid exposure suppresses BDNF transcription in the orbitofrontal cortex (Gourley et al., 2009) and reduces TrkB and ERK1/2 signaling in the hippocampus (Gourley et al., 2008). Although studies indicate that reduced activity-dependent BDNF secretion probably does not by itself cause spine loss or dendritic atrophy (Hill et al., 2005; Magarinos et al., 2011), it is likely that altered BDNF signaling plays a role through interactions with other factors. Stress—especially chronic, uncontrollable stress—is an important risk factor for depression, PTSD, and other anxiety disorders, and stress effects on glucocorticoid oscillations may contribute to this effect.
Stress—especially chronic, uncontrollable stress—is an important risk factor for many neuropsychiatric disorders. The underlying mechanisms are complex and multifactorial, but they involve correlated changes in structural and functional measures of neuronal connectivity within cortical microcircuits and across neuroanatomically distributed brain networks. Here, we review evidence from animal models and human neuroimaging studies implicating stress-associated changes in functional connectivity in the pathogenesis of PTSD, depression, and other neuropsychiatric conditions. Changes in fMRI measures of corticocortical connectivity across distributed networks may be caused by specific structural alterations that have been observed in the prefrontal cortex, hippocampus, and other vulnerable brain regions. These effects are mediated in part by glucocorticoids, which are released from the adrenal gland in response to a stressor and also oscillate in synchrony with diurnal rhythms. Recent work indicates that circadian glucocorticoid oscillations act to balance synapse formation and pruning after learning and during development, and chronic stress disrupts this balance. We conclude by considering how disrupted glucocorticoid oscillations may contribute to the pathophysiology of depression and PTSD in vulnerable individuals, and how circadian rhythm disturbances may affect non-psychiatric populations, including frequent travelers, shift workers, and patients undergoing treatment for autoimmune disorders.
Activity-dependent alterations in the sensitivity to BDNF-TrkB signaling may promote excessive dendritic arborization and spinogenesis in fragile X syndrome in order to compensate for compromised postsynaptic activity
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Fragile X syndrome (FXS), the most common cause of inherited human mental retardation, results from the loss of function of fragile X mental retardation protein (FMRP). To date, most researchers have thought that FXS neural pathologies are primarily caused by extreme dendritic branching and spine formation. With this rationale, several researchers attempted to prune dendritic branches and reduce the number of spines in FXS animal models. We propose that increased dendritic arborization and spinogenesis in FXS are developed rather as secondary compensatory responses to counteract the compromised postsynaptic activity during uncontrollable metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD). When postsynaptic and electrical activities become dampened in FXS, dendritic trees can increase their sensitivity to brain-derived neurotrophic factor (BDNF) by using the molecular sensor called eukaryotic elongation factor 2 (eEF2) and taking advantage of the tight coupling of mGluR and BDNF-TrkB signaling pathways. Then, this activity-dependent elevation of the BDNF signaling can strategically alter dendritic morphologies to foster branching and develop spine structures in order to improve the postsynaptic response in FXS. Our model suggests a new therapeutic rationale for FXS: correcting the postsynaptic and electrical activity first, and then repairing structural abnormalities of dendrites. Then, it may be possible to successfully fix the dendritic morphologies without affecting the survival of neurons. Our theory may also be generalized to explain aberrant dendritic structures observed in other neurobehavioral diseases, such as tuberous sclerosis, Rett syndrome, schizophrenia, and channelopathies, which accompany high postsynaptic and electrical activity.
Dynamic plasticity: The role of glucocorticoids, brain-derived neurotrophic factor and other trophic factors
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While BDNF-overexpressing mice had lower basal CORT level, there was no difference in the CORT response to stress across genotypes (Govindarajan et al., 2006). Conversely, selective deletion of BDNF from the forebrain structures resulted in a thinner cortex in adulthood (Gorski et al., 2003b), but no difference in pyramidal cell spine density (Hill et al., 2005). Conditional deletion mice were impaired on some learning tasks and showed deficiencies in long term potentiation (LTP) (Monteggia et al., 2004), but had no change in anxiety-like behaviors (Gorski et al., 2003a).
Brain-derived neurotrophic factor (BDNF) is a secreted protein that has been linked to numerous aspects of plasticity in the central nervous system (CNS). Stress-induced remodeling of the hippocampus, prefrontal cortex and amygdala is coincident with changes in the levels of BDNF, which has been shown to act as a trophic factor facilitating the survival of existing and newly born neurons. Initially, hippocampal atrophy after chronic stress was associated with reduced BDNF, leading to the hypothesis that stress-related learning deficits resulted from suppressed hippocampal neurogenesis. However, recent evidence suggests that BDNF also plays a rapid and essential role in regulating synaptic plasticity, providing another mechanism through which BDNF can modulate learning and memory after a stressful event. Numerous reports have shown BDNF levels are highly dynamic in response to stress, and not only vary across brain regions but also fluctuate rapidly, both immediately after a stressor and over the course of a chronic stress paradigm. Yet, BDNF alone is not sufficient to effect many of the changes observed after stress. Glucocorticoids and other molecules have been shown to act in conjunction with BDNF to facilitate both the morphological and molecular changes that occur, particularly changes in spine density and gene expression. This review briefly summarizes the evidence supporting BDNF’s role as a trophic factor modulating neuronal survival, and will primarily focus on the interactions between BDNF and other systems within the brain to facilitate synaptic plasticity. This growing body of evidence suggests a more nuanced role for BDNF in stress-related learning and memory, where it acts primarily as a facilitator of plasticity and is dependent upon the coactivation of glucocorticoids and other factors as the determinants of the final cellular response.
Factoring neurotrophins into a neurite-based pathophysiological model of schizophrenia
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In contrast to the essential role of NT-3 in forming thalamocortical connections (Ma et al., 2002) and the crucial action of NT-4/5 on dendrite growth in layer VI (Wirth et al., 2003), BDNF, interestingly, does not seem to be indispensable for the early establishment of proper brain connectivity. An 80% reduction in BDNF expression during embryogenesis or adult life does not alter the structure of dendrites, the number of spines, or the neuronal soma size of pyramidal neurons in the prelimbic or anterior cingulate cortices (Hill et al., 2005). Similar findings are evident in layer IV of the somatosensory cortex of transgenic mice carrying a 50% reduction in BDNF protein (Genoud et al., 2004).
Neurotrophins are growth factors that, through variations in concentration and changes in receptor expression, regulate the formation of axons and dendrites during development and throughout adult life. Here we review these growth factors, particularly in the context of schizophrenia, a psychiatric disorder characterized by neurodevelopmental abnormalities. We first discuss emerging information derived from physiologically relevant organotypic cultures and in vivo studies regarding the effects of neurotrophins on the neuronal structure including pruning and GABAergic neurons. We then review postmortem studies of neurotrophin levels and their receptors in brains of individuals with schizophrenia, and compare them with what is known about neurotrophin effects on neuronal structure. This comparison indicates that only some neuropathological defects encountered in patients with schizophrenia can be explained by the single action of neurotrophins on dendrites and axons. However, we propose that a number of inconsistent findings and apparently unrelated results in the schizophrenia field can be reconciled if neurons are considered structurally plastic cells capable of extending and retracting dendrites and axons throughout life.
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BDNF stimulates the growth of dendrites and increases spine density on dendrites of cortical pyramidal neurons (McAllister et al., 1996). The expression of BDNF mRNA and protein is lower in the PFC of subjects with SZ (for a review see Roth et al., 2009) and BDNF levels and spine density on the basilar dendrites of deep layer III pyramidal neurons are positively correlated in SZ patients (Hill et al., 2005). It is important to mention that in SZ patients, reduced BDNF expression in pyramidal neurons is accompanied by a significant decrease in TrkB mRNA levels in GABAergic interneurons (Lewis et al., 2005).
It is becoming increasingly clear that a dysfunction of the GABAergic/glutamatergic network in telencephalic brain structures may be the pathogenetic mechanism underlying psychotic symptoms in schizophrenia (SZ) and bipolar (BP) disorder patients. Data obtained in Costa’s laboratory (1996–2009) suggest that this dysfunction may be mediated primarily by a downregulation in the expression of GABAergic genes (e.g., glutamic acid decarboxylase₆₇ [GAD₆₇] and reelin) associated with DNA methyltransferase (DNMT)-dependent hypermethylation of their promoters.
A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that induce DNA-demethylation when administered at doses that facilitate chromatin remodeling.
The benefits elicited by combining VPA with antipsychotics in the treatment of BP disorder suggest that an investigation of the epigenetic interaction of these drugs is warranted.
Our studies in mice suggest that when associated with VPA, clinically relevant doses of clozapine elicit a synergistic potentiation of VPA-induced GABAergic promoter demethylation. Olanzapine and quetiapine (two clozapine congeners) also facilitate chromatin remodeling but at doses higher than used clinically, whereas haloperidol and risperidone are inactive. Hence, the synergistic potentiation of VPA’s action on chromatin remodeling by clozapine appears to be a unique property of the dibenzepines and is independent of their action on catecholamine or serotonin receptors.
By activating DNA-demethylation, the association of clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be considered a promising treatment strategy for normalizing GABAergic promoter hypermethylation and the GABAergic gene expression downregulation detected in the postmortem brain of SZ and BP disorder patients.
This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

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Brief reportsAnalysis of pyramidal neuron morphology in an inducible knockout of brain-derived neurotrophic factor

Background

Methods

Results

Conclusions

Section snippets

Methods and materials

Results

Discussion

Neuroscience

Brain Res

Neuron

Neuron

Pyramidal cells of the frontal lobeAll the more spinous to think with

J Neurosci

Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia

J Neurol Neurosurg Psychiatry

Altered synapse formation in the adult somatosensory cortex of brain-derived neurotrophic factor heterozygote mice

J Neurosci

Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia

Arch Gen Psychiatry

Relationship of brain-derived neurotrophic factor and its receptor trkB to altered inhibitory prefrontal circuitry in schizophrenia

J Neurosci

Brief reports
Analysis of pyramidal neuron morphology in an inducible knockout of brain-derived neurotrophic factor