Developmental Exposure to Polychlorinated Biphenyls Prevents Recovery from Noise-Induced Hearing Loss and Disrupts the Functional Organization of the Inferior Colliculus.

Exposure to combinations of environmental toxins is growing in prevalence; and therefore, understanding their interactions is of increasing societal importance. Here, we examined the mechanisms by which two environmental toxins, polychlorinated biphenyls (PCBs) and high-amplitude acoustic noise, interact to produce dysfunction in central auditory processing. PCBs are well established to impose negative developmental impacts on hearing. However, it is not known whether developmental exposure to this ototoxin alters the sensitivity to other ototoxic exposures later in life. Here, male mice were exposed to PCBs in utero, and later as adults were exposed to 45 min of high-intensity noise. We then examined the impacts of the two exposures on hearing and the organization of the auditory midbrain using two-photon imaging and analysis of the expression of mediators of oxidative stress. We observed that developmental exposure to PCBs blocked hearing recovery from acoustic trauma. In vivo two-photon imaging of the inferior colliculus (IC) revealed that this lack of recovery was associated with disruption of the tonotopic organization and reduction of inhibition in the auditory midbrain. In addition, expression analysis in the inferior colliculus revealed that reduced GABAergic inhibition was more prominent in animals with a lower capacity to mitigate oxidative stress. These data suggest that combined PCBs and noise exposure act nonlinearly to damage hearing and that this damage is associated with synaptic reorganization, and reduced capacity to limit oxidative stress. In addition, this work provides a new paradigm by which to understand nonlinear interactions between combinations of environmental toxins.SIGNIFICANCE STATEMENT Exposure to common environmental toxins is a large and growing problem in the population. This work provides a new mechanistic understanding of how the prenatal and postnatal developmental changes induced by polychlorinated biphenyls (PCBs) could negatively impact the resilience of the brain to noise-induced hearing loss (NIHL) later in adulthood. The use of state-of-the-art tools, including in vivo multiphoton microscopy of the midbrain helped in identifying the long-term central changes in the auditory system after the peripheral hearing damage induced by such environmental toxins. In addition, the novel combination of methods employed in this study will lead to additional advances in our understanding of mechanisms of central hearing loss in other contexts.

Developmental exposure to polychlorinated biphenyls prevents recovery from noise-induced hearing loss and disrupts the functional organization of the inferior colliculus.

Exposure to combinations of environmental toxins is growing in prevalence, and therefore understanding their interactions is of increasing societal importance. Here, we examined the mechanisms by which two environmental toxins - polychlorinated biphenyls (PCBs) and high-amplitude acoustic noise - interact to produce dysfunction in central auditory processing. PCBs are well-established to impose negative developmental impacts on hearing. However, it is not known if developmental exposure to this ototoxin alters the sensitivity to other ototoxic exposures later in life. Here, male mice were exposed to PCBs in utero, and later as adults were exposed to 45 minutes of high-intensity noise. We then examined the impacts of the two exposures on hearing and the organization of the auditory midbrain using two-photon imaging and analysis of the expression of mediators of oxidative stress. We observed that developmental exposure to PCBs blocked hearing recovery from acoustic trauma. In vivo two-photon imaging of the inferior colliculus revealed that this lack of recovery was associated with disruption of the tonotopic organization and reduction of inhibition in the auditory midbrain. In addition, expression analysis in the inferior colliculus revealed that reduced GABAergic inhibition was more prominent in animals with a lower capacity to mitigate oxidative stress. These data suggest that combined PCBs and noise exposure act nonlinearly to damage hearing and that this damage is associated with synaptic reorganization, and reduced capacity to limit oxidative stress. In addition, this work provides a new paradigm by which to understand nonlinear interactions between combinations of environmental toxins. Significance statement: Exposure to common environmental toxins is a large and growing problem in the population. This work provides a new mechanistic understanding of how the pre-and postnatal developmental changes induced by polychlorinated biphenyls could negatively impact the resilience of the brain to noise-induced hearing loss later in adulthood. The use of state-of-the-art tools, including in vivo multiphoton microscopy of the midbrain helped in identifying the long-term central changes in the auditory system after the peripheral hearing damage induced by such environmental toxins. In addition, the novel combination of methods employed in this study will lead to additional advances in our understanding of mechanisms of central hearing loss in other contexts.

V-NeuroStack: Open-source 3D time stack software for identifying patterns in neuronal data.

Understanding functional correlations between the activities of neuron populations is vital for the analysis of neuronal networks. Analyzing large-scale neuroimaging data obtained from hundreds of neurons simultaneously poses significant visualization challenges. We developed V-NeuroStack, a novel network visualization tool to visualize data obtained using calcium imaging of spontaneous activity of neurons in a mouse brain slice as well as in vivo using two-photon imaging. V-NeuroStack creates 3D time stacks by stacking 2D time frames for a time-series dataset. It provides a web interface to explore and analyze data using both 3D and 2D visualization techniques. Previous attempts to analyze such data have been limited by the tools available to visualize large numbers of correlated activity traces. V-NeuroStack's 3D view is used to explore patterns in dynamic large-scale correlations between neurons over time. The 2D view is used to examine any timestep of interest in greater detail. Furthermore, a dual-line graph provides the ability to explore the raw and first-derivative values of activity from an individual or a functional cluster of neurons. V-NeuroStack can scale to datasets with at least a few thousand temporal snapshots. It can potentially support future advancements in in vitro and in vivo data capturing techniques to bring forth novel hypotheses by allowing unambiguous visualization of massive patterns in neuronal activity data.

Patterns of Unilateral and Bilateral Projections From Layers 5 and 6 of the Auditory Cortex to the Inferior Colliculus in Mouse.

The auditory cortex sends massive projections to the inferior colliculus, but the organization of this pathway is not yet well understood. Previous work has shown that the corticocollicular projection emanates from both layers 5 and 6 of the auditory cortex and that neurons in these layers have different morphological and physiological properties. It is not yet known in the mouse if both layer 5 and layer 6 project bilaterally, nor is it known if the projection patterns differ based on projection location. Using targeted injections of Fluorogold into either the lateral cortex or dorsal cortex of the inferior colliculus, we quantified retrogradely labeled neurons in both the left and right lemniscal regions of the auditory cortex, as delineated using parvalbumin immunostaining. After dorsal cortex injections, we observed that approximately 18-20% of labeled cells were in layer 6 and that this proportion was similar bilaterally. After lateral cortex injections, only ipsilateral cells were observed in the auditory cortex, and they were found in both layer 5 and layer 6. The ratio of layer 5:layer 6 cells after lateral cortex injection was similar to that seen after dorsal cortex injection. Finally, injections of different tracers were made into the two inferior colliculi, and an average of 15-17% of cells in the auditory cortex were double-labeled, and these proportions were similar in layers 5 and 6. These data suggest that (1) only the dorsal cortex of the inferior colliculus receives bilateral projections from the auditory cortex, (2) both the dorsal and lateral cortex of the inferior colliculus receive similar layer 5 and layer 6 auditory cortical input, and (3) a subpopulation of individual neurons in both layers 5 and 6 branch to innervate both dorsal cortices of the inferior colliculus.

Evidence for Layer-Specific Connectional Heterogeneity in the Mouse Auditory Corticocollicular System.

The auditory cortex (AC) sends long-range projections to virtually all subcortical auditory structures. One of the largest and most complex of these-the projection between AC and inferior colliculus (IC; the corticocollicular pathway)-originates from layer 5 and deep layer 6. Though previous work has shown that these two corticocollicular projection systems have different physiological properties and network connectivities, their functional organization is poorly understood. Here, using a combination of traditional and viral tracers combined with in vivo imaging in both sexes of the mouse, we observed that layer 5 and layer 6 corticocollicular neurons differ in their areas of origin and termination patterns. Layer 5 corticocollicular neurons are concentrated in primary AC, while layer 6 corticocollicular neurons emanate from broad auditory and limbic areas in the temporal cortex. In addition, layer 5 sends dense projections of both small and large (>1 µm2 area) terminals to all regions of nonlemniscal IC, while layer 6 sends small terminals to the most superficial 50-100 µm of the IC. These findings suggest that layer 5 and 6 corticocollicular projections are optimized to play distinct roles in corticofugal modulation. Layer 5 neurons provide strong, rapid, and unimodal feedback to the nonlemniscal IC, while layer 6 neurons provide heteromodal and limbic modulation diffusely to the nonlemniscal IC. Such organizational diversity in the corticocollicular pathway may help to explain the heterogeneous effects of corticocollicular manipulations and, given similar diversity in corticothalamic pathways, may be a general principle in top-down modulation.SIGNIFICANCE STATEMENT We demonstrate that a major descending system in the brain is actually two systems. That is, the auditory corticocollicular projection, which exerts considerable influence over the midbrain, comprises two projections: one from layer 5 and the other from layer 6. The layer 6 projection is diffusely organized, receives multisensory inputs, and ends in small terminals; while the layer 5 projection is derived from a circumscribed auditory cortical area and ends in large terminals. These data suggest that the varied effects of cortical manipulations on the midbrain may be related to effects on two disparate systems. These findings have broader implications because other descending systems derive from two layers. Therefore, a duplex organization may be a common motif in descending control.

Corticothalamic gating of population auditory thalamocortical transmission in mouse.

The mechanisms that govern thalamocortical transmission are poorly understood. Recent data have shown that sensory stimuli elicit activity in ensembles of cortical neurons that recapitulate stereotyped spontaneous activity patterns. Here, we elucidate a possible mechanism by which gating of patterned population cortical activity occurs. In this study, sensory-evoked all-or-none cortical population responses were observed in the mouse auditory cortex in vivo and similar stochastic cortical responses were observed in a colliculo-thalamocortical brain slice preparation. Cortical responses were associated with decreases in auditory thalamic synaptic inhibition and increases in thalamic synchrony. Silencing of corticothalamic neurons in layer 6 (but not layer 5) or the thalamic reticular nucleus linearized the cortical responses, suggesting that layer 6 corticothalamic feedback via the thalamic reticular nucleus was responsible for gating stochastic cortical population responses. These data implicate a corticothalamic-thalamic reticular nucleus circuit that modifies thalamic neuronal synchronization to recruit populations of cortical neurons for sensory representations.

Aging and Central Auditory Disinhibition: Is It a Reflection of Homeostatic Downregulation or Metabolic Vulnerability?

Aging-related changes have been identified at virtually every level of the central auditory system. One of the most common findings across these nuclei is a loss of synaptic inhibition with aging, which has been proposed to be at the heart of several aging-related changes in auditory cognition, including diminished speech perception in complex environments and the presence of tinnitus. Some authors have speculated that downregulation of synaptic inhibition is a consequence of peripheral deafferentation and therefore is a homeostatic mechanism to restore excitatory/inhibitory balance. As such, disinhibition would represent a form of maladaptive plasticity. However, clinical data suggest that deafferentation-related disinhibition tends to occur primarily in the aged brain. Therefore, aging-related disinhibition may, in part, be related to the high metabolic demands of inhibitory neurons relative to their excitatory counterparts. These findings suggest that both deafferentation-related maladaptive plastic changes and aging-related metabolic factors combine to produce changes in central auditory function. Here, we explore the arguments that downregulation of inhibition may be due to homeostatic responses to diminished afferent input vs. metabolic vulnerability of inhibitory neurons in the aged brain. Understanding the relative importance of these mechanisms will be critical for the development of treatments for the underlying causes of aging-related central disinhibition.

Visual hallucinations, thalamocortical physiology and Lewy body disease: A review.

One of the core diagnostic criteria for Dementia with Lewy Bodies (DLB) is the presence of visual hallucinations. The presence of hallucinations, along with fluctuations in the level of arousal and sleep disturbance, point to potential pathological mechanisms at the level of the thalamus. However, the potential role of thalamic dysfunction in DLB, particularly as it relates to the presence of formed visual hallucinations is not known. Here, we review the literature on the pathophysiology of DLB with respect to modern theories of thalamocortical function and attempt to derive an understanding of how such hallucinations arise. Based on the available literature, we propose that combined thalamic-thalamic reticular nucleus and thalamocortical pathology may explain the phenomenology of visual hallucinations in DLB. In particular, diminished α7 cholinergic activity in the thalamic reticular nucleus may critically disinhibit thalamocortical activity. Further, concentrated pathological changes within the posterior regions of the thalamus may explain the predilection for the hallucinations to be visual in nature.

γ-Tocotrienol Suppression of the Warburg Effect Is Mediated by AMPK Activation in Human Breast Cancer Cells.

Cancer cell metabolism is characterized by aerobic glycolysis or the "Warburg effect". Enhanced Akt signaling is associated with activation of various downstream enzymes involved in the glycolytic process, whereas activation of 5'-AMP-activated kinase (AMPK) acts to terminate energy expending mechanisms and decrease glycolytic enzyme expression. Studies were conducted to determine if the anticancer effects of γ-tocotrienol, are mediated through a suppression in aerobic glycolysis. Results show that treatment with 0-7 µM γ-tocotrienol throughout a 4-day culture period resulted in a dose-responsive increase in AMPK activation, and corresponding decrease in Akt activity in human MCF-7 and MDA-MB-231 breast cancer cells. γ-Tocotrienol treatment was also found to induce a dose-responsive decrease in phosphorylated-Fox03 (inactivated), a transcription factor that acts to inhibit in the levels of glycolytic enzyme, and this decrease was associated with a reduction in glycolytic enzyme levels and activity, as well as glucose consumption in these cells. PCR microarray analysis shows that γ-tocotrienol treatment decreases the expression of genes associate with metabolic signaling and glycolysis in MCF-7 and MDA-MB-231 breast cancer cells. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are mediated, at least in part, by a suppression in the Warburg effect.

A novel mutual information estimator to measure spike train correlations in a model thalamocortical network.

The impact of thalamic state on information transmission to the cortex remains poorly understood. This limitation exists due to the rich dynamics displayed by thalamocortical networks and because of inadequate tools to characterize those dynamics. Here, we introduce a novel estimator of mutual information and use it to determine the impact of a computational model of thalamic state on information transmission. Using several criteria, this novel estimator, which uses an adaptive partition, is shown to be superior to other mutual information estimators with uniform partitions when used to analyze simulated spike train data with different mean spike rates, as well as electrophysiological data from simultaneously recorded neurons. When applied to a thalamocortical model, the estimator revealed that thalamocortical cell T-type calcium current conductance influences mutual information between the input and output from this network. In particular, a T-type calcium current conductance of ~40 nS appears to produce maximal mutual information between the input to this network (conceptualized as afferent input to the thalamocortical cell) and the output of the network at the level of a layer 4 cortical neuron. Furthermore, at particular combinations of inputs to thalamocortical and thalamic reticular nucleus cells, thalamic cell bursting correlated strongly with recovery of mutual information between thalamic afferents and layer 4 neurons. These studies suggest that the novel mutual information estimator has advantages over previous estimators and that thalamic reticular nucleus activity can enhance mutual information between thalamic afferents and thalamorecipient cells in the cortex. NEW & NOTEWORTHY In this study, a novel mutual information estimator was developed to analyze information flow in a model thalamocortical network. Our findings suggest that this estimator is a suitable tool for signal transmission analysis, particularly in neural circuits with disparate firing rates, and that the thalamic reticular nucleus can potentiate ascending sensory signals, while thalamic recipient cells in the cortex can recover mutual information in ascending sensory signals that is lost due to thalamic bursting.

Effect of temperature on FAD and NADH-derived signals and neurometabolic coupling in the mouse auditory and motor cortex.

Tight coupling of neuronal metabolism to synaptic activity is critical to ensure that the supply of metabolic substrates meets the demands of neuronal signaling. Given the impact of temperature on metabolism, and the wide fluctuations of brain temperature observed during clinical hypothermia, we examined the effect of temperature on neurometabolic coupling. Intrinsic fluorescence signals of the oxidized form of flavin adenine dinucleotide (FAD) and the reduced form of nicotinamide adenine dinucleotide (NADH), and their ratios, were measured to assess neural metabolic state and local field potentials were recorded to measure synaptic activity in the mouse brain. Brain slice preparations were used to remove the potential impacts of blood flow. Tight coupling between metabolic signals and local field potential amplitudes was observed at a range of temperatures below 29 °C. However, above 29 °C, the metabolic and synaptic signatures diverged such that FAD signals were diminished, but local field potentials retained their amplitude. It was also observed that the declines in the FAD signals seen at high temperatures (and hence the decoupling between synaptic and metabolic events) are driven by low FAD availability at high temperatures. These data suggest that neurometabolic coupling, thought to be critical for ensuring the metabolic health of the brain, may show temperature dependence, and is related to temperature-dependent changes in FAD supplies.

Phosphodiesterase 11A (PDE11A), Enriched in Ventral Hippocampus Neurons, is Required for Consolidation of Social but not Nonsocial Memories in Mice.

The capacity to form long-lasting social memories is critical to our health and survival. cAMP signaling in the ventral hippocampal formation (VHIPP) appears to be required for social memory formation, but the phosphodiesterase (PDE) involved remains unknown. Previously, we showed that PDE11A, which degrades cAMP and cGMP, is preferentially expressed in CA1 and subiculum of the VHIPP. Here, we determine whether PDE11A is expressed in neurons where it could directly influence synaptic plasticity and whether expression is required for the consolidation and/or retrieval of social memories. In CA1, and possibly CA2, PDE11A4 is expressed throughout neuronal cell bodies, dendrites (stratum radiatum), and axons (fimbria), but not astrocytes. Unlike PDE2A, PDE9A, or PDE10A, PDE11A4 expression begins very low at postnatal day 7 (P7) and dramatically increases until P28, at which time it stabilizes to young adult levels. This expression pattern is consistent with the fact that PDE11A is required for social long-term memory (LTM) formation during adolescence and adulthood. Male and female PDE11 knockout (KO) mice show normal short-term memory (STM) for social odor recognition (SOR) and social transmission of food preference (STFP), but no LTM 24 h post training. Importantly, PDE11A KO mice show normal LTM for nonsocial odor recognition. Deletion of PDE11A may impair memory consolidation by impairing requisite protein translation in the VHIPP. Relative to WT littermates, PDE11A KO mice show reduced expression of RSK2 and lowered phosphorylation of S6 (pS6-235/236). Together, these data suggest PDE11A is selectively required for the proper consolidation of recognition and associative social memories.

Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.

A large percentage of human breast cancers are characterized by excessive or aberrant HER2 activity. Lipid rafts are specialized microdomains within the plasma membrane that are required for HER2 activation and signal transduction. Since the anticancer activity of γ-tocotrienol is associated with suppression in HER2 signaling, studies were conducted to examine the effects of γ-tocotrienol on HER2 activation within the lipid raft microdomain in HER2-positive SKBR3 and BT474 human breast cancer cells. Treatment with 0-5µM γ-tocotrienol induced a significant dose-dependent inhibition in cancer cell growth after a 5-day culture period, and these growth inhibitory effects were associated with a reduction in HER2 dimerization and phosphorylation (activation). Phosphorylated HER2 was found to be primarily located in the lipid raft microdomain of the plasma membrane in vehicle-treated control groups, whereas γ-tocotrienol treatment significantly inhibited this effect. Assay of plasma membrane subcellular fractions showed that γ-tocotrienol also accumulates exclusively within the lipid raft microdomain. Hydroxypropyl-ß-cyclodextrin (HPßCD) is an agent that disrupts lipid raft integrity. Acute exposure to 3mM HPßCD alone had no effect, whereas an acute 24-h exposure to 20µM γ-tocotrienol alone significantly decreased SKBR3 and BT474 cell viability. However, combined treatment with these agents greatly reduced γ-tocotrienol accumulation in the lipid raft microdomain and cytotoxicity. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are associated with its accumulation in the lipid raft microdomain and subsequent interference with HER2 dimerization and activation in SKBR3 and BT474 human breast cancer cells.

Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow.

Estradiol Regulates Dorsal Vagal Complex Signal Transduction Pathway Transcriptional Reactivity to the AMPK Activator 5-Aminoimidazole-4-Carboxamide-Riboside (AICAR).

The ovarian hormone estradiol (E) regulates effects of hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) on caudal dorsal vagal complex (cDVC) neuron genomic activation and systemic glucostasis. The present study examined the hypothesis that cDVC signal transduction pathways exhibit distinctive E-dependent reactivity to activation of this sensor. RT-PCR microarray analysis was performed on RNA extracted from the cDVC of E- or oil (O)-implanted ovariectomized (OVX) adult female rats injected into the caudal fourth ventricle with the AMP mimetic 5-aminoimidazole-4-carboxamide-riboside (AICAR) (A) or saline (S). Microarray results show that the majority of marker genes differentially expressed in the E/S versus O/S cDVC were upregulated, as only myc (TGFß; WNT pathways), bcl2 (Hedgehog pathway), and serpine (hypoxia pathway) mRNA profiles were downregulated by E. Several JAK/STAT and NFκB signaling pathway marker gene profiles were upregulated in O/A but unchanged in E/A; additional NFκB genes were inhibited by A in E but not O. Hypoxia and p53 pathways contain genes that were inhibited or stimulated in O/A, but unaltered in E/A. Conversely, TGFß, p53, and NOTCH pathways each contained marker genes that were correspondingly modified or maintained in E/A versus O/A. Moreover, several oxidative stress pathway genes were suppressed in O/A while elevated or unchanged in E/A. Hedgehog, PPAR, and WNT signaling pathways were characterized by numerous examples of A-induced reversal of E augmentation of marker gene expression coinciding with opposite or no drug effects in O. Data presented here demonstrate that E exerts distinctive effects on cDVC signal transduction pathway marker gene reactivity to activated AMPK. Further research is needed to determine if observed changes in signal pathway marker gene transcription correlate with adjustments in gene product protein expression, and to characterize the role of aforementioned signaling pathways in E-sensitive cellular and systemic responses to hindbrain AMPK activation.

Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-ß in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.

Estradiol regulates effects of hindbrain activator 5-aminoimidazole-4-carboxamide-riboside administration on hypothalamic adenosine 5'-monophosphate-activated protein kinase activity and metabolic neurotransmitter mRNA and protein expression.

Hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) activation alters hypothalamic neuronal genomic activity in an estradiol (E)-dependent manner. This study examines the premise that E regulates metabolic effector neuron reactivity to hindbrain AMPK. Paraventricular (PVH), arcuate (ARH), and ventromedial (VMH) nuclei were micropunched from brains of E- or oil (O)-implanted ovariectomized female rats that had been injected, into the fourth ventricle, with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR; A) or saline (S) and analyzed by quantitative polymerase chain reaction and Western blotting for neurotransmitter mRNA and protein expression. PVH corticotrophin-releasing hormone gene and protein profiles were decreased in O/A and E/A animals. ARH pro-opiomelanocortin (POMC) mRNA and protein were both elevated in O/A but were diminished or unchanged, respectively, in E/A animals; ARH neuropeptide Y (NPY) transcription was inhibited in O/A and E/A animals, but neuropeptide content was augmented in E/A only. VMH SF-1 mRNA and protein were reduced in O and E animals. AICAR did not alter AMPK protein in any structure but elevated PVH (↑E), did not alter ARH, and decreased VMH (↓O,↓E) pAMPK. Results demonstrate hypothalamic metabolic neurotransmitter and AMPK reactivity to hindbrain AMPK activation, including E-dependent adjustments in POMC and NPY transcription and protein expression. Dissimilar POMC (↑O vs. ↔E) and NPY (↓O vs. ↑E) neuropeptide responses to caudal fourth ventricle AICAR indicate E regulation of hindbrain AMPK signaling and/or target receptivity, implying that ARH-controlled metabolic responses may differ in the presence vs. absence of E. Evidence for variable changes in hypothalamic AMPK activity resulting from hindbrain sensor manipulation suggests that individual (or region-based groups of) AMPK-expressing neuron populations are uniquely impacted by hindbrain AMPK.

Energy metabolism and hindbrain AMPK: regulation by estradiol.

Nerve cell energy status is screened within multiple classically defined hypothalamic and hindbrain components of the energy balance control network, including the hindbrain dorsal vagal complex (DVC). Signals of caudal DVC origin have a physiological role in glucostasis, e.g., maintenance of optimal supply of the critical substrate fuel, glucose, through control of motor functions such as fuel consumption and gluco-counterregulatory hormone secretion. A2 noradrenergic neurons are a likely source of these signals as combinatory laser microdissection/high-sensitivity Western blotting reveals expression of multiple biomarkers for metabolic sensing, including adenosine 5'-monophosphate-activated protein kinase (AMPK). Hypoglycemia elicits estradiol-dependent sex differences in A2 AMPK activation as phospho-AMPK (pAMPK) expression is augmented in male and ovariectomized (OVX) female, but not estrogen-replaced, OVX rats. This dichotomy may reflect, in part, estradiol-mediated up-regulation of glycolytic and tricarboxylic acid cycle enzyme expression during hypoglycemia. Our new model for short-term feeding abstinence has physiological relevance to planned (dieting) or unplanned (meal delay) interruption of consumption in modern life, which is negatively correlated with appetite control and obesity, and is useful for investigating how estrogen may mitigate the effects of disrupted fuel acquisition on energy balance via actions within the DVC. Estradiol reduces DVC AMPK activity after local delivery of the AMP mimic, 5-aminoimidazole-4-carboxamide-riboside, or cessation of feeding for 12 h but elevates pAMPK expression when these treatments are combined. These data suggest that estrogen maintains cellular energy stability over periods of suspended fuel acquisition and yet optimizes, by DVC AMPK-dependent mechanisms, counter-regulatory responses to metabolic challenges that occur during short-span feeding abstinence.

Hindbrain lactostasis regulates hypothalamic AMPK activity and metabolic neurotransmitter mRNA and protein responses to hypoglycemia.

Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolosensory loci operate autonomously or interact to coordinate central nervous system (CNS) reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite l-lactate during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal AMPK activity, coincident with exacerbation of hypoglycemia. Hindbrain lactate repletion also blunted hypoglycemic upregulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 phosphoAMPK (pAMPK), elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus.

Caudal fourth ventricular administration of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside regulates glucose and counterregulatory hormone profiles, dorsal vagal complex metabolosensory neuron function, and hypothalamic Fos expression.

This study investigated the hypothesis that estrogen controls hindbrain AMP-activated protein kinase (AMPK) activity and regulation of blood glucose, counterregulatory hormone secretion, and hypothalamic nerve cell transcriptional status. Dorsal vagal complex A2 noradrenergic neurons were laser microdissected from estradiol benzoate (E)- or oil (O)-implanted ovariectomized female rats after caudal fourth ventricular (CV4) delivery of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR), for Western blot analysis. E advanced AICAR-induced increases in A2 phospho-AMPK (pAMPK) expression and in blood glucose levels and was required for augmentation of Fos, estrogen receptor-α (ERα), monocarboxylate transporter-2, and glucose transporter-3 protein in A2 neurons and enhancement of corticosterone secretion by this treatment paradigm. CV4 AICAR also resulted in site-specific modifications in Fos immunolabeling of hypothalamic metabolic structures, including the paraventricular, ventromedial, and arcuate nuclei. The current studies demonstrate that estrogen regulates AMPK activation in caudal hindbrain A2 noradrenergic neurons during pharmacological replication of energy shortage in this area of the brain, and that this sensor is involved in neural regulation of glucostasis, in part, through control of corticosterone secretion. The data provide unique evidence that A2 neurons express both ERα and -ß proteins and that AMPK upregulates cellular sensitivity to ERα-mediated signaling during simulated energy insufficiency. The results also imply that estrogen promotes glucose and lactate uptake by these cells under those conditions. Evidence for correlation between hindbrain AMPK and hypothalamic nerve cell genomic activation provides novel proof for functional connectivity between this hindbrain sensor and higher order metabolic brain loci while demonstrating a modulatory role for estrogen in this interaction.