Kv1.1 subunits localize to cardiorespiratory brain networks in mice where their absence induces astrogliosis and microgliosis.

Cardiorespiratory collapse following a seizure is a suspected cause of sudden unexpected death in epilepsy (SUDEP), the leading cause of epilepsy-related mortality. In the commonly used Kcna1 gene knockout (Kcna1-/-) mouse model of SUDEP, cardiorespiratory profiling reveals an array of aberrant breathing patterns that could contribute to risk of seizure-related mortality. However, the brain structures mediating these respiratory abnormalities remain unknown. We hypothesize that Kv1.1 deficiency in respiratory control centers of the brain contribute to respiratory dysfunction in Kcna1-/- mice leading to increased SUDEP risk. Thus, in this study, we first used immunohistochemistry to map expression of Kv1.1 protein in cardiorespiratory brain regions of wild-type Kcna1+/+ (WT) mice. Next, GFAP and Iba1 immunostaining was used to test for the presence of astrogliosis and microgliosis, respectively, in the cardiorespiratory centers of Kcna1-/- mice, which could be indicative of seizure-related brain injury that could impair breathing. In WT mice, we detected Kv1.1 protein in all cardiorespiratory centers examined, including the basolateral amygdala, dorsal respiratory group, dorsal motor nucleus of vagus, nucleus ambiguus, ventral respiratory column, and pontine respiratory group, as well as chemosensory centers including the retrotrapezoid and median raphae nuclei. Extensive gliosis was observed in the same areas in Kcna1-/- mice suggesting that seizure-associated brain injury could contribute to respiratory abnormalities.

Genetic ablation or pharmacological inhibition of Kv1.1 potassium channel subunits impairs atrial repolarization in mice.

Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1-/-) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1-/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1-/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1-/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.

Epilepsy-associated Kv1.1 channel subunits regulate intrinsic cardiac pacemaking in mice.

The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking.

GABAergic inhibition through synergistic astrocytic neuronal interaction transiently decreases vasopressin neuronal activity during hypoosmotic challenge.

The neuropeptide vasopressin is crucial to mammalian osmotic regulation. Local hypoosmotic challenge transiently decreases and then increases vasopressin secretion. To investigate mechanisms underlying this transient response, we examined the effects of hypoosmotic challenge on the electrical activity of rat hypothalamic supraoptic nucleus (SON) vasopressin neurons using patch-clamp recordings. We found that 5 min exposure of hypothalamic slices to hypoosmotic solution transiently increased inhibitory postsynaptic current (IPSC) frequency and reduced the firing rate of vasopressin neurons. Recovery occurred by 10 min of exposure, even though the osmolality remained low. The γ-aminobutyric acid (GABA)A receptor blocker, gabazine, blocked the IPSCs and the hypoosmotic suppression of firing. The gliotoxin l-aminoadipic acid blocked the increase in IPSC frequency at 5 min and the recovery of firing at 10 min, indicating astrocytic involvement in hypoosmotic modulation of vasopressin neuronal activity. Moreover, ß-alanine, an osmolyte of astrocytes and GABA transporter (GAT) inhibitor, blocked the increase in IPSC frequency at 5 min of hypoosmotic challenge. Confocal microscopy of immunostained SON sections revealed that astrocytes and magnocellular neurons both showed positive staining of vesicular GATs (VGAT). Hypoosmotic stimulation in vivo reduced the number of VGAT-expressing neurons, and increased co-localisation and molecular association of VGAT with glial fibrillary acidic protein that increased significantly by 10 min. By 30 min, neuronal VGAT labelling was partially restored, and astrocytic VGAT was relocated to the ventral portion while it decreased in the somatic zone of the SON. Thus, synergistic astrocytic and neuronal GABAergic inhibition could ensure that vasopressin neuron firing is only transiently suppressed under hypoosmotic conditions.

Kv1.1 potassium channel subunit deficiency alters ventricular arrhythmia susceptibility, contractility, and repolarization.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.

Decreased bioavailability of hydrogen sulfide links vascular endothelium and atrial remodeling in atrial fibrillation.

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Bollgard II cotton: compositional analysis and feeding studies of cottonseed from insect-protected cotton (Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins.

Bollgard II cotton event 15985 producing the Cry1Ac and Cry2Ab2 proteins has been developed by genetic modification to broaden the spectrum of insects to which the plant is tolerant and to provide an insect resistance management tool to impede the onset of resistance. The purpose of this study was to evaluate the composition and nutrition of Bollgard II cotton, relative to the use for food and animal feed, compared to that of conventional cotton varieties. Compositional analyses were conducted to measure proximate, fiber, amino acid, fatty acid, gossypol, and mineral contents of cottonseed from a total of 14 U.S. field sites over two years. Compositional analysis results showed that the cottonseed and cottonseed oil from Bollgard II cotton were comparable in their composition to those of the conventional control cotton line and other commercial varieties. The composition data are supported by nutritional safety studies conducted with dairy cows, catfish, and quail. Results from these studies showed that Bollgard II performed similarly to the conventional control cotton varieties. These data demonstrate that Bollgard II cotton is compositionally and nutritionally equivalent to conventional cotton varieties. These data support the conclusion that Bollgard II cotton is as safe and nutritious as conventional cotton for food and feed use.

Metabotropic glutamate receptors and dendrodendritic synapses in the main olfactory bulb.

The main olfactory bulb (MOB) is the first site of synaptic processing in the central nervous system for odor information that is relayed from olfactory receptor neurons in the nasal cavity via the olfactory nerve (ON). Glutamate and ionotropic glutamate receptors (iGluRs) play a dominant role at ON synapses. Similarly, glutamate and iGluRs mediate dendrodendritic transmission between several populations of neurons within the MOB network. Neuroanatomical studies demonstrate that metabotropic glutamate receptors (mGluRs) are densely expressed through the MOB network, and they are particularly abundant at dendrodendritic synapses. Until recently, the physiological roles of mGluRs in the MOB were poorly understood. Over the past several years, mGluRs have been shown to play surprisingly powerful neuromodulatory roles at ON synapses and in dendrodendritic neurotransmission in the MOB. This chapter focuses on recent advances in our understanding of mGluR-mediated signaling components at dendrodendritic synapses.

Sensory deafferentation transsynaptically alters neuronal GluR1 expression in the external plexiform layer of the adult mouse main olfactory bulb.

Altered distribution of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 has been linked to stimulation-dependent changes in synaptic efficacy, including long-term potentiation and depression. The main olfactory bulb (OB) remains plastic throughout life; how GluR1 may be involved in this plasticity is unknown. We have previously shown that neonatal naris occlusion reduces numbers of interneuron cell bodies that are immunoreactive for GluR1 in the external plexiform layer (EPL) of the adult mouse OB. Here, we show that immunoreactivity of mouse EPL interneurons for GluR1 is also dramatically reduced following olfactory deafferentation in adulthood. We further show that expression of glutamic acid decarboxylase (GAD) 65, 1 of 2 GAD isoforms expressed by adult gamma-aminobutyric acidergic interneurons, is reduced, but to a much smaller extent, and that in double-labeled cells, immunoreactivity for the Ca(2+)-binding protein parvalbumin (PV) is also reduced. In addition, GluR1 expression is reduced in presumptive tufted cells and interneurons that are negative for GAD65 and PV. Consistent with previous reports, sensory deafferentation resulted in little neuronal degeneration in the adult EPL, indicating that these differences were not likely due to death of EPL neurons. Together, these results suggest that olfactory input regulates expression of the GluR1 AMPA receptor subunit by tufted cells that may in turn regulate GluR1 expression by interneurons within the OB EPL.

Group I metabotropic glutamate receptors are differentially expressed by two populations of olfactory bulb granule cells.

In the main olfactory bulb, several populations of granule cells (GCs) can be distinguished based on the soma location either superficially, interspersed with mitral cells within the mitral cell layer (MCL), or deeper, within the GC layer (GCL). Little is known about the physiological properties of superficial GCs (sGCs) versus deep GCs (dGCs). Here, we used patch-clamp recording methods to explore the role of Group I metabotropic glutamate receptors (mGluRs) in regulating the activity of GCs in slices from wildtype and mGluR-/- mutant mice. In wildtype mice, bath application of the selective Group I mGluR agonist DHPG depolarized and increased the firing rate of both GC subtypes. In the presence of blockers of fast synaptic transmission (APV, CNQX, gabazine), DHPG directly depolarized both GC subtypes, although the two GC subtypes responded differentially to DHPG in mGluR1-/- and mGluR5-/- mice. DHPG depolarized sGCs in slices from mGluR5-/- mice, although it had no effect on sGCs in slices from mGluR1-/- mice. By contrast, DHPG depolarized dGCs in slices from mGluR1-/- mice but had no effect on dGCs in slices from mGluR5-/- mice. Previous studies showed that mitral cells express mGluR1 but not mGluR5. The present results therefore suggest that sGCs are more similar to mitral cells than dGCs in terms of mGluR expression.

Distribution of GluR1 is altered in the olfactory bulb following neonatal naris occlusion.

The olfactory system is well suited for studies of glutamate receptor plasticity. The sensory neurons are glutamatergic, and they turn over throughout life, and the olfactory bulb neurons that process their inputs express many of the known glutamate receptor subunits. Neonatal naris occlusion alters olfactory bulb development and the expression of certain neuroactive substances and receptors, at least in part due to loss of the sensory inputs. We therefore postulated that neonatal naris occlusion might alter glutamate receptor expression during postnatal development. Single nares of newborn mice were occluded on postnatal days 1-2, and the distribution of glutamate receptor subunits was evaluated using immunoperoxidase methods. Light microscopic examination on postnatal day 6 failed to reveal adult-like staining of neuronal cell bodies in the olfactory bulbs. By day 12, cell bodies that were immunoreactive (-IR) for the GluR1 subunit were visible in the external plexiform layer (EPL) of both sides. By day 18, many of the GluR1-IR cell bodies could be identified as cell types that had previously been reported to express homomeric GluR1 receptors. Analysis of single, mid-dorsal sections from 18-25-day-old mice showed that the medial EPL of the occluded side had a significantly lower density of these cell bodies. The GluR1 staining of the adjacent mitral cell layer (MCL) was also heavier on the occluded side, but no gross differences in staining for other glutamate receptor subunits were observed. Neonatal naris occlusion therefore appears to provide a new model for studying expression of GluR1 receptors during the development of a discrete population of olfactory bulb neurons.