Patricia A. Glazebrook

Differential Distribution and Function of Hyperpolarization-Activated Channels in Sensory Neurons and Mechanosensitive Fibers

Sensory neurons express hyperpolarization-activated currents (IH) that differ in magnitude and kinetics within the populations. We investigated the structural basis for these differences and explored the functional role of the IH channels in sensory neurons isolated from rat nodose ganglia. Immunohistochemical studies demonstrated a differential distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) protein (HCN1, HCN2, HCN4) in sensory neurons and peripheral terminals. HCN2 and HCN4 immunoreactivity was present in all nodose neurons. In contrast, only 20% of the total population expressed HCN1 immunoreactivity. HCN1 did not colocalize with IB4 (a marker for C-type neurons), and only 15% of HCN1-positive neurons colocalized with immunoreactivity for the vanilloid receptor VR1, another protein associated primarily with C-type neurons. Therefore, most HCN1-containing neurons were A-type neurons. In further support, HCN1 was present in the mechanosensitive terminals of myelinated but not unmyelinated sensory fibers, whereas HCN2 and HCN4 were present in receptor terminals of both myelinated and unmyelinated fibers. In voltage-clamp studies, cell permeant cAMP analogs shifted the activation curve for IH to depolarized potentials in C-type neurons but not A-type neurons. In current-clamp recording, CsCl, which inhibits only IH in nodose neurons, hyperpolarized the resting membrane potential from –63 ± 1 to –73 ± 2 mV and nearly doubled the input resistance from 1.3 to 2.2 GΩ. In addition, action potentials were initiated at lower depolarizing current injections in the presence of CsCl. At the sensory receptor terminal, CsCl decreased the threshold pressure for initiation of mechanoreceptor discharge. Therefore, elimination of the IH increases excitability of both the soma and the peripheral sensory terminals.

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage‐gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0‐3) and in 85‐90 % of the neurons from older animals (postnatal days 21‐60). In voltage clamp studies, α‐dendrotoxin (α‐DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K+ currents of the sensory neurons. α‐DTX blocked current in both A‐ and C‐type neurons. The current had characteristics of a delayed rectifier with activation positive to −50 mV and little inactivation during 250 ms pulses. In current‐clamp experiments α‐DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A‐ and C‐type neurons. However, there were prominent effects on excitability. α‐DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after‐hyperpolarization and increased the frequency/pattern of discharge of A‐ and C‐type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K+ currents function in response to the loss of the α‐DTX‐sensitive current to effect these changes in action potential wave shape and discharge.

TRPC channels as signal transducers

The study of the TRPC cation channels as signal transducers in sensory neurons is in its infancy. Mechanoreceptors that monitor arterial pressure are prime candidates for the involvement of TRPC channels as either primary mechanical transducers or as modulators of the transduction process. Their activity patterns can be regulated by growth factors such as BDNF and by a variety of ligands that activate Gq-coupled receptors, mechanisms that have been shown in heterologous expression systems to activate TRPC channels. We investigated the distribution of TRPC1 and TRPC3–7 in nodose sensory neurons and in their peripheral axons that terminate as mechanosensitive receptors in the aortic arch of the rat. Using immunocytochemical techniques we identified these six TRPC proteins in the soma of the nodose neurons but only TRPC1 and TRPC3–5 were found to distribute to the peripheral axons and the mechanosensory terminals. TRPC1 and TRPC3 extended into the low threshold complex sensory endings with very strong labeling. In contrast, TRPC4 and TRPC5 were found primarily in major branches of the receptor but immunoreactivity was weak in the region where mechanotransduction is presumed to occur. Terminals arising from unmyelinated fibers also expressed TRPC1 and TRPC3–5 but not all fibers expressed all of the channels suggesting that specific TRPC protein may be aligned with previously described subclasses of the unmyelinated C-fibers.

The KCNQ/M‐current modulates arterial baroreceptor function at the sensory terminal in rats

The ion channels responsible for the pattern and frequency of discharge in arterial baroreceptor terminals are, with few exceptions, unknown. In this study we examined the contribution of KCNQ potassium channels that underlie the M‐current to the function of the arterial baroreceptors. Labelled aortic baroreceptor neurons, immunohistochemistry and an isolated aortic arch preparation were used to demonstrate the presence and function of KCNQ2, KCNQ3 and KCNQ5 channels in aortic baroreceptors. An activator (retigabine) and an inhibitor (XE991) of the M‐current were used to establish a role for these channels in setting the resting membrane potential and in regulating the response to ramp increases in arterial pressure. Retigabine raised the threshold for activation of arterial baroreceptors and shifted the pressure–response curve to higher aortic pressures. XE991, on the other hand, produced an increase in excitability as shown by an increase in discharge at elevated pressures as compared to control. We propose that KCNQ2, KCNQ3 and KCNQ5 channels provide a hyperpolarizing influence to offset the previously described depolarizing influence of the HCN channels in baroreceptor neurons and their terminals.

KCa1.1 channel contributes to cell excitability in unmyelinated but not myelinated rat vagal afferents

High conductance calcium-activated potassium (BK(Ca)) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BK(Ca) channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BK(Ca) channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type K(Ca)1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the K(Ca)1.1 channel protein. Measures of cell size and immunostaining intensity for K(Ca)1.1 and IB4 cluster into two statistically distinct (P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated (n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated (n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BK(Ca) channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that K(Ca)1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type K(Ca) channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.

The juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity

Despite considerable effort, the identification of genes that regulate complex multigenic traits such as obesity has proven difficult with conventional methodologies. The use of a chromosome substitution strain-based mapping strategy based on deep congenic analysis overcame many of the difficulties associated with gene discovery and led to the finding that the juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity. The effects of a mild Cntnap2 mutation on body weight were highly dependent on genetic background, as both obesity-promoting and obesity-resistant effects of Cntnap2 were observed on different genetic backgrounds. The more severe effect of complete TAG1 deficiency, by decreasing food intake, completely prevented the weight gain normally associated with high-fat-diet feeding. Together, these studies implicate two novel proteins in the regulation of diet-induced obesity. Moreover, as juxtaparanodal proteins have previously been implicated in various neurological disorders, our results suggest a potential genetic and molecular link between obesity and diseases such as autism and epilepsy.

Distribution of voltage-gated potassium and hyperpolarization-activated channels in sensory afferent fibers in the rat carotid body.

The chemosensory glomus cells of the carotid body (CB) detect changes in O2 tension. Carotid sinus nerve fibers, which originate from peripheral sensory neurons located within the petrosal ganglion, innervate the CB. Release of transmitter from glomus cells activates the sensory afferent fibers to transmit information to the nucleus of the solitary tract in the brainstem. The ion channels expressed within the sensory nerve terminals play an essential role in the ability of the terminal to initiate action potentials in response to transmitter‐evoked depolarization. However, with a few exceptions, the identity of ion channels expressed in these peripheral nerve fibers is unknown. This study addresses the expression of voltage‐gated channels in the sensory fibers with a focus on channels that set the resting membrane potential and regulate discharge patterns. By using immunohistochemistry and fluorescence confocal microscopy, potassium channel subunits and HCN (hyperpolarization‐activated) family members were localized both in petrosal neurons that expressed tyrosine hydroxylase and in the CSN axons within the carotid body. Channels contributing to resting membrane potential, including HCN2 responsible in part for Ih current and the KCNQ2 and KCNQ5 subunits thought to underlie the neuronal “M current,” were identified in the sensory neurons and their axons innervating the carotid body. In addition, the results presented here demonstrate expression of several potassium channels that shape the action potential and the frequency of discharge, including Kv1.4, Kv1.5, Kv4.3, and KCa (BK). The role of these channels should be considered in interpretation of the fiber discharge in response to perturbation of the carotid body environment. J. Comp. Neurol. 510:367–377, 2008.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts

STUDY QUESTION What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? SUMMARY ANSWER Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. WHAT IS KNOWN ALREADY The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. STUDY DESIGN, SIZE, DURATION Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. PARTICIPANTS/MATERIALS, SETTING, METHODS General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. MAIN RESULTS AND THE ROLE OF CHANCE The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. LIMITATIONS, REASONS FOR CAUTION TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. WIDER IMPLICATIONS OF THE FINDINGS These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. STUDY FUNDING/COMPETING INTERESTS X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. TRIAL REGISTRATION NUMBER None.

Influence of high fat diet and resveratrol supplementation on placental fatty acid uptake in the Japanese macaque

INTRODUCTION Adequate maternal supply and placental delivery of long chain polyunsaturated fatty acids (LCPUFA) is essential for normal fetal development. In humans, maternal obesity alters placental FA uptake, though the impact of diet remains uncertain. The fatty fetal liver observed in offspring of Japanese macaques fed a high fat diet (HFD) was prevented with resveratrol supplementation during pregnancy. We sought to determine the effect of HFD and resveratrol, a supplement with insulin-sensitizing properties, on placental LCPUFA uptake in this model. METHODS J. macaques were fed control chow (15% fat, n = 5), HFD (35% fat, n = 10) or HFD containing 0.37% resveratrol (n = 5) prior to- and throughout pregnancy. At ∼ 130 d gestation (term = 173 d), placentas were collected by caesarean section. Fatty acid uptake studies using (14)C-labeled oleic acid, arachidonic acid (AA) and docosahexanoic acid (DHA) were performed in placental explants. RESULTS Resveratrol supplementation increased placental uptake of DHA (P < 0.05), while HFD alone had no measurable effect. Resveratrol increased AMP-activated protein kinase activity and mRNA expression of the fatty acid transporters FATP-4, CD36 and FABPpm (P < 0.05). Placental DHA content was decreased in HFD dams; resveratrol had no effect on tissue fatty acid profiles. DISCUSSION Maternal HFD did not significantly affect placental LCPUFA uptake. Furthermore, resveratrol stimulated placental DHA uptake capacity, AMPK activation and transporter expression. Placental handling of DHA is particularly sensitive to the dramatic alterations in the maternal metabolic phenotype and placental AMPK activity associated with resveratrol supplementation.

Expression of the P/Q (Cav2.1) calcium channel in nodose sensory neurons and arterial baroreceptors

The predominant calcium current in nodose sensory neurons, including the subpopulation of baroreceptor neurons, is the N-type channel, Cav2.2. It is also the primary calcium channel responsible for transmitter release at their presynaptic terminals in the nucleus of the solitary tract in the brainstem. The P/Q channel, Cav2.1, the other major calcium channel responsible for transmitter release at mammalian synapses, represents only 15-20% of total calcium current in the general population of sensory neurons and makes a minor contribution to transmitter release at the presynaptic terminal. In the present study we identified a subpopulation of the largest nodose neurons (capacitance>50pF) in which, surprisingly, Cav2.1 represents over 50% of the total calcium current, differing from the remainder of the population. Consistent with these electrophysiological data, anti-Cav2.1 antibody labeling was more membrane delimited in a subgroup of the large neurons in slices of nodose ganglia. Data reported in other synapses in the central nervous system assign different roles in synaptic information transfer to the P/Q-type versus N-type calcium channels. The study raises the possibility that the P/Q channel which has been associated with high fidelity transmission at other central synapses serves a similar function in this group of large myelinated sensory afferents, including arterial baroreceptors where a high frequency regular discharge pattern signals the pressure pulse. This contrasts to the irregular lower frequency discharge of the unmyelinated fibers that make up the majority of the sensory population and that utilize the N-type channel in synaptic transmission.