Diana L. Kunze

Exogenous Brain-Derived Neurotrophic Factor Rescues Synaptic Dysfunction in Mecp2-Null Mice

Postnatal deficits in brain-derived neurotrophic factor (BDNF) are thought to contribute to pathogenesis of Rett syndrome (RTT), a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2-null mice, a model of RTT, BDNF deficits are most pronounced in structures important for autonomic and respiratory control, functions that are severely affected in RTT patients. However, relatively little is known about how these deficits affect neuronal function or how they may be linked to specific RTT endophenotypes. To approach these issues, we analyzed synaptic function in the brainstem nucleus tractus solitarius (nTS), the principal site for integration of primary visceral afferent inputs to central autonomic pathways and a region in which we found markedly reduced levels of BDNF in Mecp2 mutants. Our results demonstrate that the amplitude of spontaneous miniature and evoked EPSCs in nTS neurons is significantly increased in Mecp2-null mice and, accordingly, that mutant cells are more likely than wild- type cells to fire action potentials in response to primary afferent stimulation. These changes occur without any increase in intrinsic neuronal excitability and are unaffected by blockade of inhibitory GABA currents. However, this synaptopathy is associated with decreased BDNF availability in the primary afferent pathway and can be rescued by application of exogenous BDNF. On the basis of these findings, we hypothesize that altered sensory gating in nTS contributes to cardiorespiratory instability in RTT and that nTS is a site at which restoration of normal BDNF signaling could help reestablish normal homeostatic controls.

KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons

The M‐current is a slowly activating, non‐inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M‐current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition‐enhancing drug linopirdine (1–50 μm) and its analogue, XE991 (10 μm), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M‐currents. This current activated between −60 and −55 mV, had a voltage‐dependent activation time constant of 208 ± 12 ms at −20 mV, a deactivation time constant of 165 ± 24 ms at −50 mV and V1/2 of −24 ± 2 mV, values which are consistent with previous reports for endogenous M‐currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as −60 mV. Flupirtine (10–20 μm), an M‐current activator, caused a 3–14 mV leftward shift in the current–voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M‐current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.

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.

Adaptive Depression in Synaptic Transmission in the Nucleus of the Solitary Tract after In Vivo Chronic Intermittent Hypoxia: Evidence for Homeostatic Plasticity

The respiratory system is highly pliable in its adaptation to low-oxygen (hypoxic) environments. After chronic intermittent hypoxia (CIH), alterations in the regulation of cardiorespiratory system become persistent because of changes in the peripheral chemoreceptor reflex. We present evidence for the induction of a novel form of homeostatic plasticity in this reflex pathway in the nucleus tractus solitarius (NTS), the site of termination of the chemosensory afferent fibers. CIH induces an increase in NTS postsynaptic cell activity initiated by spontaneous presynaptic transmitter release that is counterbalanced by a reduction in evoked synaptic transmission between sensory afferents and NTS second-order cells. This is accomplished via presynaptic mechanisms involving changes in neurotransmitter release and calcium/calmodulin-dependent kinase II activation.

The COOH-terminal Domain of Drosophila TRP Channels Confers Thapsigargin Sensitivity

Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd, and is relatively selective for Caversus Na and Ba. In contrast, trpl encodes a Ca-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd, and non-selective with respect to Ca, Na, and Ba. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca over Na and Ba. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca, Ba, and Na. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.

Endogenous Brain-Derived Neurotrophic Factor in the Nucleus Tractus Solitarius Tonically Regulates Synaptic and Autonomic Function

Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are highly expressed in the nucleus tractus solitarius (nTS), the principal target of cardiovascular primary afferent input to the brainstem. However, little is known about the role of BDNF signaling in nTS in cardiovascular homeostasis. We examined whether BDNF in nTS modulates cardiovascular function in vivo and regulates synaptic and/or neuronal activity in isolated brainstem slices. Microinjection of BDNF into the rat medial nTS (mnTS), a region critical for baroreflex control of sympathetic outflow, produced dose-dependent increases in mean arterial pressure (MAP), heart rate (HR), and lumbar sympathetic nerve activity (LSNA) that were blocked by the tyrosine kinase inhibitor K252a. In contrast, immunoneutralization of endogenous BDNF (anti-BDNF), or microinjection of K252a alone, decreased MAP, HR, and LSNA. The effects of anti-BDNF were abolished by blockade of ionotropic glutamate receptors, indicating a role for glutamate signaling in the response to BDNF. In vitro, BDNF reduced the amplitude of miniature EPSCs as well as solitary tract (TS) evoked EPSC amplitude and action potential discharge (APD) in second-order nTS neurons. BDNF effects on EPSCs were independent of GABAergic signaling and abolished by AMPA receptor blockade. In contrast, K252a increased spontaneous EPSC frequency and TS evoked EPSC amplitude. BDNF also attenuated APD evoked by injection of depolarizing current into second-order neurons, indicating reduced intrinsic neuronal excitability. Our data demonstrate that BDNF signaling in mnTS plays a tonic role in regulating cardiovascular function, likely via modulation of primary afferent glutamatergic excitatory transmission and neural activity.

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.

Dopamine Inhibits N-Type Channels in Visceral Afferents to Reduce Synaptic Transmitter Release Under Normoxic and Chronic Intermittent Hypoxic Conditions

Glutamatergic synaptic currents elicited in second-order neurons in the nucleus of the solitary tract (nTS) by activation of chemosensory and other visceral afferent fibers are severely reduced following 10 days of chronic intermittent hypoxia (CIH). The mechanism by which this occurs is unknown. A strong candidate for producing the inhibition is dopamine, which is also released from the presynaptic terminals and which we have shown exerts a tonic presynaptic inhibition on glutamate release. We postulated that tonic activation of the D2 receptors inhibits presynaptic calcium currents to reduce transmitter release and that in CIH this occurs in conjunction with an increase in the dopamine inhibitory response due to the increase in presynaptic D2 receptors or an increase in dopamine release further suppressing the evoked excitatory postsynaptic current (eEPSC). Thus we predicted that blockade of the D2 receptors would return the EPSC to values of animals maintained under normoxic conditions. We found that dopamine and quinpirole, the selective D2-like agonist, inhibit calcium currents via the D2 receptors by acting on the N-type calcium channel in presynaptic neurons and their nTS central terminals. However, in brain slice studies from CIH animals, although the D2 antagonist sulpiride increased the CIH-reduced amplitude of synaptic currents, EPSCs were not restored to normal levels. This indicates that while the dopamine inhibitory effect remains intact in CIH, most of the reduction in the eEPSC amplitude occurs via alternative mechanisms.

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.

Distribution of transient receptor potential channels in the rat carotid chemosensory pathway

Glomus cells in the carotid body respond to decreases in oxygen tension of the blood and transmit this sensory information in the carotid sinus nerve to the brain via neurons in the petrosal ganglion. G‐protein‐coupled membrane receptors linked to phospholipase C may play an important role in this response through the activation of the cation channels formed by the transient receptor potential (TRP) proteins. In the present study, expression of TRPC proteins in the rat carotid body and petrosal ganglion was examined using immunohistochemical techniques. TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 were present in neurons throughout the ganglion. TRPC1 was expressed in only 28% of petrosal neurons, and of this population, 45% were tyrosine hydroxylase (TH)‐positive, accounting for essentially all the TH‐expressing neurons in the ganglion. Because TH‐positive neurons project to the carotid body, this result suggests that TRPC1 is selectively associated with the chemosensory pathway. Confocal images through the carotid body showed that TRPC1/3/4/5/6 proteins localize to the carotid sinus nerve fibers, some of which were immunoreactive to an anti‐neurofilament (NF) antibody cocktail. TRPC1 and TRPC3 were present in both NF‐positive and NF‐negative fibers, whereas TPRC4, TRPC5, and TRPC6 expression was primarily localized to NF‐negative fibers. Only TRPC1 and TRPC4 were localized in the afferent nerve terminals that encircle individual glomus cells. TRPC7 was not expressed in sensory fibers. All the TRPC proteins studied were present in type I glomus cells. Although their role as receptor‐activated cation channels in the chemosensory pathway is yet to be established, the presence of TRPC channels in glomus cells and sensory nerves of the carotid body suggests a role in facilitating and/or sustaining the hypoxic response. J. Comp. Neurol. 464:404–413, 2003.