John H. Schild

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.

Electrophysiological and pharmacological validation of vagal afferent fiber type of neurons enzymatically isolated from rat nodose ganglia.

An unavoidable consequence of enzymatic dispersion of sensory neurons from intact ganglia is loss of the axon and thus the ability to classify afferent fiber type based upon conduction velocity (CV). An intact rat nodose ganglion preparation was used to randomly sample neurons (n=76) using the patch clamp technique. Reliable electrophysiological and chemophysiological correlates of afferent fiber type were established for use with isolated neuron preparations. Myelinated afferents (approximately 25%) formed two groups exhibiting strikingly different functional profiles. One group (n=10) exhibited CVs in excess of 10 m/s and narrow (<1 ms) action potentials (APs) while the other (n=9) had CVs as low as 4m/s and broad (>2 ms) APs that closely approximated those identified as unmyelinated afferents (n=57) with CVs less than 1m/s. A cluster analysis of select measures from the AP waveforms strongly correlated with CV, producing three statistically unique populations (p<0.05). These groupings aligned with our earlier hypothesis (Jin et al., 2004) that a differential sensitivity to the selective purinergic and vanilloid receptor agonists can be used as reliable pharmacological indicators of vagal afferent fiber type. These metrics were further validated using an even larger population of isolated (n=240) nodose neurons. Collectively, these indicators of afferent fiber type can be used to provide valuable insight concerning the relavence of isolated cellular observations to integrated afferent function of visceral organ systems.

Patch clamp electrophysiology in nodose ganglia of adult rat

The patch clamp technique is widely utilized for studying the electrophysiological properties of enzymatically isolated sensory neurons. Unfortunately, dissociation of the ganglion severs the afferent fibers. As a result, isolated neurons can only be broadly categorized according to somatic action potential waveforms, ion channel subtypes, chemical sensitivities and cell diameter. Such restricted classifications contrast with the continuum of conduction velocities (CVs), discharge patterns, sensory modalities and functional properties of visceral and spinal afferents. Previous reports of patch clamp recordings using intact ganglion have been limited to young animal preparations. This raises concerns regarding postnatal development and impedes the use of chronic models of disease or injury, which often necessitate the use of a more mature animal preparation. Here, we present a methodology for preparing nodose ganglion from adult rat (250-400 g) for study using the patch clamp technique. Successful whole cell recordings were obtained from approximately 50% of the cells selected for study, the majority of which had intact afferent fibers. Measures of somatic discharge and afferent fiber CV at both room and physiological temperatures were consistent with investigations using sharp microelectrodes. Voltage clamp recordings of whole cell Na(+), Ca(2+) and K(+) ion channel currents were comparable to those obtained using isolated neuron preparations. The ability to classify voltage- and ligand-gated ion channel type with afferent fiber CV in an adult preparation adds a valuable new dimension to cellular investigations of the diverse functional and chemical properties of the peripheral afferent nervous system.

Electrophysiological and neuroanatomical evidence of sexual dimorphism in aortic baroreceptor and vagal afferents in rat.

Evidence for sexual dimorphism in autonomic control of cardiovascular function is both compelling and confounding. Across healthy and disease populations sex-associated differences in neurocirculatory hemodynamics are far too complex to be entirely related to sex hormones. As an initial step toward identifying additional physiological mechanisms, we investigated whether there is a sex bias in the relative expression of low-threshold-myelinated and high-threshold-unmyelinated aortic baroreceptor afferents in rats. These two types of afferent fibers have markedly different reflexogenic effects upon heart rate and blood pressure and thus the potential impact upon baroreflex dynamics could be substantial. Our results, using a combination of a patch-clamp study of fluorescently identified aortic baroreceptor neurons (ABN) and morphometric analysis of aortic baroreceptor nerve fibers, demonstrate that females exhibit a greater percentage of myelinated baroreceptor fibers (24.8% vs. 18.7% of total baroreceptor fiber population, P < 0.01) and express a functional subtype of myelinated ABN rarely found in age-matched males (11% vs. 2.3%, n = 107, P < 0.01). Interestingly, this neuronal phenotype is more prevalent in the general population of female vagal afferent neurons (17.7% vs. 3.8%, n = 169, P < 0.01), and ovariectomy does not alter its expression but does lessen neuronal excitability. These data suggest there are fundamental neuroanatomical and electrophysiological differences between aortic baroreceptor afferents of female and male rats. Possible explanations are presented as to how such a greater prevalence of low-threshold myelinated afferents could be a contributing factor to the altered baroreflex sensitivity and vagal tone of females compared with males.

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.

Unmyelinated visceral afferents exhibit frequency dependent action potential broadening while myelinated visceral afferents do not.

Sensory information arising from visceral organ systems is encoded into action potential trains that propagate along afferent fibers to target nuclei in the central nervous system. These information streams range from tight patterns of action potentials that are well synchronized with the sensory transduction event to irregular, patternless discharge with no clear correlation to the sensory input. In general terms these afferent pathways can be divided into unmyelinated and myelinated fiber types. Our laboratory has a long standing interest in the functional differences between these two types of afferents in terms of the preprocessing of sensory information into action potential trains (synchrony, frequency, duration, etc.), the reflexogenic consequences of this sensory input to the central nervous system and the ionic channels that give rise to the electrophysiological properties of these unique cell types. The aim of this study was to determine whether there were any functional differences in the somatic action potential characteristics of unmyelinated and myelinated vagal afferents in response to different rates of sensory nerve stimulation. Our results showed that activity and frequency-dependent widening of the somatic action potential was quite prominent in unmyelinated but not myelinated vagal afferents. Spike broadening often leads to increased influx of Ca(2+) ions that has been associated with a diverse range of modulatory mechanisms both at the cell body and central synaptic terminations (e.g. increased neurotransmitter release.) We conclude that our observations are indicative of fundamentally different mechanisms for neural integration of sensory information arising from unmyelinated and myelinated vagal afferents.

Differential distribution of voltage-gated channels in myelinated and unmyelinated baroreceptor afferents

Voltage gated ion channels (VGC) make possible the frequency coding of arterial pressure and the neurotransmission of this information along myelinated and unmyelinated fiber pathways. Although many of the same VGC isoforms are expressed in both fiber types, it is the relative expression of each that defines the unique discharge properties of myelinated A-type and unmyelinated C-type baroreceptors. For example, the fast inward Na⁺ current is a major determinant of the action potential threshold and the regenerative transmembrane current needed to sustain repetitive discharge. In A-type baroreceptors the TTX-sensitive Na(v)1.7 VGC contributes to the whole cell Na⁺ current. Na(v)1.7 is expressed at a lower density in C-type neurons and in conjunction with TTX-insensitive Na(v)1.8 and Na(v)1.9 VGC. As a result, action potentials of A-type neurons have firing thresholds that are 15-20 mV more negative and upstroke velocities that are 5-10 times faster than unmyelinated C-type neurons. A more depolarized threshold in conjunction with a broader complement of non-inactivating K(V) VGC subtypes produces C-type action potentials that are 3-4 times longer in duration than A-type neurons and at markedly lower levels of cell excitability. Unmyelinated baroreceptors also express KCa1.1 which provides approximately 25% of the total outward K⁺ current. KCa1.1 plays a critically important role in shaping the action potential profile of C-type neurons and strongly impacts neuronal excitability. A-type neurons do not functionally express the KCa1.1 channel despite having a whole cell Ca(V) current quite similar to that of C-type neurons. As a result, A-type neurons do not have the frequency-dependent braking forces of KCa1.1. Lack of a KCa current and only a limited complement of non-inactivating K(V) VGC in addition to a hyperpolarization activated HCN1 current that is nearly 10 times larger than in C-type neurons leads to elevated levels of discharge in A-type neurons, a hallmark of myelinated baroreceptors. Interestingly, HCN2 and HCN4 expression levels are comparable in both fiber types. Collectively, such apportion of VGC constrains the neural coding of myelinated A-type baroreceptors to low threshold, high frequency, high fidelity discharge but with a limited capacity for neuromodulation of afferent bandwidth. Unmyelinated C-type baroreceptors require greater depolarizing forces for spike initiation and have a low frequency discharge profile that is often poorly correlated with the physiological stimulus. But the complement of VGC in C-type neurons provides far greater capacity for neuromodulation of cell excitability than can be obtained from A-type baroreceptors.

Functional impact of the hyperpolarization-activated current on the excitability of myelinated A-type vagal afferent neurons in the rat.

1. The hyperpolarization‐induced, cation‐selective current Ih is widely observed in peripheral sensory neurons of the vagal and dorsal root ganglia, but the peak magnitude and voltage‐ and time‐dependent properties of this current vary widely across afferent fibre type.

Gender Differences in Histamine-Induced Depolarization and Inward Currents in Vagal Ganglion Neurons in Rats

Evidence has shown gender differences regarding the critical roles of histamine in the prevalence of asthma, anaphylaxis, and angina pectoris. Histamine depolarizes unmyelinated C-type neurons without any effects on myelinated A-type vagal ganglion neurons (VGNs) in male rats. However, little is known if VGNs from females react to histamine in a similar manner. Membrane depolarization and inward currents were tested in VGNs isolated from adult rats using a whole-cell patch technique. Results from males were consistent with the literature. Surprisingly, histamine-induced depolarization and inward currents were observed in both unmyelinated C-type and myelinated A- and Ah-type VGNs from female rats. In Ah-type neurons, responses to 1.0 μM histamine were stronger in intact females than in males and significantly reduced in ovariectomized (OVX) females. In C-type neurons, histamine-induced events were significantly smaller (pA/pF) in intact females compared with males and this histamine-induced activity was dramatically increased by OVX. Female A-types responded to histamine, which was further increased following ovariectomy. Histamine at 300 nM depolarized Ah-types in females, but not Ah-types in OVX females. In contrast, the sensitivity of A- and C-types to histamine was upregulated by OVX. These data demonstrate gender differences in VGN chemosensitivity to histamine for the first time. Myelinated Ah-types showed the highest sensitivity to histamine across female populations, which was changed by OVX. These novel findings improve the understanding of gender differences in the prevalence of asthma, anaphylaxis, and pain. Changes in sensitivity to histamine by OVX may explain alterations in the prevalence of certain pathophysiological conditions when women reach a postmenopausal age.