John D. Tompkins

Neurally released pituitary adenylate cyclase‐activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability

Intracellular recordings were made in vitro from guinea‐pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase‐activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non‐cholinergic receptor‐mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC1 antagonist PACAP6‐38 (500 nm), the sEPSP evoked by 20 Hz stimulation was reduced by ∼50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nm PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6‐38 (500 nm) diminished the increase in excitability caused by 1 nm PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones.

Experiments were done using guinea‐pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium‐induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca2+]i) with the Ca2+‐sensitive dye fluo‐3, we demonstrated that 10 mm caffeine activated ryanodine receptors and caused a rise in [Ca2+]i in both Ca2+‐containing and Ca2+‐deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μm thapsigargin or caffeine and 20 μm ryanodine blocked subsequent caffeine‐induced elevations of [Ca2+]i. Treatment with thapsigargin, ryanodine or 200 μm Cd2+ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μm tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca2+ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea‐pig sympathetic stellate neurones.

VIP and PACAP Effects on Mouse Major Pelvic Ganglia Neurons

Major pelvic ganglia (MPG) neurons innervate urogenital organs and components of the lower bowel. Immunoreactivity for vasoactive intestinal polypeptide (VIP) has previously been observed in the MPG, and VIP knockout animals have impaired micturition reflexes suggesting a role for this neuropeptide in urogenital function. Here, we investigate the presence and action of VIP and a related neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the pelvic ganglia of male mice. An abundance of VIP-immunoreactive (IR) neurons and nerve fibers were observed in the ganglion, whereas PACAP immunoreactivity was not seen. Extracts from acutely isolated MPG contained transcripts for the VPAC1, VPAC2, and PAC1 receptors. Local application of VIP, PACAP, or maxadilan to isolated pelvic ganglion neurons shortened the duration of the afterhyperpolarization (AHP) of action potentials elicited by brief intracellular depolarization. All three peptides also increased neuronal excitability within a subpopulation of the sampled neurons. Bath application of apamin, a peptide antagonist of SK channels, shortened the duration of the AHP indicating that AHP duration in pelvic neurons is determined principally by SK-channel activity. The results suggest that VIP has a role in the neural control of pelvic organ function and activation of VPAC and/or PAC1 receptors can modulate the activity of the autonomic neurons innervating pelvic organs.

Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni(2+)) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Synaptic transmission at parasympathetic neurons of the major pelvic ganglion from normal and diabetic male mice.

Bladder and erectile dysfunction are common urologic complications of diabetes and are associated with reduced parasympathetic autonomic control. To determine whether disruption of ganglionic neurotransmission contributes to the loss of function, we investigated synaptic transmission at parasympathetic, major pelvic ganglion (MPG) neurons in control and chronically (20 wk) diabetic mice. In contrast to what has been reported for sympathetic neurons, diabetes did not cause an interruption of synaptic transmission at parasympathetic MPG neurons from streptozotocin-treated C57BL/6J (STZ) or db/db mice. Cholinergically mediated excitatory postsynaptic potentials (EPSPs) were suprathreshold during 5-s trains of 5-, 10-, and 20-Hz stimuli. Asynchronous neurotransmitter release, observed as miniature EPSPs (mEPSPs) during and after stimulation, permitted quantitative assessment of postganglionic, cholinergic receptor sensitivity. mEPSP amplitude following tetanic stimulation (recorded at -60 mV) was reduced in STZ (4.95 ± 0.4 vs. 3.71 ± 0.3 mV, P = 0.03), but not db/db mice. The number of posttetanic mEPSPs was significantly greater in db/db mice at all frequencies tested. Assessment of basic electrophysiological properties revealed that parasympathetic MPG neurons from db/db mice had less negative membrane potentials, lower input resistances, and shorter afterhyperpolarizations relative to their control. MPG neurons from STZ had longer afterhyperpolarizations but were otherwise similar to controls. Membrane excitability, measured by the membrane responsiveness to long-duration (1 s), suprathreshold depolarizing pulses, was unchanged in either model. The present study indicates that, while parasympathetic neurotransmission at the MPG is intact in chronically diabetic mice, obese, type 2 diabetic animals exhibit an altered presynaptic regulation of neurotransmitter release.

Autonomic dysfunction and plasticity in micturition reflexes in human α‐synuclein mice

Although often overshadowed by the motor dysfunction associated with Parkinsons disease (PD), autonomic dysfunction including urinary bladder and bowel dysfunctions are often associated with PD and may precede motoric changes; such autonomic dysfunction may permit early detection and intervention. Lower urinary tract symptoms are common in PD patients and result in significant morbidity. This studies focus on nonmotor symptoms in PD using a transgenic mouse model with overexpression of human α‐synuclein (hSNCA), the peptide found in high concentrations in Lewy body neuronal inclusions, the histopathologic hallmark of PD. We examined changes in the physiological, molecular, chemical, and electrical properties of neuronal pathways controlling urinary bladder function in transgenic mice. The results of these studies reveal that autonomic dysfunction (i.e., urinary bladder) can precede motor dysfunction. In addition, mice with hSNCA overexpression in relevant neuronal populations is associated with alterations in expression of neurotransmitter/neuromodulatory molecules (PACAP, VIP, substance P, and neuronal NOS) within neuronal pathways regulating bladder function as well as with increased NGF expression in the urinary bladder. Changes in the electrical and synaptic properties of neurons in the major pelvic ganglia that provide postganglionic innervation to urogenital tissues were not changed as determined with intracellular recording. The urinary bladder dysfunction observed in transgenic mice likely reflects changes in peripheral (i.e., afferent) and/or central micturition pathways or changes in the urinary bladder. SYN‐OE mice provide an opportunity to examine early events underlying the molecular and cellular plasticity of autonomic nervous system pathways underlying synucleinopathies.

Calcium Influx through Channels Other than Voltage-Dependent Calcium Channels Is Critical to the Pituitary Adenylate Cyclase-Activating Polypeptide-Induced Increase in Excitability in Guinea Pig Cardiac Neurons

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) effects on intracellular calcium ([Ca2+]i) and excitability have been studied in adult guinea pig intracardiac neurons. PACAP increased excitability, but did not elicit Ca2+ release from intracellular stores. Exposure to a Ca2+‐deficient solution did not deplete [Ca2+]i stores but did eliminate the PACAP‐induced increase in excitability. We postulate that Ca2+ influx is required for the PACAP‐induced increase in excitability.

Source and Action of Pituitary Adenylate Cyclase-Activating Polypeptide in Guinea Pig Intrinsic Cardiac Ganglia

The mammalian parasympathetic cardiac ganglia form a complex intrinsic cardiac nervous system presumed to contain multiple neuron types and are innervated by extrinsic fibers from multiple sources, each containing specific neurotransmitters and neuropeptides. In the guinea pig, the preganglionic parasympathetic cholinergic fibers contain the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), and essentially all cardiac neurons express the PACAP selective PAC1 receptor. Application of exogenous PACAP depolarizes and enhances the excitability of guinea pig cardiac neurons. The mechanism by which PACAP enhances excitability is not established. However, Ca(superscript 2+) influx through PACAP-activated nonselective cation channels is required for the PACAP-induced increase in excitability of guinea pig cardiac neurons. In addition, a PACAP-induced shift in the voltage dependence of activation of the cyclic nucleotide-gated, hyperpolarization-activated current, I(subscript h), most likely participates in the peptide-induced increase in neuronal excitability. The release of endogenous PACAP by repetitive stimulation of preganglionic fibers in vitro contributes to the generation of a slow excitatory postsynaptic potential and also can enhance cardiac neuron excitability. We hypothesize that PACAP released during ongoing vagal activity may regulate cardiac neuron excitability in vivo.