Micromolar 4-aminopyridine enhances invasion of a vertebrate neurosecretory terminal arborization: optical recording of action potential propagation using an ultrafast photodiode-MOSFET camera and a photodiode array.

Modulation of the amount of neuropeptide released from a neurosecretory tissue may be achieved by different means. These include alterations in the quantity secreted from each active nerve terminal or in the actual number of terminals activated. From the vertebrate hypothalamus, magnocellular neurons project their axons as bundles of fibers through the median eminence and infundibular stalk to arborize extensively and terminate in the neurohypophysis, where the neurohypophysial peptides and proteins are released into the circulation by a Ca-dependent mechanism. Elevating [Ca2+]o increases the magnitude of an intrinsic optical change in the neurohypophysial terminals that is intimately related to the quantity of neuropeptide released. Similarly, the addition of micromolar concentrations of 4-aminopyridine to the bathing solution enhances this change in large angle light scattering. However, we show here that, while these effects are superficially similar, they reflect different mechanisms of action. Evidence from intrinsic optical signals (light scattering) and extrinsic (potentiometric dye) absorption changes suggests that calcium increases the amount of neuropeptide released from each active terminal in the classical manner, while 4-aminopyridine exerts its secretagogue action by enhancing the invasion of action potentials into the magno-cellular neuron's terminal arborization, increasing the actual number of terminals activated. Physiologically, electrical invasion of the complex terminal arborization in the neurohypophysis may represent an extremely sensitive control point for modulation of peptide secretion. This would be especially effective in a neurohaemal organ like the posterior pituitary, where, in contrast with a collection of presynaptic terminals, the precise location of release is less important than the quantity released.

Electrophysiological characterization of peptidergic neurosecretory terminals

Electrical activity recorded intracellularly from peptidergic neurosecretory terminal dilatations in the sinus gland of crabs (principally Cardisoma guanhumi and C. carnifex) is described. Recordings were made from the neurohaemal organ in situ on the neural tissue of the isolated eyestalk and from isolated sinus gland-sinus gland nerve preparations. Verification that electrodes penetrated terminals was obtained by dye marking. Resting potentials ranged between −30 and −80mV. Overshooting action potentials of long duration (5–20 ms at 1/2 amplitude) relative to those of non-secretory axons (less than 2ms) were recorded in approximately 70% of stable penetrations. Action potentials occurred spontaneously at slow (less than 0.2s-1) rates in 75% of penetrations. Sequential intra- and extracellular recordings with the same microelectrode, on the same terminal, indicate impulse generation by the terminal itself. Extracellular stimulation of the axon tract evokes an all-or-none action potential at distinct threshold and latency. At rates of stimulation exceeding 5s-1, discrete fluctuations in the form of responses occur. Similar waveforms occur spontaneously and can be evoked by passing current through the electrode. They are interpreted to be electrotonically recorded activity of other parts of a complex axonal terminal arborization. Some, but not all, terminals exhibit impulse broadening (up to three-fold at 1/2 amplitude) during repetitive firing exceeding 1s-1. The same terminals show reduced impulse duration with hyperpolarization and broadened impulses with imposed depolarization. The changes are due to altered repolarization rates. Terminals sustain steady impulse firing at rates (up to 5s-1) linearly related to the imposed depolarizing current. Regenerative potentials, though of reduced rate of rise and amplitude, can be evoked by depolarizing current passed through the electrode during perfusion with salines having 1/2 normal [Na+], or containing tetrodotoxin (10(−6)moll-1). However, these block axonal conduction. Nominally Ca-free saline causes increased spontaneity and depolarization of about 5 mV in half the preparations examined, but reaching 20 mV in the others, with resultant inactivation of regenerative activity. Impulses in low-Ca saline show alterations of the falling phase, it being faster initially and then slower than normal. Thus, while the action potentials of neurosecretory axons are Na dependent, the terminals support regenerative impulses mediated by both Na and Ca.

Electrically Elicited Neurosecretory and Electrical Responses of the Isolated Crab Sinus Gland in Normal and Reduced Calcium Salines

The sinus gland (a neurohaemal organ) and its nerve have been isolated from the eyestalk of the crabs Cardisoma carnifex and Portunus sanguinolentus for studies correlating electrical responses recorded extra-cellularly from the sinus gland with hormone release. The appearance of erythrophore concentrating hormone (ECH) in the perfusate was followed by bioassay on leg segments of Ocypode pallidula. Electrical stimulation of the sinus gland nerve (175 pulses in trains of 5 at 5/s, every 10 s) results in significant amounts of hormone appearing in the perfusate, provided that a propagated compound action potential is recorded from the sinus gland. Release is normally below assayable levels in equivalent unstimulated control periods. A single preparation will release in excess of the equivalent of 20 pg of synthetic ECH in response to the standard routine of 175 stimuli. Many such secretory responses can be obtained over periods extending to as long as 30 h. Addition of tetrodotoxin (3 × 10-7 M) rapidly abolishes propagated electrical responses and secretion; the effects are reversible. Perfusion with saline having 30% (7.5 mM) of normal (25 mM) calcium reduces hormone release to about 10%, while the electrical response is often augmented. In 10% normal calcium, release is further decreased, and in 1% is indistinguishable from unstimulated release; electrical responses are reduced. Reduced calcium salines also induce spontaneous unit potentials, which persist in 30% calcium saline but subside after 10 min or longer exposure to salines of lower calcium concentration. All the effects of reduced calcium salines are reversible. Inhibition of secretion without reduction of electrical responses in 30% calcium saline provides evidence for a direct role of calcium in excitationsecretion coupling. In salines that are more calcium deficient, failure of terminal electrical responses may also contribute to reduction of secretion. In the crab sinus gland, as in other neurosecretory systems, propagation of action potentials to the terminals causes hormone release for which the presence of external calcium is essential.