Long-lasting intrinsic optical changes observed in the neurointermediate lobe of the mouse pituitary reflect volume changes in cells of the pars intermedia.

BACKGROUND/AIMS: Complex intrinsic optical changes (light scattering) are readily observed in the neurointermediate lobe of the mouse pituitary gland following electrical stimulation of the infundibular stalk. Our laboratory has previously identified three distinct phases within the light scattering signal: two rapid responses to action potential stimulation and a long duration recovery. The rapid light scattering signals, restricted to the neurohypophysial portion (posterior pituitary) of the neurointermediate lobe, consist of an E-wave and an S-wave that reflect excitation and secretion, respectively. The E-wave has the approximate shape of the action potential and includes voltage- and current-related components and is independent of Ca(2+) entry. The S-wave is related to Ca(2+) entry and exocytosis. The slow recovery phase of the light scattering signal, which we designated the R-wave, is less well characterized. METHODS: Using high temporal resolution light scattering measurements, we monitored intrinsic optical changes in the neurointermediate lobe of the mouse pituitary gland. Pharmacological interventions during the measurements were employed. RESULTS: The data presented here provide optical and pharmacological evidence suggesting that the R-wave, which comprises signals from the posterior pituitary as well as from the pars intermedia, mirrors volume changes in pars intermedia cells following a train of stimuli applied to the infundibular stalk. These volume changes were blocked by the GABA-receptor antagonists bicuculline and picrotoxin, and were mimicked by direct application of GABA in the absence of electrical stimulation. CONCLUSIONS: These results emphasize the importance of central GABAergic projections into the neurointermediate lobe, and the potential role of GABA in effecting hormone release from the pars intermedia.

A mechanical spike accompanies the action potential in Mammalian nerve terminals.

Large and rapid changes in light scattering accompany secretion from nerve terminals of the mammalian neurohypophysis (posterior pituitary). In the mouse, these intrinsic optical signals are intimately related to the arrival of the action potential E-wave and the release of arginine vasopressin and oxytocin (S-wave). Here we have used a high bandwidth atomic force microscope to demonstrate that these light-scattering signals are associated with changes in terminal volume that are detected as nanometer-scale movements of a cantilever positioned on top of the neurohypophysis. The most rapid mechanical response ("spike"), having a duration shorter than the action potential but comparable to that of the E-wave, represents a transient increase in terminal volume due to water movement associated with Na(+)-influx. The slower mechanical event ("dip"), on the other hand, depends upon Ca(2+)-entry as well as on intraterminal Ca(2+)-transients and, analogously to the S-wave, seems to monitor events associated with secretion.

Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.

Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal alpha3-, alpha4-, alpha3/alpha5-, beta2-, beta4- and alpha7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception of alpha4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of alpha3beta2-, alpha3beta4- and/or alpha7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the evoked synaptic response vary depending upon the location of the stimulating electrode with respect to the ganglion under study. The range of activity patterns that follows sequential pharmacological elimination of individual subtypes suggests that nAChRs may be capable of regulating the activity of both excitatory and inhibitory pathways, in a manner similar to that described in the central nervous system.

Changes in FAD and NADH fluorescence in neurosecretory terminals are triggered by calcium entry and by ADP production.

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.

An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.

We demonstrate that high power light-emitting diodes (LED's) exhibit low-frequency noise characteristics that are clearly superior to those of quartz tungsten halogen lamps, the non-coherent light source most commonly employed when freedom from intensity variation is critical. Their extreme stability over tens of seconds (combined with readily selectable wavelength) makes high power LED's ideal light sources for DC recording of optical changes, from living cells and tissues, that last more than a few hundred milliseconds. These optical signals (DeltaI/I(0)) may be intrinsic (light scattering, absorbance or fluorescence) or extrinsic (absorbance or fluorescence from probe molecules) and we show that changes as small as approximately 8 x 10(-5) can be recorded without signal averaging when LED's are used as monochromatic light sources. Here, rapid and slow changes in the intrinsic optical properties of mammalian peptidergic nerve terminals are used to illustrate the advantages of high power LED's compared to filament bulbs.

Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis.

The submucous plexus of the guinea pig intestine is a quasi-two-dimensional mammalian neural network that is particularly amenable to study using multiple site optical recording of transmembrane voltage (MSORTV) [Biol. Bull. 183 (1992) 344; J. Neurosci. 19 (1999) 3073]. For several years the potentiometric dye of choice for monitoring the electrical activity of its individual neurons has been di-8-ANEPPS [Neuron 9 (1992) 393], a naphthylstyryl-pyridinium dye with a propylsulfonate headgroup that provides relatively large fluorescence changes during action potentials and synaptic potentials. Limitations to the use of this dye, however, have been its phototoxicity and its low water solubility which requires the presence of DMSO and Pluronic F-127 in the staining solution. In searching for less toxic and more soluble dyes exhibiting larger fluorescence signals, we first tried the dienylstyryl-pyridinium dye RH795 [J. Neurosci. 14 (1994) 2545] which is highly soluble in water. This dye yielded relatively large signals, but it was internalized quickly by the submucosal neurons resulting in rapid degradation of the signal-to-noise ratio. We decided to synthesize a series of naphthylstyryl-pyridinium dyes (di-n-ANEPPDHQ) having the same chromophore as di-8-ANEPPS and the quaternary ammonium headgroup (DHQ) of RH795 (resulting in two positive charges versus the neutral propylsulfonate-ring nitrogen combination), and we tested the di-methyl (JPW3039), di-ethyl (JPW2081), di-propyl (JPW3031), di-butyl (JPW5029), and di-octyl (JPW5037) analogues, all of them soluble in ethanol. We found that the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms yielded the best combination of signal-to-noise ratio, moderate phototoxicity and absence of dye internalization.

Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.

Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize alpha3/alpha5, alpha7, and beta2 nAChR subunits, and the results demonstrate the prevalence of alpha3/alpha5. It is this alpha3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application.

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.

Microsecond response of a voltage-sensitive merocyanine dye: fast voltage-clamp measurements on squid giant axon.

Extrinsic absorption changes exhibited by squid giant axons stained with the voltage-sensitive merocyanine-oxazolone dye NK 2367 were measured during brief voltage-clamp steps. Experiments that employed an optical recording system having a sub-microsecond response time constant demonstrate that this dye responds to step changes in membrane voltage in less than 2 microseconds at room temperature. The optical response is independent of ionic currents and is unlikely to depend upon cytoskeletal or cytoplasmic events.

Aminoglycoside antibiotics block voltage-dependent calcium channels in intact vertebrate nerve terminals.

Intrinsic and extrinsic optical signals recorded from the intact nerve terminals of vertebrate neurohypophyses were used to investigate the anatomical site and physiological mechanism of the antagonistic effects of aminoglycoside antibiotics on neurotransmission. Aminoglycoside antibiotics blocked the intrinsic light scattering signal closely associated with neurosecretion in the mouse neurohypophysis in a concentration-dependent manner with an IC50 of approximately 60 microM and the block was relieved by increasing [Ca2+]o. The rank order potency of different aminoglycoside antibiotics for blocking neurosecretion in this preparation was determined to be: neomycin greater than gentamicin = kanamycin greater than streptomycin. Optical recordings of rapid changes in membrane potential using voltage-sensitive dyes revealed that aminoglycoside antibiotics decreased the Ca(2+)-dependent after-hyperpolarization of the normal action potential and both the magnitude and after-hyperpolarization of the regenerative Ca2+ spike. The after-hyperpolarization results from a Ca-activated potassium conductance whose block by aminoglycoside antibiotics was also reversed by increased [Ca2+]o. These studies demonstrate that the capacity of aminoglycoside antibiotics to antagonize neurotransmission can be attributed to the block of Ca channels in the nerve terminal.

Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons.

1. Left upper quadrant (LUQ) cells isolated from the abdominal ganglion of Aplysia were maintained in culture to study how the cellular and synaptic properties of individual neurons contribute to the generation of patterns of electrical activity by neuronal ensembles. 2. Conventional microelectrodes were used to examine the spiking characteristics of individually cultured LUQ cells in vitro and to characterize their synaptic interactions. 3. In vitro, in contrast to in situ, LUQ neurons innervate other LUQ neurons. Intracellular recordings from pairs of LUQ cells showed that the prevalent type of postsynaptic potential was purely inhibitory. The other type of response was a dual-action postsynaptic potential, with inhibition followed by a delayed, slow excitation. 4. We established a set of criteria for the use of multiple-site optical recording techniques, in combination with impermeant probes of membrane potential, to observe the patterns of electrical activity generated by ensembles of co-cultured LUQ cells. 5. The spiking activity of individual cells within the neuronal ensembles was detected by means of the change in optical absorption of cells that were vitally stained with the dye RH155. The change in absorption was typically delta A congruent to 4 X 10(-4) per spike. We achieved a signal-to-noise (peak-to-peak) ratio of approximately 10 for a 50 X 50-microns photodetector field and an incident intensity of approximately 10 mW/cm2, close to the theoretical limit. 6. These conditions permitted, for the first time, continuous optical recording from cultured neurons for periods of up to 3 h with no discernible photodynamic damage or photobleaching. This long-term optical recording permitted examination of the different patterns of electrical activity generated by individual neuronal ensembles under several different experimental conditions. 7. An elaborate tracery of regenerated neurites present in these cultures resulted in individual photodetectors recording simultaneously the activity of multiple neurons. We reconstructed the temporal firing patterns for individual neurons within ensembles even with all the neurons active simultaneously and determined the functional connections in the ensemble by analyzing the temporal relationships between firing patterns of individual neurons. Excitatory as well as inhibitory functional interactions could be observed within the neuronal ensemble, the latter after the tonic activity of the neurons was increased by reducing the extracellular [Mg2+]. 8. Examination of the optical data from ensembles constructed from identified cells having characteristic physiological responses allowed us to address the question of how cellular and synaptic properties affect the patterns of electrical activity generated by neuronal ensembles.(ABSTRACT TRUNCATED AT 400 WORDS)

Foreign connections are formed in vitro by Aplysia californica interneuron L10 and its in vivo followers and non-followers.

Aplysia californica interneurone L10 forms a set of presynaptic connections with many postsynaptic 'follower' cells in the abdominal ganglion. These followers do not connect back to L10. The present study tests whether the direction and sign of these connections are obligatory and are reconstructed when neuronal processes regenerate in vitro. L10 was co-cultured with one of six different followers and two non-followers. 1. In vitro connections that preserve the sign of those formed in vivo were made by L10 onto neurones L11, L12 and L13. The connections consisted of inhibitory postsynaptic potentials (IPSPs) with characteristic fast and slow components. 2. In vitro connections that did not preserve the sign of connections found in vivo were made by L10 onto R15, R16 and L7. Neurones R15 and R16 receive excitatory inputs from L10 in vivo and L7 receives a dual-action input in vivo, with inhibition followed by excitation. A purely inhibitory connection from L10 was formed in vitro onto all these cells. 3. Connections that have never been observed in vivo in terms of both direction and sign were formed in vitro. Followers L7, L11, L12, L13 and R16 and non-follower L14A formed novel connections onto L10. All these connections were inhibitory and some were strong. For example, IPSPs with a magnitude of 20 mV were observed in L10 following a single action potential in L13. Our results show that identified Aplysia neurones can form stereotyped specific connections in vitro. The specificity is different from that in the intact ganglion. The ubiquity of novel connections suggests that restrictions imposed on synaptogenesis in the animal are distinct from those regulating synapse formation in culture.

Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to omega-conotoxin and relatively insensitive to dihydropyridines. Optical studies with and without voltage-sensitive dyes.

Extrinsic absorption changes exhibited by potentiometric dyes have established the ionic basis of the action potential in synchronously activated populations of nerve terminals in the intact neurohypophyses of amphibia and mammals (Salzberg et al., 1983; Obaid et al., 1983, 1985b). Also, large and rapid changes in light scattering, measured as transparency, have been shown to follow membrane depolarization and to be intimately associated with the release of neuropeptides from the nerve terminals of the mouse neurohypophysis (Salzberg et al., 1985; Gainer et al., 1986). We report some experiments that help to define the pharmacological profile of the calcium channels present in intact neurosecretory terminals of vertebrates. For these, we used the peptide toxin omega-conotoxin GVIA (1-5 microM) and the dihydropyridine compounds Bay-K 8644 and nifedipine (2-5 microM), together with the after-hyperpolarization of the nerve terminal action potential. This undershoot depends upon the activation of a calcium-mediated potassium channel, as suggested by its sensitivity to [Ca++]o and charybdotoxin. omega-conotoxin GVIA substantially reduced the after-hyperpolarization in neurosecretory terminals of Xenopus, while neither of the dihydropyridine compounds had any effect under conditions that mimic natural stimulation. The effects of these calcium channel modifiers on the action potential recorded optically from the terminals of the Xenopus neurohypophysis were faithfully reflected in the behavior of the light-scattering changes observed in the neurohypophysis of the CD-1 mouse. omega-conotoxin GVIA (5 microM) reduced the size of the intrinsic optical signal associated with secretion by 50%, while the dihydropyridines had little effect. These observations suggest that the type of calcium channel that dominates the secretory behavior of intact vertebrate nerve terminals is at least partially blocked by omega-conotoxin GVIA and is insensitive, under normal conditions, to dihydropyridines.

Effects of barium and bicarbonate on glial cells of Necturus optic nerve. Studies with microelectrodes and voltage-sensitive dyes.

We have studied the effects of Ba++, a known K+ channel blocker, on the electrophysiological properties of the glial cells of Necturus optic nerve. The addition of Ba++ reversibly depolarized glial cells by 25-50 mV; the half maximal deplorization was obtained with a Ba++ concentration of approximately 0.3 mM. In the presence of Ba++, the sensitivity of the membrane to changes in K+ was reduced and there was evidence of competition between K+ and Ba++ for the K+ channel. These effects, which were accompanied by a large increase in the input resistance of the glial cells, indicate that Ba++ blocks the K+ conductance in glial cells of Necturus optic nerve. With the K+ conductance reduced, we were able to investigate the presence of other membrane conductances. We found that in the presence of Ba++, the addition of HCO3- caused a Na+-dependent hyperpolarization that was sensitive to the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid). Removal of Na+ resulted in a HCO3- -dependent, SITS-sensitive depolarization. These results are consistent with the presence in the glial membrane of an electrogenic Na+/HCO3- cotransporter in which Na+, HCO3-, and net negative charge are transported in the same direction. In Cl- -free solutions, the Ba++-induced depolarization increased, suggesting a small permeability to Cl-. Using voltage-sensitive dyes and a photodiode array for multiple site optical recording, the distribution of potential changes in response to square pulses of intracellularly injected current were recorded before and after the addition of increased and the decay of amplitude as a function of distance decreased. Such results indicate that Ba++ increases the membrane resistance more than the resistance of the intercellular junctions.

Optical studies of the secretory event at vertebrate nerve terminals.

Potentiometric probes are small (300-500 Mr) amphipatic molecules that bind to, but do not cross, cell membranes and behave as fast linear transducers of membrane voltage. Their optical properties, particularly absorbance and fluorescence, respond to changes in potential in less than 2 microseconds, and they may be used to follow electrical events in membranes which are inaccessible to microelectrodes. We have used these dyes to study the properties of the action potential in the neurosecretory terminals of vertebrate neurohypophyses and, in particular, to investigate the behaviour of the local population of calcium channels. These channels are sensitive to the peptide toxin omega-conotoxin GVIA, derived from the venom of the marine snail Conus geographicus, but insensitive to dihydropyridine channel modulators. In the neurohypophysis of the mouse, it is possible to demonstrate that the calcium channels that are blocked by omega-conotoxin are those that are required for secretion of peptide hormones. In the terminals of the neurohypophysis, excitation is coupled to secretion, and the secretory event is accompanied by large and rapid changes in light scattering. These intrinsic optical signals provide a millisecond time-resolved monitor of events in the terminal that follow the entry of calcium, and may precede the release of hormones. We will consider how the changes in light scattering can be related to secretion, and how the extrinsic (absorption) and intrinsic optical signals may provide complementary information about excitation-secretion coupling.

Optical recording of electrical activity from parallel fibres and other cell types in skate cerebellar slices in vitro.

1. A reliable and simple fish brain slice preparation was obtained from the cerebellum of the skate, and its properties were described. 2. A potentiometric oxonol dye, RH-482, and multiple site optical recording of transmembrane voltage (MSORTV) were used to reveal the electrophysiological properties of the parallel fibre action potential and to measure its conduction (0.13 m/s). The parallel fibre action potential was blocked in the presence of tetrodotoxin (TTX) and prolonged by tetraethylammonium (TEA), suggesting that the upstroke depends upon sodium entry and the repolarization upon potassium efflux. An after-hyperpolarization results from a calcium-dependent potassium conductance. 3. A second potentiometric dye, RH-155, differing only slightly from RH-482, exhibited a high affinity for glial cell membrane, and could be used to monitor changes in extracellular potassium concentration by detecting changes in glial membrane potential. 4. Calcium channel blockers such as cadmium ions blocked the optical signal that reflected the extracellular accumulation of potassium. 5. Interventions that modified the extracellular volume, and thereby affected the accumulation of potassium, produced large changes in the optical signal that monitored glial depolarization. Hypertonic and hypotonic bathing solutions resulted in decreases and increases, respectively, in the magnitude of the extrinsic absorption change that tracked potassium accumulation. 6. Blocking sodium-potassium pump activity by means of ouabain prolonged the time course of the optical signal that was related to potassium accumulation in the extracellular space. 7. Extracellular potassium accumulation was revealed to be critically dependent upon intracellular calcium ions.

Laser light scattering determination of size and dispersity of synaptosomes and synaptic vesicles isolated from squid (Loligo pealei) optic lobes.

Quasi-elastic laser light scattering has been used to investigate the size and dispersity of synaptosomes and synaptic vesicles isolated from optic lobes of the squid Loligo pealei. Synaptosomal fractions were highly polydisperse (mu2/gamma -2 = 0.5) and the mean diameter (-d) ranged from 0.5-2.0 microns. Size distribution histograms yielded two major components - smaller particles (-d approximately 300-700 nm) and a larger group of particles (-d approximately 1,500-5,000 nm). The heterogeneity of the synaptosomal particles detected in solution is in agreement with published data obtained using electron microscopy. Purified synaptic vesicle fractions also yielded complex particle size distribution data. A component with a mean diameter in the range 150-250 nm was detected, though a smaller particle (-d approximately 40-110 nm) dominated the scattering signal. This smaller particle closely resembles in size the electron lucent vesicles seen in the majority of squid optic lobe nerve terminals when examined by electron microscopy. Osmotically-induced shrinkage and swelling of the synaptosomes was detected. Depolarization by veratridine (1.0 x 10(-4) M) did not result in a detectable change in the size of synaptosomal particles.