Immunocytochemical identification of proctolinlike immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana (L.)

Summary In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl₂ iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.For the preparation of tritiated proctolin we thank Dr. S. Reißmann, WB Biochemie, Sektion Biologie, FSU JenaThe authors wish to thank G. Schörlitz, Film- und Bildstelle, FSU Jena, for photographs of whole mount preparations and Ms. A. Zinßer and Mrs. B. Cosack for excellent technical assistance.     

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.     

Responses in the First or Second Somatosensory Cortical Area in Cats during Transient Inactivation of the Other Ipsilateral Area with Lidocaine Hydrochloride

Simultaneous recordings were obtained from the primary and secondary somatosensory cortical areas (SI and SII) in cats anesthetized with ketamine or pentobarbital. A total of 40 individual neurons were studied (29 in SII and 11 in SI) before, during, and following injections of microliter quantities of lidocaine hydrochloride in the other ipsilateral cortical area. Activity in the cortex injected with the local anesthetic was monitored with single-neuron, multi-neuron, or evoked potential responses to determine the time course of inactivation within 0.5-2 mm of the injection sites. Recording sites in both cortical locations were in the representations of the distal forelimb. Responses were elicited by transcutaneous electrical stimulation across the receptive fields with needle electrodes. Short-latency responses were synchronously activated, and, in those circumstances where single neurons were isolated in both areas, no overall differences in latency were noted. Anesthetization of either cortical area never blocked access of somatosensory information to the intact area, even when the injected cortex was completely silenced in the vicinity of the injection mass. In 15 SII neurons and 7 SI neurons, changes were seen in short-latency evoked responses to stimulation of their receptive fields or in background activity following local anesthesia of the other area through several cycles of injection and recovery. In 7 of these 15 SII cells, changes were noted in the timing and/or firing rates of the short-latency responses; changes were noted in the short-latency responses of 2 of these 7 SI cells while SII was silenced. In 11 SII and 6 SI cells, “background” activity that was recorded during the interstimulus intervals either increased (most cases) or decreased during local anesthesia of the other area. The results are discussed in reference to the hypothesis that primary sensory cortical areas feed information forward to secondary areas, and these feed back modulatory controls to the primary regions.     

Glutamate Transmission in the Rostral Ventrolateral Medullary Sympathetic Premotor Pathway

1. The aim of these studies was to test the hypothesis that glutamate is the principal excitatory neurotransmitter in the sympathetic premotor pathway from the rostral ventrolateral medulla (RVLM) to the sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord.2. Iontophoretic and pressure ejection of glutamate receptor agonists and antagonists was made onto antidromically identified splanchnic and adrenal SPNs before and during electrical stimulation of the RVLM in urethane/chloralose-anesthetized, artificially ventilated rats.3. SPNs were excited by both NMDA and non-NMDA glutamate receptor agonists. Blockade of glutamate receptors in the IML interrupted the ability of electrical activation of sympathetic premotor neurons in the RVLM to excite SPNs. Within the IML, antergradely labeled terminals of RVLM neurons were found to contain glutamate immunoreactivity and to make asymmetric synapses on local dendrites.4. These data support a significant role for glutamate neurotransmission in mediating the tonic and phasic excitation of SPNs by the sympathetic premotor pathway from the RVLM. It seems likely that stimulation of the RVLM produces glutamate release from both C1 and non-PNMT-containing axon terminals in the IML.     

Exogenous zinc improves blood fluidity but has no effect on the mechanisms of vascular response to acetylcholine iontophoresis in humans

Recent findings in cellular signaling function of zinc through the mobilization intracellular calcium or by inducing ATP release suggest that extracellular zinc plays an important role in many physiological functions. However, such an extracellular signaling action of zinc for most cells is not known. Therefore, we investigated whether zinc plays any role in endothe-lium-dependent acetylcholine (ACh)-induced vasodilatation in microvascular beds. Transdermal iontophoresis was used to transport ACh through the forearm skin and cutaneous perfusion was measured using a laser Doppler flowmeter (LDF). Experiments were repeated using (1) zinc instead of ACh to test the effect of zinc ions alone and (2) concomitant iontophoresis of ACh and zinc to explore the effect of zinc on ACh-induced vasodilatation. Although zinc augments blood flow, curve-fitting to LDF signals indicate that zinc has no effect on the neural and endothelial component of ACh-induced vasodilatation. Additionally, no effect of Zn²⁺ on blood flow was found during its iontophoresis alone. Therefore, it is suggested from the Fourier analysis of LDF signals that the Zn⁺ might influence blood fluidity by its action on red blood cells deformability/aggregability during a high-blood-flow condition, which might, in turn, decrease blood viscosity and improve blood flow in vivo.     

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert,Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.     

Tracing of neurosecretory neurons in crayfish optic ganglia by cobalt iontophoresis

Summary The axonal connections between the medulla terminalis ganglionic X-organ (MTGXO) and the sinus gland are traced by iontophoretic application of cobalt dye to the neurosecretory system in the eyestalks of the crayfish, Orconectes limosus. The MTGXO consists of about 15 large perikarya, forming a distinct subgroup of neurosecretory cells in the medulla terminalis and giving rise to a prominent fibre bundle. Additional axons reaching the sinus gland from the medulla interna, the medulla externa and the optic nerve are less conspicuous.Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 87, Projekt A 3).Part of the work has been presented at the 9th Conference of European Comparative Endocrinologists in Giessen, August 1977Thanks are due to Dr. H.G. Wolff of the Universität Köln for his advice during the initial stage of this work     

Studies on Transdermal Delivery Enhancement of Zidovudine

The purpose of this study was to investigate physicochemical characteristics and in vitro release of zidovudine from monolithic film of Eudragit RL 100 and ethyl cellulose. Films included 2.5% or 5% (w/w) zidovudine of the dry polymer weight were prepared in various ratios of polymers by solvent evaporation method from methanol/acetone solvent mixture. The release studies were carried out by vertical Franz cells (2.2 cm² area, 20 ml receptor fluid). Ex vivo studies were done on Wistar rat skin within the films F6 (Eudragit RL100) and F7 (Eudragit RL100/Ethylcellulose, 1:1) consisting 5% (w/w) zidovudine in comparison with the same amount of free drug. Either iontophoresis (0.1 and 0.5 mA/cm² direct currents, Ag/AgCl electrodes) or dimethyl sulfoxide (pretreatment of 1% and 5%, w/w, solutions) were used as enhancers. Films consisting of ethyl cellulose under the ratio of 50% (w/w) gave similar release profiles, and the highest in vitro cumulative released amount was achieved with F6 film which gave the closest results with the free drug. This result could be due to the high swelling capacity and re-crystallization inhibition effect of RL 100 polymer which also influenced the film homogenization. All the films were fitted to Higuchi release kinetics. It was also observed that both 0.5-mA/cm² current and 5% (w/w) dimethyl sulfoxide applications significantly increased the cumulative permeated amount of zidovudine after 8 h; however, the flux enhancement ratio was higher for 0.5-mA/cm² current application, especially within F6 film. Thus, it was concluded that Eudragit RL100 film (F6) could be further evaluated for the transdermal application of zidovudine.     

Simultaneous transdermal extraction of glucose and lactate from human subjects by reverse iontophoresis

This study investigated the possibility of simultaneously extracting glucose and lactate from human subjects, at the same skin location, using transdermal reverse iontophoresis. Transdermal monitoring using iontophoresis is made possible by the skin's permeability to small molecules and the nanoporous and microporous nature of the structure of skin. The study was intended to provide information which could be used to develop a full, biosensor-based, monitoring system for multiple parameters from transdermal extraction. As a precursor to the human study, in vitro reverse iontophoresis experiments were performed in an artificial skin system to establish the optimum current waveforms to be applied during iontophoresis. In the human study, a bipolar DC current waveform (with reversal of the electrode current direction every 15 minutes) was applied to ten healthy volunteers via skin electrodes and utilized for simultaneous glucose and lactate transdermal extraction at an applied current density of 300 microA/cm2. Glucose and lactate were successfully extracted through each subject's skin into the conducting gel that formed part of each iontophoresis electrode. The results suggest that it will be possible to noninvasively and simultaneously monitor glucose and lactate levels in patients using this approach and this could have future applications in diagnostic monitoring for a variety of medical conditions.