Simultaneous Electrophysiological Recordings Research Articles

Voltage-Dependent Sodium Channel Function Is Regulated Through Membrane Mechanics

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na + channel α subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na + channels involve mechanical destabilization. Our observation that the β1 subunit causes similar changes in function of the Na + channel α subunit suggests that β1 may act through interaction with cytoskeleton.

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation.

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation. The effects of combined O2/glucose deprivation were investigated on large aspiny (LA) interneurons recorded from a striatal slice preparation by means of simultaneous electrophysiological and optical recordings. LA interneurons were visually identified and impaled with sharp microelectrodes loaded with the calcium (Ca2+)-sensitive dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a membrane hyperpolarization coupled to a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, this [Ca2+]i elevation was more pronounced in dendritic branches rather than in the somatic region. The O2/glucose-deprivation-induced membrane hyperpolarization reversed its polarity at the potassium (K+) equilibrium potential. Both membrane hyperpolarization and [Ca2+]i rise were unaffected by TTX or by a combination of ionotropic glutamate receptors antagonists, D-2-amino-5-phosphonovaleric acid and 6cyano-7-nitroquinoxaline-2, 3-dione. Sulfonylurea glibenclamide, a blocker of ATP-sensitive K+ channels, markedly reduced the O2/glucose-deprivation-induced membrane hyperpolarization but failed to prevent the rise in [Ca2+]i. Likewise, charybdotoxin, a large K+-channel (BK) inhibitor, abolished the membrane hyperpolarization but did not produce detectable changes of [Ca2+]i elevation. A combination of high-voltage-activated Ca2+ channel blockers significantly reduced both the membrane hyperpolarization and the rise in [Ca2+]i. In a set of experiments performed without dye in the recording electrode, either intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid or external barium abolished the membrane hyperpolarization induced by O2/glucose deprivation. The hyperpolarizing effect on membrane potential was mimicked by oxotremorine, an M2-like muscarinic receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an increase but rather to a decrease of the basal [Ca2+]i. Furthermore glibenclamide did not reduce the oxotremorine- and baclofen-induced membrane hyperpolarization. In conclusion, the present results suggest that in striatal LA cells, O2/glucose deprivation activates a membrane hyperpolarization that does not involve ligand-gated K+ conductances but is sensitive to barium, glibenclamide, and charybdotoxin. The increase in [Ca2+]i is partially due to influx through voltage-gated high-voltage-activated Ca2+ channels.

A rapid wavelength changer based on liquid crystal shutters for use in ratiometric microspectrophotometry.

Many of the fluorescent indicator molecules most useful for measuring intracellular concentrations of ions of biological importance, such as Ca2+ or H+, require illumination first at one wavelength, at which the fluorescence depends strongly on the concentration of the ion, and then at another wavelength (e.g. the isosbestic point), so that a ratio can be obtained. Existing wavelength changers are mechanical and involve moving filters, mirrors or gratings. These systems have the disadvantage that they introduce mechanical shocks that can interfere with simultaneous electrophysiological recording. In addition, they require special electrical driving systems and are relatively expensive, especially if they are capable of switching rapidly. We describe a new wavelength changer based on liquid crystal shutters which has the following advantages: (1) it has no mechanical moving parts; (2) it can switch rapidly (@1 ms) and in any desired pattern (off - on1 - off - on2 - off, etc.); (3) it is driven by a low-power 15-V pulse; and (4) it is substantially cheaper than existing wavelength changers. Its limitations are that it does not pass wavelengths shorter than about 400 nm and transmission in the range 430-700 nm is only 20-40%.

Imaging Spreading Depression and Associated Intracellular Calcium Waves in Brain Slices

Spreading depression (SD) was analyzed in hippocampal and neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or imaging of intracellular calcium dynamics. The goal was to determine the roles of intracellular calcium (Ca2+int) waves in the generation and propagation of SD. Imaging of intrinsic optical signals in the hippocampus showed that ouabain consistently induced SD, which characteristically started in the CA1 region, propagated at 15-35 micrometer/sec, and traversed across the hippocampal fissure to the dentate gyrus. In the dendritic regions of both CA1 and the dentate gyrus, SD caused a transient increase in light transmittance, characterized by both a rapid onset and a rapid recovery. In contrast, in the cell body regions the transmittance increase was prolonged. Simultaneous imaging of intracellular calcium and intrinsic optical signals revealed that a slow Ca2+int increase preceded any change in transmittance. Additionally, a wave of increased Ca2+int typically propagated many seconds ahead of the change in transmittance. These calcium increases were also observed in individual astrocytes injected with calcium orange, indicating that Ca2+int waves were normally associated with SD. However, when hippocampal slices were incubated in calcium-free/EGTA external solutions, SD was still observed, although Ca2+int waves were completely abolished. Under these conditions SD had a comparable peak increase in transmittance but a slower onset and a faster recovery. These results demonstrate that although there are calcium dynamics associated with SD, these increases are not necessary for the initiation or propagation of spreading depression.

Giant Depolarizing Potentials: the Septal Pole of the Hippocampus Paces the Activity of the Developing Intact Septohippocampal ComplexIn Vitro

In neonatal hippocampal slices, recurrent spontaneous giant depolarizing potentials (GDPs) provide neuronal synchronized firing and Ca2+ oscillations. To investigate the possible role of GDPs in the synchronization of neuronal activity in intact neonatal limbic structures, we used multiple simultaneous electrophysiological recordings in the recently described preparation of intact neonatal septohippocampal complex in vitro. Combined whole-cell (in single or pairs of cells) and extracellular field recordings (one to five simultaneous recording sites) from the CA3 hippocampal region and various parts of the septum indicated that spontaneous GDPs, which can be initiated anywhere along the longitudinal hippocampal axis, are most often initiated in the septal poles of hippocampus and propagate to medial septum and temporal poles of both hippocampi simultaneously. GDPs were abolished in the medial septum but not in the hippocampus after surgical separation of both structures, suggesting hippocampal origin of GDPs. The preferential septotemporal orientation of GDP propagation observed in the intact hippocampus was associated with a corresponding gradient of GDP frequency in isolated portions of hippocampus. Accordingly, most GDPs propagated in the septotemporal direction in both septal and temporal hippocampal isolated halves, and whereas GDP frequency remained similar in the septal part of hippocampus after its surgical isolation, it progressively decreased in more temporally isolated portions of the hippocampus. Because GDPs provide most of the synaptic drive of neonatal neurons, they may modulate the development of neuronal connections in the immature limbic system.

Microcutting of living brain slices by a pulsed ultrafine water jet which allows simultaneous electrophysiological recordings (micromingotome)

Up to now microsurgical dissections in living nervous tissue (e.g. in slices or cell cultures) are performed either by micro-scalpels or by laser beams. As an alternative technique, a device for cutting with an ultrafine pulsed water jet was developed to allow precise, visually controled dissections in neuronal circuits even during electrophysiological recordings. Water is ejected by pressure (20–30 bar) from patch pipettes with tip diameters of 10–12 μm. By means of a piezo-element the pipette and the water jet are forced to oscillate vertically with a frequency of 200–400 Hz with an adjustable amplitude. These oscillations facilitate the transsection of neuronal connections even in thick slice preparations. Best results were obtained when the tip of the pipette was about 500 μm above the surface of the submerged slice tissue. This micromingotome offers the following advantages: (i) histological studies show that the water jet cleans the cutting surface, thus avoiding debris and its uncontrolable effects on cells underneath; (ii) the arrangement enables ongoing electrophysiological recordings from hippocampal slices during the cutting procedure and thus facilitates studies of the functions of neuronal connections; (iii) the device allows even disconnection in cultured nervous tissue overgrowing polyamid grids with 50 μm wide meshes.

Myenteric neurons of the rat descending colon: electrophysiological and correlated morphological properties.

Conventional intracellular electrophysiological recordings were made from 502 myenteric neurons of the rat descending colon. Myenteric neurons could be classified into three groups on the basis of distinct electrophysiological properties. The first group of neurons (51% of all neurons) fired tetrodotoxin-sensitive action potentials in response to direct somal depolarization and the majority (98%) of this group generated fast cholinergic excitatory synaptic potentials in response to focal stimulation and were therefore designated S/Type 1 neurons. The second group (40%) of neurons fired tetrodotoxin-insensitive action potentials which were followed by long-lasting membrane afterhyperpolarizations, hence were termed AH neurons. These neurons did not receive fast cholinergic synaptic inputs but ionophoretic application of acetylcholine induced rapid nicotinic cholinoceptor-mediated depolarizations. The final group of neurons (9%), named Type 3 neurons, received fast cholinergic synaptic inputs but could never be made to fire action potentials. Rundown in amplitude of successive fast excitatory synaptic potentials evoked by a short train of presynaptic nerve stimuli was observed in only a small proportion of neurons (8/37; 22%) with the majority of neurons (29/37; 78%) showing no such decrease in amplitude, even at frequencies of stimulation as high as 10 Hz. Superfusion of 5-hydroxytryptamine could induce both an inhibition and a facilitation of cholinergic fast synaptic transmission. Evidence was adduced that these presynaptic inhibitory and facilitatory actions appeared to be mediated via 5-hydroxytryptamine 1A and 5-hydroxytryptamine 4 receptors, respectively. Muscarinic slow excitatory synaptic potentials were not detected (9/9 neurons tested) and non-cholinergic slow excitatory synaptic potentials following repetitive focal presynaptic nerve stimulation were observed in only 39/502 (8%) of all neurons. In those neurons in which a demonstrable change in membrane input resistance was detectable, slow excitatory potentials were accompanied by an increased input resistance. In addition, in a small subset (4%) of S/Type 1 neurons, slow membrane hyperpolarizations accompanied by an increased membrane input resistance were observed following tetanic presynaptic nerve stimulation. Superfusion of 5-hydroxytryptamine induced both membrane depolarizations and hyperpolarizations. Membrane depolarizations were observed in 40% of all neuronal types (34% of S/Type 1 neurons, 58% of AH neurons and 11% of Type 3 neurons) and were accompanied by an increased membrane input resistance and occasionally, in S/Type 1 and AH neurons, by anodal break excitation or spontaneous action potential firing. Membrane hyperpolarizations were observed in S/Type 1 neurons (5%) only and were accompanied, unexpectedly, by an increased membrane input resistance. In those neurons that responded both to application of 5-hydroxytryptamine and tetanic presynaptic nerve stimulation, 5-hydroxytryptamine always mimicked the slow synaptic response indicating that 5-hydroxytryptamine may function as a slow synaptic mediator in some myenteric neurons. Myenteric neurons identified by intracellular injection of the neuronal marker Neurobiotin TM were found to conform to the morphological classification schemes proposed for myenteric neurons of the guinea-pig and porcine intestine, that is, Dogiel Types I and II and Stach Type IV neurons were present. Simultaneous electrophysiological recording and intracellular staining techniques revealed that a correlation existed between the electrophysiological and morphological properties of myenteric neurons of the rat colon, with electrophysiological classified S/Type 1 neurons having Dogiel Type I morphologies (95/108 neurons; 88%) and electrophysiological classified AH neurons having Dogiel Type II morphologies (87/94 neurons; 93%)...

Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices.

1. Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by activation of GABAA receptors (synaptic stimulation or application of the GABAA agonist isoguvacine) were studied on pyramidal cells and interneurons from hippocampal slices of rats from two age groups (postnatal days (P) 2-5 and P12-13) using the fluorescent dye fluo-3 and a confocal laser scanning microscope. Cells were loaded with the dye either intracellularly, using patch pipettes containing fluo-3 in the internal solution, or extracellularly, using pressure pulses applied to an extracellular pipette containing the permeant dye fluo-3 AM. 2. Interneurons and pyramidal cells from P2-5 slices loaded with fluo-3 AM responded by an increase in [Ca2+]i to isoguvacine and to glutamate, in contrast to cells from P12-13 slices which responded to glutamate but not to isoguvacine. 3. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the GABAA receptor antagonist bicuculline (20 microM), suggesting the specific involvement of GABAA receptors. The sodium channel blocker tetrodotoxin (TTX, 1 microM in the bath) did not prevent the isoguvacine-induced rise in [Ca2+]i. 4. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the calcium channel blocker D600 (50 microM) suggesting the involvement of voltage-dependent Ca2+ channels. 5. Electrical stimulation of afferent fibres induced a transient increase in [Ca2+]i in neonatal pyramidal cells and interneurons (P5) loaded non-invasively with fluo-3 AM. This elevation of [Ca2+]i was reversibly blocked by bicuculline (20 microM) but not by APV (50 microM) and CNQX (10 microM). 6. During simultaneous electrophysiological recording in the current-clamp mode and [Ca2+]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibres, in the presence of APV (50 microM) and CNQX (10 microM), caused synaptic depolarization accompanied by a few action potentials and a transient increase in [Ca2+]i. In voltage clamp (-70 mV) however, there was no increase in [Ca2+]i following synaptic stimulation, showing that it is depolarization dependent. 7. Using a non-invasive method of [Ca2+]i monitoring, we demonstrate here that in neonatal (P2-5) hippocampus, GABA is an excitatory neurotransmitter which can cause an elevation of [Ca2+]i in interneurons and pyramidal cells via activation of voltage-dependent Ca2+ channels. This action may underlie the trophic role of GABA in hippocampal development.

Fluorescence imaging of extracellular purinergic receptor sites and putative ecto-ATPase sites on isolated cochlear hair cells

Fluorescence imaging of extracellular adenosine-5'-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea pig organ of Corti was achieved using the fluorescent analog of ATP, 2'-(or-3')-O-(trinitrophenyl)adenosine-5'- triphosphate (TNP-ATP; 30-75 microM). This analog, which fluoresces on binding to these sites, was pressure applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution of extracellular binding sites, including the stereocilia, cuticular plate, and the basolateral margins of the cells, but particularly in infracuticular and infranuclear regions. In support of extracellular binding, simultaneous electrophysiological recordings demonstrated that rapid washout of TNP-ATP-induced fluorescence was dependent upon cell integrity. Suramin, a nonselective P2 purinoceptor antagonist, coapplied with TNP-ATP, reduced the fluorescence observed on the stereocilia and apical surface of the cuticular plate only. This implies that binding sites on the apical surface of hair cells are P2 receptors, consistent with previous electrophysiological evidence for localization of P2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P2 purinoceptors was confirmed by its antagonism of the inward current elicited by ATP (10 microM) in voltage-clamped hair cells. Fluorescence from the basolateral margin was significantly quenched when TNP-ATP was applied in divalent cation-free solution. Because divalent cations are required for ATPase activity, this finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The divalent cation-free condition had no significant effect on the ATP-gated P2 purinoceptor conductance. We propose that there are two classes of ATP binding sites on cochlear hair cells: apically located P2 purinoceptors gating nonselective cation channels and basolaterally located ecto-ATPases that may be involved in purine turnover.

Live Computerized Videomicroscopy of Cerebral Microvessels in Brain Slices

A model system for studying cerebral microvasculature is presented in which submerged in vitro brain slices are examined by computerized videomicroscopy. Brain slices are superfused continuously with artificial cerebrospinal fluid, while blood vessels are monitored using a transmission light microscope with water immersion objectives. The responses to well-characterized vasoactive compounds indicate that basic physiological characteristics are maintained in this preparation. This model system represents a simple and rapid technique for studying cerebrovascular responses under conditions in which vessels are surrounded by their normal cellular microenvironment. An additional advantage of this technique is the ability to perform simultaneous electrophysiological recordings in identified neurons. This will facilitate the study of interactions between neuronal and vascular elements and may help elucidate mechanisms underlying the local regulation of cerebral microvasculature.

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca 2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca 2+] i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K + resulted in rapid elevation of [Ca 2+] i to 500–1,000 nM showing recovery to baseline [Ca 2+] i over several minutes. The increases in [Ca 2+] i caused by K + depolarization were completely abolished by the removal of extracellular [Ca 2+], and were reduced by ∼80% by the ‘L-type’ Ca 2+ channel blocker, nimodipine (1 μM). [Ca 2+] i was also increased by the excitatory amino and l-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg 2+, large fluctuations in [Ca 2+] i were observed that were blocked by removal of extracellular Ca 2+, by tetrodotoxin (TTX), or by antagonists of N-methyl d-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg 2+ and TTX, NMDA caused dose-dependent increases in [Ca 2+] i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca 2+] i in the absence of extracellular Ca 2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca 2+ stores. Thapsigargin (2 μM) had little effect on [Ca 2+] i, or on the recovery from K + depolarization. Removal of extracellular Na + had little effect on basal [Ca 2+] i or on responses to high K +, suggesting that Na +/Ca 2+ exchange mechanisms do not play a significant role in the short-term control of [Ca 2+] i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca 2+] i; however, [Ca 2+] i recovered as normal from high K + stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca 2+] i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca 2+] i associated with action potential activity was measured. The amplitude of the [Ca 2+] i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca 2+] i from the modest Ca 2+ loads imposed on the neuron by action potential trains follows a simple exponential decay ( τ = 3–5s).

Sampling fluid from slice chambers by microsiphoning

We have developed a simple technique that continuously samples perfusion fluid from an in vitro slice chamber and permits simultaneous electrophysiological recordings from a slice. This procedure uses a microsiphon that diverts approximately 1.5% of the total effluent of artificial cerebrospinal fluid from an interface slice chamber into a collection tube at a lower level outside the chamber. The tip of the microsiphon may be moved anywhere within the chamber without disturbing the slice. For optimum sampling of the release of substances from one area of a slice, the tip is positioned immediately downstream from the location being studied. Concentrations of several amino acids were measured to identify significant spatial and temporal characteristics of the technique and to demonstrate that spontaneous and induced release of amino acids from slices can be measured with adequate sensitivity and time resolution.

Ion fluxes inAcetabularia: Vesicular shuttle

Ion flux relations in the unicellular marine alga Acetabularia have been investigated by uptake and washout kinetics of radioactive tracers (22Na+, 42K+, 36Cl- and 86Rb+) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K+ with influx and efflux of about 100 nmol.m-2.sec-1. K+ passes three- to sevenfold more easily than Rb+ does. Under normal conditions, Cl- (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mumol.m-2.sec-1, and a cytoplasmic as well as vacuolar [Cl-] of about 420 mM ([Cl-]o = 529 mM). At 4 degrees C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl-], are significantly reduced. Na+ ([Na+]i: about 70 mM, [Na+]o: 461 mM), which is of minor electrophysiological relevance compared to K+, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mumol.m-2.sec-1 for influx and efflux). Some results with Na+ and Cl- are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normal Acetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.

NMDA-, kainate- and quisqualate-stimulated release of taurine from electrophysiologically monitored rat hippocampal slices

While excitatory amino acids (EAAs) are known to evoke the release of taurine in the hippocampus, we have found that taurine is localized primarily in dendrites and only to a lesser extent in terminals in this region. To determine whether taurine is released as a neurotransmitter by non-toxic concentrations of EAAs, or exclusively as a neuroprotectant in response to excitotoxicity, we monitored the release of amino acids from hippocampal slices during simultaneous electrophysiological recording in the CA1 region to assess tissue viability. N-methyl-D-aspartate (NMDA) was the most potent of the EAA agonists tested for stimulating release of taurine. Exposure of slices to 120 microM NMDA increased the concentration of taurine in the perfusate to 1325% of its basal value. Kainate (KA) at a concentration of 128 microM increased taurine to 543% of baseline while quisqualate (Quis) at a concentration of 120 microM increase taurine to only 202% of its baseline value. Release of taurine in response to NMDA and KA peaked during the period when the concentration of the agonist was declining in the bath and did not return to its baseline value until 20 min after removal of the agonist. Increases in release of taurine were associated with concentrations of NMDA, KA, and Quis that caused an incomplete recovery of the CA1 field potential. These results suggest that taurine is primarily released by concentrations of glutamate receptor agonists that exhibit evidence of excitotoxicity in the CA1 region.

A new perfusion method adapted to rainbow trout ( Salmo gairdneri) brain pituitary preparation for simultaneous electrophysiological recording and hormone secretion determination

1. 1. The development of an in vitro brain-pituitary perfusion technique allowed both the measurement of gonadotropin release and the recording of electrical activity in various brain areas of the rainbow trout. 2. 2. GnRH sensitive neurons were found in the nucleus preopticus (NPO). 3. 3. Gonadotropin release was evoked after microinjections of LHRH-A into the pituitary or into the NPO. 4. 4. Norepinephrine has been demonstrated to have a stimulatory effect on gonadotropin release.

AN AUTOMATIC PELLET DISPENSER FOR PRECISE CONTROL OF FEEDING TOPOGRAPHY IN GRANIVOROUS BIRDS

Design and construction of an automatic pellet dispenser for granivorous birds are described. The dispenser permits rapid pneumatic delivery of pellets (five pellets per second maximum) to one controlled position and does not interfere with simultaneous electrophysiological recording. In addition, the device continuously indicates presence or absence of a pellet in the delivery position. This automatic dispenser proved very effective in our studies of stereotyped topographies of feeding in granivorous birds, such as pigeons and chickens.

Simultaneous recording of substantia nigra neurons and voltammetric release of dopamine in the caudate of behaving cats

Simultaneous electrophysiological recordings of single dopamine-containing neurons in the pars compacta of the substantia nigra and the voltammetric release of dopamine in the caudate were made in the behaving cat. Unit activity showed no significant changes during sleep and small changes during active waking, while the release of dopamine in post-synaptic target regions of the caudate nucleus decreased by approximately 35% during sleep and increased approximately 50% during movement. These data demonstrate that recording the electrophysiological activity of single dopamine-containing neurons alone does not accurately reflect the functional state of the central dopamine system. The present study is the first report on the simultaneous measurement of the post-synaptic release of a neurotransmitter and the electrophysiological recording of neurons identified to contain that transmitter substance.

A device for inverting the visual field of animals

A device for inverting the visual field of small animals is described. Binocular lenses are mounted in a lightweight, aluminum frame which, in turn, is secured to a surgically attached, dental acrylic skull cap. The construction of the device, and its usefulness when simultaneous electrophysiological recordings are desired, is described by illustrating the details of fabricating such a device for a cat.

The neural control of cercal grooming behaviour in the cockroach, Periplaneta americana

The pro- and mesothoracic ganglia were found to inhibit cercal grooming behaviour controlled by the metathoracic ganglion. After nerve cord transection between the meso- and metathoracic ganglia, repetitive grooming movements with the metathoracic legs spontaneously occur. In experiments where the head and thoracic ganglia were serially excised, maximum activity occurred when all ganglia anterior to the metathoracic were removed. Stimulation of cercal afferents has long been known to induce evasive behaviour involving giant interneurons, but in animals with the nerve cord sectioned between the meso- and metathoracic ganglia, cercal stimulation increased cercal grooming behaviour. With asymmetrical cercal stimulation, the homolateral metathoracic leg engages in significantly more cercal grooming activity than the heterolateral metathoracic leg, thus indicating that this spontaneous activity can be modified by sensory input. When cercal afferents are stimulated with the anterior thoracic ganglia present, the metathoracic legs are coordinated in evasive running movements. With the anterior thoracic ganglia removed, the same type of cercal stimulation induces increased grooming activity with the metathoracic legs rather than running movements. The ventral nerve cord posterior to the head ganglia is preset, therefore, for escape rather than grooming activity when the cerci are stimulated, with cercal grooming continuously inhibited by the anterior thoracic ganglia. Simultaneous electrophysiological recordings from the metathoracic nerve 5 and the abdominal nerve cord showed that the efferent motor rhythm for grooming behaviour is independent of ventilative activity, also known to originate in the metathoracic ganglion. Spike patterns during grooming movements showed that different groups of levator and depressor neurons were alternatively active. Carbon dioxide concentrations which stimulated the metathoracic ventilative output caused a breakdown of cercal grooming activity, thus indicating a basic difference in the neural mechanism producing the two rhythms.