Hylan C. Moises

Muscarinic responses of rat basolateral amygdaloid neurons recorded in vitro.

1. Intracellular recordings were obtained from pyramidal‐type neurons in the basolateral amygdaloid nucleus (BLA) in slices of rat ventral forebrain and used to compare the actions of exogenously applied cholinomimetics to the effects produced by electrical stimulation of amygdalopetal cholinergic afferents from basal forebrain. 2. Bath application of carbachol depolarized pyramidal cells with an associated increase in input resistance (Ri), reduced the slow after‐hyperpolarization (AHP) that followed a series of current‐evoked action potentials and blocked spike frequency accommodation. All of these effects were reversed by the muscarinic antagonist atropine but not by the nicotinic antagonist hexamethonium. 3. Electrical stimulation of amygdaloid afferents within the external capsule evoked a series of synaptic potentials consisting of a non‐cholinergic fast excitatory postsynaptic potential (EPSP), followed by early and late inhibitory postsynaptic potentials (IPSPs). Each of these synaptic potentials was reduced by carbachol in an atropine‐sensitive manner. 4. Local application of carbachol to pyramidal cells produced a short‐latency hyperpolarization followed by a prolonged depolarization. The hyperpolarization and depolarization to carbachol were blocked by atropine but not hexamethonium. 5. The carbachol‐induced hyperpolarization was associated with a decrease in Ri and had a reversal potential nearly identical to that of the early IPSP. The inhibitory response was blocked by perfusion of medium containing tetrodotoxin (TTX), bicuculline or picrotoxin, while the subsequent depolarization was unaffected. On the basis of these data, it is concluded that the muscarinic hyperpolarization is mediated through the rapid excitation of presynaptic GABAergic interneurons in the slice. 6. The findings that the carbachol‐induced depolarization was associated with an increase in Ri, often had a reversal potential below ‐80 mV, was sensitive to changes in extracellular potassium concentration and was blocked by intracellular ionophoresis of the potassium channel blocker caesium suggest that it resulted from a muscarinic blockade of one or more potassium conductances. 7. Repetitive stimulation of sites within the slice containing cholinergic afferents evoked a series of fast EPSPs followed by IPSPs. These non‐cholinergic potentials were followed by a slow EPSP that lasted from 10 s‐4 min. The slow EPSP was enhanced by eserine and blocked by atropine. It was also blocked by TTX or cadmium, indicating that it was dependent on spike propagation and calcium‐dependent release of acetylcholine (ACh). 8. Stimulation of cholinergic afferents in the slice mimicked other effects produced by carbachol including blockade of the slow AHP and accommodation of action potential discharge and these actions were potentiated by eserine and blocked by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro

The purpose of the present study was to characterize the ionic and pharmacological basis of the actions of synaptically released and exogenously applied GABA in basolateral amygdaloid pyramidal cells in vitro. Stimulation of forebrain afferents to pyramidal neurons in the basolateral amygdala evoked an excitatory postsynaptic potential followed by early and late inhibitory postsynaptic potentials. The early inhibitory postsynaptic potential had a reversal potential near -70 mV, was sensitive to changes in the chloride gradient across the membrane and was blocked by the GABAA antagonists picrotoxin and bicuculline methiodide but not by the GABAB antagonists phaclofen or 2-hydroxysaclofen. In contrast, the late inhibitory postsynaptic potential had a reversal potential of approximately -95 mV and was markedly reduced or abolished by GABAB antagonists. Pressure application of GABA to the surface of the slice typically elicited a triphasic response in basolateral amygdaloid pyramidal neurons consisting of a short-latency hyperpolarization that preceded or was superimposed on a membrane depolarization followed by a longer latency hyperpolarization. Each of the responses was associated with an increase in membrane conductance. Determinations of the reversal potential, ionic dependency and sensitivity to pharmacological blockade of each component of the GABA-induced response revealed that the initial hyperpolarizing (Erev approximately -70 mV) and depolarizing (Erev approximately -55 mV) responses were mediated by a GABAA-mediated increase in chloride conductance, whereas the late hyperpolarizing response (Erev approximately -82 mV) to GABA arose from a GABAB-mediated increase in potassium conductance. Experiments in which GABA was applied at various locations on the cell suggested that the short-latency hyperpolarization resulted from activation of somatic GABA receptors, whereas the depolarizing and late hyperpolarizing responses were generated primarily in the dendrites. In contrast to the complex membrane response profile elicited by GABA, pressure ejection of the GABAB agonist baclofen produced only membrane hyperpolarizations. Taken together, these results suggest that inhibitory responses that are recorded in basolateral amygdaloid pyramidal cells are mediated by activation of both GABAA and GABAB receptors. Consistent with findings elsewhere in the CNS, the early inhibitory postsynaptic potential and initial hyperpolarization and depolarizing response to local GABA application appear to involve a GABAA-mediated increase in chloride conductance, whereas the late inhibitory postsynaptic potential and the late hyperpolarizing response to GABA arise from a GABAB-mediated increase in potassium conductance.

Blood to Brain Sodium Transport and Interstitial Fluid Potassium Concentration during Early Focal Ischemia in the Rat

During partial ischemia, sodium and potassium ions exchange across the blood–brain barrier, resulting in a net increase in cations and brain edema. Since this exchange is likely mediated by specific transporters such as Na,K–ATPase in the capillary endothelium and because brain capillary Na,K–ATPase activity is stimulated by increased extracellular potassium in vitro, this study was designed to determine if the rate of blood to brain sodium transport is increased in ischemic tissue having an elevated interstitial fluid potassium concentration ([K]ISF) in vivo. Sprague-Dawley rats were studied between 2–3 h after occlusion of the right middle cerebral artery. To identify where cortical tissue with an elevated [K]ISF could be sampled for transport studies, the regional pattern of cerebral blood flow and [K]ISF was obtained in a group of 17 rats using hydrogen clearance and potassium-selective microelectrode techniques. We observed severely elevated [K]ISF (> 10 mM) when CBF was less than 20 ml 100 g−1 min−1 and mildly elevated levels at CBF between 20–45 ml 100 g−1 min−1. In a second group of seven rats, permeability-surface area products (PS products) for 22Na and [3H]α-aminoisobutyric acid ([3H]AIB) were determined in ischemic cortex with elevated [K]ISF and in nonischemic cortex. The PS products for AIB were similar in both tissues (2.2 ± 0.7 and 2.1 ± 0.4 μl/g/min) while the PS products for sodium was significantly increased in the ischemic tissue (1.5 ± 0.2 and 2.4 ± 1.1 μl/g/min). This study demonstrates that blood to brain sodium transport is increased in ischemic tissue at early times before the BBB is disrupted. Stimulation of the Na,K pumps in the capillary endothelium by elevated [K]ISF may mediate this effect.

Muscarinic inhibition of M-current and a potassium leak conductance in neurones of the rat basolateral amygdala.

1. Voltage‐clamp recordings using a single microelectrode were obtained from pyramidal neurones of the basolateral amygdala (BLA) in slices of the rat ventral forebrain. Slow inward current relaxations during hyperpolarizing voltage steps from a holding potential of ‐40 mV were identified as the muscarinic‐sensitive M‐current (IM), a time‐ and voltage‐dependent potassium current previously identified in other neuronal cell types. 2. Activation of IM was voltage dependent with a threshold of approximately ‐70 mV. At membrane potentials positive to this, the steady‐state current‐voltage (I‐V) relationship showed substantial outward rectification, reflecting the time‐ and voltage‐dependent opening of M‐channels. The underlying conductance (gM) also increased sharply with depolarization. 3. The reversal potential for IM was ‐84 mV in medium containing 3.5 mM K+. This was shifted positively by 27 mV when the external K+ concentration was raised to 15 mM. 4. The time courses of M‐current activation and deactivation were fitted by a single exponential. The time constant for IM decay, measured at 24 degrees C, was strongly dependent on membrane potential, ranging from 330 ms at ‐40 mV to 12 ms at ‐100 mV. 5. Bath application of carbachol (0.5‐40 microM) inhibited IM, as evidenced by the reduction or elimination of the slow inward M‐current relaxations evoked during hyperpolarizing steps from a holding potential of ‐40 mV. The outward rectification of the steady‐state I‐V relationship at membrane potentials positive to ‐70 mV was also largely eliminated. The inhibition of IM by carbachol was dose dependent and antagonized by atropine. 6. Carbachol produced an inward current shift at a holding potential of ‐40 mV that was only partially attributable to inhibition of IM. An inward current shift was also produced by carbachol at membrane potentials negative to ‐70 mV, where IM is inactive. These effects were dose dependent and antagonized by atropine. They were attributed to the muscarinic inhibition of a voltage‐insensitive potassium leak conductance (ILeak). 7. In most cells, carbachol reduced the slope of the instantaneous I‐V relationship obtained from a holding potential of ‐70 mV so that it crossed the control I‐V plot at the reversal potential for ILeak. This was found to be ‐108 mV in 3.5 mM K+ saline, shifting to ‐66 mV in 15 mM K+ saline.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological effects of dynorphin peptides on hippocampal pyramidal cells in rat

Single-unit extracellular recording was carried out in rats to characterize the effects of dynorphin and several structurally related peptides on hippocampal pyramidal cell activity. Dynorphin, applied electrophoretically or by pneumatic pressure, produced a dose-dependent depression of both spontaneous and glutamate-evoked discharge in a majority (63%) of CA1 and CA3 cells tested. In addition, a small number of cells in both cellular fields responded to the peptide with a prolonged elevation in firing. The inhibitory effects of dynorphin were not blocked by naloxone. Moreover, administration of des-tyrosine-dynorphin depressed the firing of pyramidal cells in a manner similar to that of the parent compound. Ethylketocyclazocine produced a mixed pattern of excitatory and inhibitory effects, whereas naloxone-sensitive elevations in firing were most often observed with the application of dynorphin-(1-8). Application of [Leu5]enkephalin produced only facilitations in pyramidal cell firing. The possibility is raised that biologically significant non-opiate actions, in addition to potent opiate-mediated effects, may occur upon release of pro-dynorphin peptides in the hippocampus.

Gastrointestinal-projecting neurones in the dorsal motor nucleus of the vagus exhibit direct and viscerotopically organized sensitivity to orexin

Orexin (hypocretin)‐containing projections from lateral hypothalamus (LH) are thought to play an important role in the regulation of feeding behaviour and energy balance. In rodent studies, central administration of orexin peptides increases food intake, and orexin neurones in the LH are activated by hypoglycaemia during fasting. In addition, administration of orexins into the fourth ventricle or the dorsal motor nucleus of the vagus (DMV) has been shown to stimulate gastric acid secretion and motility, respectively, via vagal efferent pathways. In this study, whole‐cell recordings were obtained from DMV neurones in rat brainstem slices to investigate the cellular mechanism(s) by which orexins produce their gastrostimulatory effects. To determine whether responsiveness to orexins might be differentially expressed among distinct populations of preganglionic vagal motor neurones, recordings were made from neurones whose projections to the gastrointestinal tract had been identified by retrograde labelling following apposition of the fluorescent tracer DiI to the gastric fundus, corpus or antrum/pylorus, the duodenum or caecum. Additionally, the responses of neurones to orexins were compared with those produced by oxytocin, which acts within the DMV to stimulate gastric acid secretion, but inhibits gastric motor function. Bath application of orexin‐A or orexin‐B (30–300 nm) produced a slow depolarization, accompanied by increased firing in 47 of 102 DMV neurones tested, including 70 % (30/43) of those that projected to the gastric fundus or corpus. In contrast, few DMV neurones that supplied the antrum/pylorus (3/13), duodenum (4/18) or caecum (1/13) were responsive to these peptides. The depolarizing responses were concentration dependent and persisted during synaptic isolation of neurones with TTX or Cd2+, indicating they resulted from activation of postsynaptic orexin receptors. They were also associated with a small increase in membrane resistance, and in voltage‐clamp recordings orexin‐A induced an inward current that reversed near the estimated equilibrium potential for K+, indicating the depolarization was due in large part to a reduction in K+ conductance. Orexins did not affect synaptically evoked excitation, but did reduce membrane excitability in a subset of gastric‐projecting DMV neurones by enhancing GABA‐mediated synaptic input. Lastly, although many DMV neurones responded to orexins and oxytocin with excitation, for the most part these peptides modulated excitability of distinct populations of gastric‐projecting vagal motor neurones. These results indicate that orexins act preferentially within the DMV to directly excite vagal motor neurones that project to gastric fundus and corpus. In this way, release of endogenous orexins from descending hypothalamic projections into the DMV may mediate the increase in gastric acid secretion and motor activity associated with the cephalic phase of feeding.

Dynorphin (1–17): Lack of analgesia but evidence for non-opiate electrophysiological and motor effects

Abstract Dynorphin and an opiate-inactive fragment des-Tyr-dynorphin produced similar effects on EEG, motor function and hippocampal unit firing. Naloxone had no effect on the actions of dynorphin in these systems and dynorphin failed to produce analgesia upon central administration. These results suggest that dynorphin has a pharmacological character that differs significantly from the classic narcotics.

μ-Opioid receptor activation inhibits N- and P-type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus

1 The whole‐cell voltage‐clamp technique was used to examine opioid regulation of Ba2+ currents (IBa) through voltage‐sensitive Ca2+ channels in isolated magnocellular supraoptic neurones (MNCs). The effects of local application of μ‐, δ‐ or κ‐opioid receptor selective agonists were examined on specific components of high voltage‐activated (HVA) IBa, pharmacologically isolated by use of Ca2+ channel‐subtype selective antagonists. 2 The μ‐opioid receptor selective agonist, DAMGO, suppressed HVA IBa (in 64/71 neurones) in a naloxone‐reversible and concentration‐dependent manner (EC50= 170 nm, Emax= 19.5 %). The DAMGO‐induced inhibition was rapid in onset, associated with kinetic slowing and voltage dependent, being reversed by strong depolarizing prepulses. Low‐voltage activated (LVA) IBa was not modulated by DAMGO. 3 Administration of κ‐ (U69 593) or δ‐selective (DPDPE) opioid receptor agonists did not affect IBa. However, immunostaining of permeabilized MNCs with an antibody specific for κ1‐opioid receptors revealed the presence of this opioid receptor subtype in a large number of isolated somata. 3 μ‐Opioid‐induced inhibition in IBa was largely abolished after blockade of N‐type and P‐type channel currents by ω‐conotoxin GVIA (1 μm) and ω‐agatoxin IVA (100 nm), respectively. Quantitation of antagonist effects on DAMGO‐induced reductions in IBa revealed that N‐ and P‐type channels contributed roughly equally to the μ‐opioid sensitive portion of total IBa. 4 These results indicate that μ‐opioid receptors are negatively coupled to N‐ and P‐type Ca2+ channels in the somatodendritic regions of MNCs, possibly via a membrane‐delimited G‐protein‐dependent pathway. They also support a scheme in which opioids may act in part to modulate cellular activity and regulate neurosecretory function by their direct action on the neuroendocrine neurones of the hypothalamic supraoptic neucleus.

μ-Opioid and GABAB receptors modulate different types of Ca2+ currents in rat nodose ganglion neurons

Whole-cell patch-clamp recordings were obtained from nodose ganglion neurons acutely dissociated from 10-30-day-old rats to characterize the Ca2+ channel types that are modulated by GABA(B) and mu-opioid receptors. Five components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nifedipine, omega-agatoxin IVA and omega-conotoxin MVIIC. Administration of the mu-opioid agonist H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (0.3-1 mM) or the GABA(B) agonist baclofen in saturating concentrations suppressed high-threshold Ca2+ currents by 49.9+/-2.4% (n=69) and 18.7+/-2.1% (n=35), respectively. The inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol exceeded that by baclofen in virtually all neurons that responded to both agonists (67%), and occlusion experiments revealed that responses to mu-opioid and GABA(B) receptor activation were not linearly additive. In addition, administration of staurosporine, a non-selective inhibitor of protein kinase A and C, did not affect the inhibitory responses to either agonist or prevent the occlusion of baclofen-induced current inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol. Blockade of N-type channels by omega-conotoxin GVIA eliminated current suppression by baclofen in all cells tested (n=11). Mu-opioid-induced inhibition in current was abolished by omega-conotoxin GVIA in 12 of 30 neurons tested, but was only partially reduced in the remaining 18 neurons. In the latter cells administration of omega-agatoxin IVA reduced, but did not eliminate the mu-opioid sensitive current component that persisted after blockade of N-type channels. This residual component of mu-opioid-sensitive current was blocked completely by omega-conotoxin MVIIC in nine neurons, whereas responses to H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol were still recorded in the remaining cells after administration of these Ca2+ channel toxins and nifedipine. Dihydropyridine-sensitive (L-type) current was not affected by activation of mu-opioid or GABA(B) receptors in any of the neurons. These data indicate that in nodose ganglion neurons mu-opioid receptors are negatively coupled to N-, P- and Q-type channels as well as to a fourth, unidentified toxin-resistant Ca2+ channel. In contrast, GABA(B) receptors are coupled only to N-type channels. Furthermore, the results do not support a role for either protein kinase C or A in the modulatory pathway(s) coupling mu-opioid and GABA(B) receptors to Ca2+ channels, but rather lend credence to the notion that the signalling mechanisms utilized by these two receptors might simply compete for inhibitory control of a common pool of N-type channels.

Ca2+ and frequency dependence of exocytosis in isolated somata of magnocellular supraoptic neurones of the rat hypothalamus

In addition to action potential‐evoked exocytotic release at neurohypophysial nerve terminals, the neurohormones arginine vasopressin (aVP) and oxytocin (OT) undergo Ca2+‐dependent somatodendritic release within the supraoptic and paraventricular hypothalamic nuclei. However, the cellular and molecular mechanisms that underlie this release have not been elucidated. In the present study, the whole‐cell patch‐clamp technique was utilized in combination with high‐time‐resolved measurements of membrane capacitance (Cm) and microfluorometric measurements of cytosolic free Ca2+ concentration ([Ca2+]i) to examine the Ca2+ and stimulus dependence of exocytosis in the somata of magnocellular neurosecretory cells (MNCs) isolated from rat supraoptic nucleus (SON). Single depolarizing steps (≥20 ms) that evoked high‐voltage‐activated (HVA) Ca2+ currents (ICa) and elevations in intracellular Ca2+ concentration were accompanied by an increase in Cm in a majority (40/47) of SON neurones. The Cm responses were composed of an initial Ca2+‐independent, transient component and a subsequent, sustained phase of increased Cm (termed ΔCm) mediated by an influx of Ca2+, and increased with corresponding prolongation of depolarizing step durations (20–200 ms). From this relationship we estimated the rate of vesicular release to be 1533 vesicles s−1. Delivery of neurone‐derived action potential waveforms (APWs) as stimulus templates elicited ICa and also induced a ΔCm, provided APWs were applied in trains of greater than 13 Hz. A train of APWs modelled after the bursting pattern recorded from an OT‐containing neurone during the milk ejection reflex was effective in supporting an exocytotic ΔCm in isolated MNCs, indicating that the somata of SON neurones respond to physiological patterns of neuronal activity with Ca2+‐dependent exocytotic activity.