Morphine and opioid peptides reduce paraventricular neuronal activity: studies on the rat hypothalamic slice preparation.

Extracellular discharges of neurons in the paraventricular nucleus (PVN) were recorded from slices of rat hypothalamus in vitro. PVN neurons (n = 14) were identified by the criteria of (i) phasic activity patterns and (ii) antidromic invasion from the neurohypophysial tract. Neurons not displaying either of these features were considered unidentified with respect to physiological function (n = 85). The majority of unidentified neurons responded to bath application of morphine (1 microM), [D-Ala2,Met5]enkephalin (1 microM) or beta-endorphin (0.01-1 microM) with a prompt, reversible, dose-related reduction in spike discharge frequency. Naloxone (1 microM) antagonized the opioid-induced depressions in some, but not all, cases. At the concentrations tested, no tachyphylaxis to the effects of the opioids was observed. The opioid effects on putative neurohypophysial neurons were less pronounced; while 2 were depressed, the remaining 12 displayed no change in frequency or pattern of discharge to micromolar concentrations of morphine, [D-Ala2,Met5]enkephalin, or beta-endorphin. Our results indicate that opioids depress neuronal activity in the rat PVN via an interaction with a specific opiate receptor but that this effect is more pronounced on unidentified neurons than on putative neurohypophysial neurons in the slice.     

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.     

Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity.

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate localizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cell populations, though in differing proportions. In the hippocampus, localizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity. Hippocampal pyramidal cells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.     

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.     

Existence of nitric oxide synthase in rat hippocampal pyramidal cells.

It has been proposed that nitric oxide (NO) serves as a key retrograde messenger during long-term potentiation at hippocampal synapses, linking induction of long-term potentiation in postsynaptic CA1 pyramidal cells to expression of long-term potentiation in presynaptic nerve terminals. However, nitric oxide synthase (NOS), the proposed NO-generating enzyme, has not yet been detected in the appropriate postsynaptic cells. We here demonstrate specific NOS immunoreactivity in the CA1 region of hippocampal sections by using an antibody specific for NOS type I and relatively gentle methods of fixation. NOS immunoreactivity was found in dendrites and cell bodies of CA1 pyramidal neurons. Cultured hippocampal pyramidal cells also displayed specific immunostaining. Control experiments showed no staining with preimmune serum or immune serum that was blocked with purified NOS. These results demonstrate that CA1 pyramidal cells contain NOS, as required were NO involved in retrograde signaling during hippocampal synaptic plasticity.     

Active summation of excitatory postsynaptic potentials in hippocampal CA3 pyramidal neurons

The manner in which the thousands of synaptic inputs received by a pyramidal neuron are summed is critical both to our understanding of the computations that may be performed by single neurons and of the codes used by neurons to transmit information. Recent work on pyramidal cell dendrites has shown that subthreshold synaptic inputs are modulated by voltage-dependent channels, raising the possibility that summation of synaptic responses is influenced by the active properties of dendrites. Here, we use somatic and dendritic whole-cell recordings to show that pyramidal cells in hippocampal area CA3 sum distal and proximal excitatory postsynaptic potentials sublinearly and actively, that the degree of nonlinearity depends on the magnitude and timing of the excitatory postsynaptic potentials, and that blockade of transient potassium channels linearizes summation. Nonlinear summation of synaptic inputs could have important implications for the computations performed by single neurons and also for the role of the mossy fiber and perforant path inputs to hippocampal area CA3.     

Enhancement of synaptic potentials in rabbit CA1 pyramidal neurons following classical conditioning

A synaptic potential elicited by high-frequency stimulation of the Schaffer collaterals was enhanced in hippocampal CA1 pyramidal cells from rabbits that were classically conditioned relative to cells from control rabbits. In addition, confirming previous reports, the after-hyperpolarization was reduced in cells from conditioned animals. We suggest that reduced after-hyperpolarization and enhanced synaptic responsiveness in cells from conditioned animals work in concert to contribute to the functioning of hippocampal CA1 pyramidal cells during classical conditioning.     

Enhanced sensitivity of hippocampal pyramidal neurons from mdx mice to hypoxia-induced loss of synaptic transmission.

The gene at the Duchenne/Becker muscular dystrophy locus encodes dystrophin, a member of a protein superfamily that links the actin cytoskeleton to transmembrane plasmalemmal proteins. In mature skeletal myocytes, the absence of dystrophin is associated with decreased membrane stability, altered kinetics of several calcium channels, and increased intracellular calcium concentration. In the central nervous system, dystrophin is restricted to specific neuronal populations that show heightened susceptibility to excitotoxic damage and is localized in proximal dendrites and the neuronal somata. We report that CA1 pyramidal neurons in a hippocampal slice preparation from a dystrophin-deficient mouse genetic model of Duchenne muscular dystrophy (the mdx mouse) exhibit significant increased susceptibility to hypoxia-induced damage to synaptic transmission. This selective vulnerability was substantially ameliorated by pretreatment with diphenylhydantoin, an anticonvulsant that blocks both sodium-dependent action potentials and low-threshold transient calcium conductances. These findings suggest that dystrophin deficiency could predispose susceptible neuronal populations to cumulative hypoxic insults that may contribute to the development of cognitive deficits in Duchenne/Becker muscular dystrophy patients and that the effects of such periods of hypoxia may be pharmacologically remediable.     

Conditioning-specific membrane changes of rabbit hippocampal neurons measured in vitro.

Intracellular recordings were made from hippocampal CA1 pyramidal neurons within brain slices of nictitating membrane conditioned, pseudoconditioned, and naive adult male albino rabbits. All neurons included (26 conditioned, 26 pseudoconditioned, and 28 naive) had stable penetration and at least 60 mV action potential amplitudes. Mean input resistances were approximately equal to 60 mu omega for the three groups. A marked reduction in the afterhyperpolarization (AHP) following an impulse was apparent for conditioned (x = -0.98 mV) as compared to the pseudoconditioned (x = -1.7 mV) and naive (x = -2.0 mV) neurons. The AHP has been attributed previously to activation of a Ca2+-dependent outward K+ current. The distribution of AHP amplitudes for the conditioned group included a new lower range of values for which there was little overlap with the other groups. The conditioning-specific reduction of AHP may be due to reduction of ICa2+-K+ as shown previously for conditioned Hermissenda neurons. This conditioning-induced biophysical alteration of the CA1 pyramidal cell must be stored by mechanisms intrinsic to the hippocampal slice and cannot be explained as a consequence of changes of presynaptic input arising elsewhere in the brain. Our experiments demonstrate the feasibility of analyzing cellular mechanisms of associative learning in mammalian brain with the in vitro brain slice technique.     

GABAB-receptor-activated K+ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures.

GABAB receptors are a subclass of receptors for gamma-amino-n-butyric acid (GABA) that are also activated by the antispastic drug beta-p-chlorophenyl-GABA (baclofen). One effect of baclofen is to inhibit excitatory transmission from CA3 to CA1 hippocampal pyramidal cells. To identify the ionic mechanism of GABAB-receptor-mediated depression, we have studied the effect of baclofen and GABA on ionic currents in voltage-clamped CA3 pyramidal cell somata in rat hippocampal slice cultures. Baclofen (10 microM) induced an inwardly rectifying outward current that reversed at -74 +/- 4.3 mV (mean +/- SD). This appeared to be a K+ current since (i) its reversal potential showed the expected shift when extracellular K+ concentration was changed and (ii) it was blocked by external Ba2+ or internal Cs+. The action of baclofen was closely imitated by GABA after the GABAA-mediated Cl- current had been abolished with pitrazepin (10 microM); under these conditions, GABA (100 microM) also produced an inwardly rectifying, Ba2+-sensitive current with a reversal potential identical to that of the baclofen-induced current. When outward currents were blocked with internal Cs+, the residual inward voltage-dependent Ca2+ current was not changed by baclofen. It is concluded that the primary effect of GABAB-receptor activation in these neurones is to increase K+ permeability rather than to reduce Ca2+ permeability.     

A postsynaptic transient K(+) current modulated by arachidonic acid regulates synaptic integration and threshold for LTP induction in hippocampal pyramidal cells

Voltage-gated ion channels in the dendrites and somata of central neurons can modulate the impact of synaptic inputs. One of the ionic currents contributing to such modulation is the fast inactivating A-type potassium current (I(A)). We have investigated the role of I(A) in synaptic integration in rat CA1 pyramidal cells by using arachidonic acid (AA) and heteropodatoxin-3 (HpTX3), a selective blocker of the Kv4 channels underlying much of the somatodendritic I(A). AA and HpTX3 each reduced I(A) by 60-70% (measured at the soma) and strongly enhanced the amplitude and summation of excitatory postsynaptic responses, thus facilitating action potential discharges. HpTX3 also reduced the threshold for induction of long-term potentiation. We conclude that the postsynaptic I(A) is activated during synaptic depolarizations and effectively regulates the somatodendritic integration of high-frequency trains of synaptic input. AA, which can be released by such input, enhances synaptic efficacy by suppressing I(A), which could play an important role in frequency-dependent synaptic plasticity in the hippocampus.     

Identification of pyramidal cells as the critical elements in hippocampal neuronal plasticity during learning.

The activity of single neurons recorded from rabbit hippocampus during classical conditioning of the nictitating membrane reflex was studied. All cells were first categorized according to their responses after fornix stimulation--i.i., antidromic activation, orthodromic activation, or no activation. The majority of cells that were antidromically activated--pyramidal cells--showed a highly positive correlation between the pattern of unit discharge and the topography of the nicititating membrane response within trial periods. Units that were orthodromically driven by fornix stimulation tended to inhibit during the presentation of trial stimuli, whereas most non-activated cells maintained low spontaneous levels of activity at all times. Thus, the major output neurons of the hippocampus appear to be the neuroanatomical substrate for the large and rapidly developing neuronal plasticity induced by this classical conditioning paradigm.     

Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro.

Pyramidal neurons in the rat hippocampus contain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to which the adrenal steroid corticosterone binds with differential affinity. We have used intracellular recording techniques to examine MR-mediated effects on membrane properties of CA1 pyramidal neurons in hippocampal slices from adrenalectomized rats. Low doses of corticosterone (1 nM) applied by perfusion for 20 min decreased the spike accommodation observed during a depolarizing current pulse (0.5 nA for 500 ms) and the amplitude of the subsequent afterhyperpolarization without affecting other membrane properties tested. The decrease became apparent ca. 15 min after steroid perfusion was started and reached its peak value 10-20 min after the steroid perfusion was terminated. The steroid effect was blocked by the MR antagonist spironolactone and mimicked by the natural MR ligand aldosterone (1 nM). Neurons recorded 30-90 min after termination of aldosterone application still displayed a decreased spike accommodation. However, 30-90 min after corticosterone application, the decrease in spike accommodation/afterhyperpolarization appeared to be reversed. Higher doses of corticosterone (greater than or equal to 30 nM) induced a significant increase in accommodation and amplitude of the afterhyperpolarization, as was previously observed for selective GR ligands. The data indicate that MR and GR activations induce opposite actions on the spike accommodation/afterhyperpolarization of CA1 pyramidal neurons, an important intrinsic mechanism of these neurons to regulate their response to excitatory input. We suggest that occupation of both MR and GR by the endogenous ligand corticosterone will result in an initial MR-mediated enhanced cellular excitability, which is gradually reversed and overridden by a GR-mediated suppression of cellular activity.     

Stress and the endogenous opioid system. I. Physiology and pharmacology of opioid peptides

In this article is given a survey on physiology and pharmacology of the opioid system. Opioid peptides are naturally occurring morphine-like acting metabolites of glycoprotein precursors. Of the opioid peptides proved hitherto in the organism met-, leu-enkephalin, dynorphin and beta-endorphin are characterized more in detail. Opioids react directly with opioid receptors. The opioid receptors are not a homogeneous system. Their distribution is depending on organs and species. Opioid peptides and receptors were proved within as well as outside the central nervous system. The main quantity of the endogenous beta-endorphin is stored in the pituitary gland. High concentrations of met-, leu-enkephalin and dynorphin are present in the gastrointestinal tract and in the adrenal glands. Opioid peptides are inactivated by enzymatic hydrolysis, in which case the splitting of the N-terminal tyrosine is decisive. The inactivation may be performed by amino peptidases, peptidyl dipeptide hydrolases or the angiotensin converting enzyme. The effect of the opioid peptides can be inhibited by opioid antagonists (naloxone, naloxazone). Up to now there are contradictory findings as to the presence of an endogenous opioid antagonist. In general, the presence of different opioid peptides and their different receptor preference indicate multiple functions in the organism. However, their physiological function is up to now only little clarified.     

Action potentials and membrane ion channels in clonal anterior pituitary cells.

The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55mV to 8.2 Hz at -25mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na+ spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+-dependent K+ channels with unitary conductances of approximately equal to 130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 microM) led to increases in burst frequency and beta-endorphin secretion mediated by activation of beta-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.     

Modeling back propagating action potential in weakly excitable dendrites of neocortical pyramidal cells.

Simultaneous recordings from the soma and apical dendrite of layer V neocortical pyramidal cells of young rats show that, for any location of current input, an evoked action potential (AP) always starts at the axon and then propagates actively, but decrementally, backward into the dendrites. This back-propagating AP is supported by a low density (-gNa = approximately 4 mS/cm2) of rapidly inactivating voltage-dependent Na+ channels in the soma and the apical dendrite. Investigation of detailed, biophysically constrained, models of reconstructed pyramidal cells shows the following. (i) The initiation of the AP first in the axon cannot be explained solely by morphological considerations; the axon must be more excitable than the soma and dendrites. (ii) The minimal Na+ channel density in the axon that fully accounts for the experimental results is about 20-times that of the soma. If -gNa in the axon hillock and initial segment is the same as in the soma [as recently suggested by Colbert and Johnston [Colbert, C. M. & Johnston, D. (1995) Soc. Neurosci. Abstr. 21, 684.2]], then -gNa in the more distal axonal regions is required to be about 40-times that of the soma. (iii) A backward propagating AP in weakly excitable dendrites can be modulated in a graded manner by background synaptic activity. The functional role of weakly excitable dendrites and a more excitable axon for forward synaptic integration and for backward, global, communication between the axon and the dendrites is discussed.     

Regulation of dopamine release in vitro from the posterior pituitary by opioid peptides

Opioid peptides are present in both the posterior pituitary (PP) and stalk-median eminence (SME). Their effects on the dopaminergic neurons in the SME are well documented, but little is known concerning their role in the regulation of dopamine (DA) release from the PP. The objectives of this study were (1) to develop an in vitro method suitable for examining the regulation of endogenous DA release from PP and SME, and (2) to describe and compare the effects of selected opioid peptides on potassium-evoked DA release from these tissues. Tissues were dissected from ovariectomized rats and incubated in media. After equilibration, two pulses of 28 mM potassium (K+), 3 min each, were delivered 30 min apart. Test substances were administered 20 min before the second K+ stimulus. DA in the media was determined by high-performance liquid chromatography. Potassium at 28 and 56 mM elicited a marked increase in DA release from the PP and SME; this was abolished by the removal of calcium. The opioid receptor antagonist, naloxone, significantly increased the release of DA from both PP and SME by 55%. Dynorphin A elicited a significant inhibition of DA release from PP and SME by 33 and 50%, respectively. In contrast, methionine enkephalinamide decreased DA release from the SME by 50%, but was without effect in the PP. The release of DA from both PP and SME was significantly inhibited by beta-endorphin, and this was reversed by naloxone. However, beta-endorphin was fourfold more effective in the SME. N-acetyl-beta-endorphin did not alter DA release. CONCLUSIONS: (1) we have developed a simple and sensitive in vitro method for studying the effects of hormones and drugs on the release of endogenous DA from PP and SME; (2) tuberoinfundibular dopaminergic and tuberohypophyseal dopaminergic nerve terminals are subjected to a similar inhibitory control by endogenous opioid peptides, and (3) exogenously applied opioid peptides exert differential effects on the release of DA from SME and PP which could be attributable to a dissimilar distribution of opioid receptor subtypes in these two tissues.     

Effects of cannabinoids on synaptic membrane enzymes. I. In vitro studies on synaptic membranes isolated from rat brain.

The understanding of the effects of cannabinoids in human subjects has been obscured by a lack of knowledge about how the various active principles from marijuana act at the cellular level in the brain. For this reason the present study was undertaken to determine the effects of cannabinoids on the enzymes associated with the synaptic membranes. Electron micrographic analysis was performed to determine the purity of synaptic membrane preparations from rat brain, and subsequently such preparations were subjected to additions of ethanol, Tween-80, 80% glycerol, and either delta-tetrahydrocannabinol, 11-hydroxy-delta-tetrahydrocannabinol, or cannabinol. Both sodium and potassium activated ATPase (Na, K-ATPase), and Mg-ATPase were measured as the micrometer orthophosphate (P) released per minute per microgram membrane protein and these specific activities of the enzymes expressed as absolute values and as the percentage depression brought about by the cannabinoids. The ATPase spcific activities are taken from the rate curve over a 30-min incubation time. Additionally, synaptic membrane acetylcholineesterase specific activity was measured by continuous rate enzyme assay. While as low as 10 M delta-tetrahydrocannabinol showed appreciable decrements in both the membrane-bound ATPases, the other cannabinoids did not show such a great depression in enzyme activity. The specific activity of acetylcholinesterase, which is weakly bound to the membrane, showed only slight or no changes in activity with the various cannabinoids. It was additionally shown that the cannabinoids, delta-tetrahydrocannabinol in particular, bound to the synaptic membranes almost irreversibly in the in vitro system, and that the vehicle for dissolving the cannabinoids, while used as background control values when calculating the percentage decrements in enzyme specific activity, did vary the effects on the ATPase enzymes in particular. These data are discussed in relation to psychotomimetic activity of the cannabinoids.