enkephalin--ala(2)-mephe(4)-gly(5)- and biocytin

The midbrain ventral tegmental area (VTA) contains neurons largely with either a dopaminergic (DAergic) or GABAergic phenotype. Physiological and pharmacological properties of DAergic neurons have been determined using tyrosine hydroxylase (TH) immunohistochemistry but many properties overlap with non-DAergic neurons presumed to be GABAergic. This study examined properties of GABAergic neurons, non-GABAergic neurons and TH-immunopositive neurons in VTA of GAD67-GFP knock-in mice. Ninety-eight per cent of VTA neurons were either GAD-GFP or TH positive,with the latter being five times more abundant. During cell-attached patch-clamp recordings, GAD-GFP neurons fired brief action potentials that could be completely distinguished from those of non-GFP neurons. Pharmacologically, the μ-opioid agonist DAMGO inhibited firing of action potentials in 92% of GAD-GFP neurons but had no effect in non-GFP neurons. By contrast, dopamine invariably inhibited action potentials in non-GFP neurons but only did so in 8% of GAD-GFP neurons. During whole-cell recordings, the narrower width of action potential in GAD-GFP neurons was also evident but there was considerable overlap with non-GFP neurons. GAD-GFP neurons invariably failed to exhibit the potassium-mediated slow depolarizing potential during injection of positive current that was present in all non-GFP neurons. Under voltage-clamp the cationic current, I(h), was found in both types of neurons with considerable overlap in both amplitude and kinetics. These distinct cellular properties may thus be used to confidently discriminate GABAergic and DAergic neurons in VTA during in vitro electrophysiological recordings.

Topics: Action Potentials; Animals; Dopamine; Dopaminergic Neurons; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABAergic Neurons; Gene Knock-In Techniques; Glutamate Decarboxylase; Green Fluorescent Proteins; Immunohistochemistry; Lysine; Male; Mice; Patch-Clamp Techniques; Potassium; Tyrosine 3-Monooxygenase; Ventral Tegmental Area

The neurotransmitter dopamine plays important roles in motor control, learning, and motivation in mammals and probably other animals as well. The strong dopaminergic projection to striatal regions and more moderate dopaminergic projections to other regions of the telencephalon predominantly arise from midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Homologous dopaminergic cell groups in songbirds project anatomically in a manner that may allow dopamine to influence song learning or song production. The electrophysiological properties of SNc and VTA neurons have not previously been studied in birds. Here we used whole cell recordings in brain slices in combination with tyrosine-hydroxylase immunolabeling as a marker of dopaminergic neurons to determine electrophysiological and pharmacological properties of dopaminergic and nondopaminergic neurons in the zebra finch SNc and VTA. Our results show that zebra finch dopaminergic neurons possess physiological properties very similar to those of mammalian dopaminergic neurons, including broad action potentials, calcium- and apamin-sensitive membrane-potential oscillations underlying pacemaker firing, powerful spike-frequency adaptation, and autoinhibition via D2 dopamine receptors. Moreover, the zebra finch SNc and VTA also contain nondopaminergic neurons with similarities (fast-firing, inhibition by the mu-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO)) and differences (strong h-current that contributes to spontaneous firing) compared with GABAergic neurons in the mammalian SNc and VTA. Our results provide insight into the intrinsic membrane properties that regulate the activity of dopaminergic neurons in songbirds and add to strong evidence for anatomical, physiological, and functional similarities between the dopaminergic systems of mammals and birds.

Topics: Action Potentials; Animals; Cholera Toxin; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Finches; Lysine; Male; Neurons; Patch-Clamp Techniques; Quinpirole; Receptors, Dopamine D2; Receptors, Opioid, mu; Substantia Nigra; Tetrodotoxin; Tyrosine 3-Monooxygenase; Ventral Tegmental Area

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Benzeneacetamides; Calcium Channels; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Immunohistochemistry; In Vitro Techniques; Lysine; Male; Medulla Oblongata; Membrane Potentials; Narcotic Antagonists; Neurons; Oligopeptides; Patch-Clamp Techniques; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Serotonin; Substance P; Tryptophan Hydroxylase

In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a significantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by mu-, delta(1)-, and kappa-selective opioids was significantly reduced when 1% but not < or =0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a significantly higher proportion of neurons fired spontaneous action potentials with either 0.05-0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward rectification were each significantly altered for neurons recorded with 1% but not 0.05-0.2% biocytin. These effects may be due to a relatively specific blockade of diverse potassium channel types. Because efficient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Benzeneacetamides; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Lysine; Membrane Potentials; Organ Culture Techniques; Patch-Clamp Techniques; Pyrrolidines; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Substantia Gelatinosa

Using the in vivo whole cell recording procedure described previously, we recorded 73 neurons in laminae I and II in the lumbar spinal cord of the rat. Input impedances averaged 332 MOmega, which indicated that prior sharp electrode recordings contained a significant current shunt. Characterization of the adequate stimuli from the excitatory hindlimb receptive field indicated that 39 of 73 neurons were nociceptive, 6 were innocuous cooling cells, 20 responded maximally to brush, and 8 cells were not excited by stimulation of the skin of the hindlimb. The locations of 15 neurons were marked with biocytin. Nociceptive neurons were mostly found in lamina I and outer II, cooling cells in lamina I, and innocuous mechanoreceptive cells were mostly found in inner II or in the overlying white matter. The mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-Enkephalin (DAMGO) hyperpolarized 7 of 19 tested neurons with a conductance increase. This hyperpolarization was reversed by naloxone in the neurons in which it was applied. DAMGO also decreased the frequency of spontaneous PSPs in 13 neurons, 7 of which were also hyperpolarized by DAMGO. Five of the seven hyperpolarized neurons were nociceptive, responding to both heat and mechanically noxious stimuli, whereas two responded to slow, innocuous brush. These results indicate that whole cell, tight seal recordings sample a similar population of lamina I and II neurons in the rat as those found with sharp electrode recordings in cat and monkey. They further indicate that DAMGO hyperpolarizes a subset of the nociceptive neurons that have input from both heat and mechanical nociceptors and that presynaptic DAMGO effects can be observed in nociceptive neurons that are not hyperpolarized by DAMGO.

Topics: Animals; Cold Temperature; Electric Stimulation; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Histocytochemistry; Hot Temperature; Lysine; Membrane Potentials; Microelectrodes; Narcotics; Neurons; Pain; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Receptors, Opioid, mu; Receptors, Presynaptic; Spinal Cord; Substantia Gelatinosa; Synaptic Membranes

1. The actions of opioids on membrane properties of rat periaqueductal gray neurones were investigated using intracellular recordings from single neurones in brain slices. Morphological properties and anatomical location of each impaled neurone were characterized by use of intracellular staining with biocytin. The present paper primarily considers neurones which were directly hyperpolarized by opioids. The accompanying paper considers inhibition of synaptic transmission by opioids. 2. Met-enkephalin (10-30 microM) hyperpolarized 29% (38/130) of neurones. The hyperpolarization was fully antagonised by naloxone (1 microM, n = 3). The response to Met-enkephalin was not affected by agents which block synaptic neurotransmission (1 microM tetrodotoxin, and 0.1 microM tetrodotoxin + 4 mM Co2+, n = 3). 3. The specific mu-receptor agonist, D-ala-met-enkephalin-glyol (3 microM, n = 17) produced hyperpolarizations of similar amplitude to those produced by Met-enkephalin (10-30 microM). The EC50 of D-ala-met-enkephalin-glyol was 80 nM and the maximum response was achieved at 1-3 microM. The delta-receptor (D-Pen-D-Pen-enkephalin, 3 microM, n = 7) and kappa-receptor (U50488H, 3 microM, n = 5) agonists had no effect on the membrane properties of these neurones. 4. The opioid-induced hyperpolarization was associated with an increased potassium conductance. Hyperpolarizations were accompanied by a significant decrease in membrane resistance between -70 and -80 mV, and a significantly greater decrease between -110 and -140 mV (n = 16). Hyperpolarizations reversed polarity at -111 +/- 3 mV (n = 16), close to the expected equilibrium potential for potassium ions. The reversal potential of outward currents increased by 24 mV when the extracellular potassium concentration was raised from 2.5 to 6.5 mM, which is close to the value predicted by the Nernst equation (25 mV) for a potassium conductance.5. Resting inward rectification (reduced input resistance at potentials more negative than - 100 mV in the absence of opioids) was significantly greater in neurones which were hyperpolarized by opioids than in those which were not hyperpolarized. The amplitude of action potential after hyperpolarizations was significantly smaller in neurones which were hyperpolarized by opioids. Other membrane properties did not differ significantly between opioid-sensitive and -insensitive neurones.6. Neurones hyperpolarized by opioids were multipolar (58%), triangular (21%) or fusiform (5%) in sha

Topics: Amino Acid Sequence; Analgesics; Animals; Cell Polarity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; In Vitro Techniques; Lysine; Male; Membrane Potentials; Molecular Sequence Data; Narcotics; Neurons; Patch-Clamp Techniques; Periaqueductal Gray; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Intracellular recordings were obtained from neurons in the paraventricular nucleus (PVN) of guinea-pig hypothalamic slices. Passive and active properties of the neurons were determined, and when possible, recorded neurons were injected with biocytin. The effects of a mu-receptor opioid agonist [D-Ala2, Nme-Phe4, Gly5-ol]enkephalin (DAGO) were studied in order to determine which types of cells have mu receptors and to test the hypothesis that an increase in K+ conductance causes mu-receptor-mediated inhibition in the PVN. The recorded cells inside the PVN were divided into two groups, primarily on the basis of the presence or absence of a low threshold Ca2+ spike (LTS). In one group of neurons, LTS potentials could not be evoked (non-LTS cells, n = 42). In another group of neurons (n = 35), LTS potentials with one or two Na+ spikes could be initiated with depolarizing pulses superimposed on steady hyperpolarizing currents; however, these neurons did not fire robust bursts (i.e. non-bursting LTS cells). The mean time constant of non-LTS cells (19.9 +/- 1.6 ms; mean +/- SEM) was significantly shorter than that of non-bursting LTS cells (26.7 +/- 2.1 ms). There were no differences in the mean resting membrane potential, spike amplitude, spike duration, input resistance, spike threshold and pattern of synaptic inputs between the two groups. Intracellular labeling with biocytin combined with cresyl violet counter-staining demonstrated that the two types of cells were located in the PVN.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Membrane; Electric Conductivity; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Lysine; Male; Membrane Potentials; Neurons; Paraventricular Hypothalamic Nucleus; Potassium; Receptors, Opioid, mu; Synapses

Intracellular recordings were made from hypothalamic arcuate (ARC) neurons with biocytin-filled electrodes under current- and voltage-clamp in slices prepared from ovariectomized guinea pigs which were pretreated with estradiol. Forty-three neurons were identified after linking the intracellular biocytin with streptavidin-FITC and subsequently were examined for beta-endorphin immunoreactivity. Ten of these neurons were immunoreactive for beta-endorphin. beta-Endorphin neurons displayed the following passive membrane properties: RMP:-56 +/- 2 mV; Rin: 439 +/- 66 M omega; tau: 17.5 +/- 2.4 ms; and often fired spontaneously (5.9 +/- 2.2 Hz). These membrane characteristics were not different from identified neurons in the ARC that were not immunoreactive for beta-endorphin. beta-Endorphin neurons exhibited instantaneous inward rectification and time-dependent rectification. The mu-opioid agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) decreased spontaneous firing, induced membrane hyperpolarization (12 +/- 2 mV; range 6-22 mV) and decreased the Rin (38 +/- 4%) of the beta-endorphin neurons. These effects of DAGO were blocked by the opioid antagonist naloxone (1 microM) and were not blocked by 1 microM TTX. DAGO-responsive cells were unaffected by either kappa- or delta-receptor opioid agonists. These results indicate that mu-receptors may be autoreceptors on ARC beta-endorphin neurons and that activation of opioid mu-receptors hyperpolarizes beta-endorphin neurons via an increase in K+ conductance. Therefore, opioid peptides may modulate opioid tone through an 'ultra-short loop' feedback control mechanism.

Topics: Action Potentials; Animals; Bacterial Proteins; beta-Endorphin; Cell Membrane; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Antibody Technique; Fluorescent Dyes; Guinea Pigs; Lysine; Membrane Potentials; Microscopy, Fluorescence; Naloxone; Neurons; Potassium Channels; Receptors, Opioid; Receptors, Opioid, mu; Streptavidin; Thiocyanates