Acute effects of lithium on dopaminergic responses: iontophoretic studies in the rat visual cortex

The interactions between lithium and cortical dopaminergic receptors were investigated using the iontophoretic technique to record and apply dopaminergic compounds, GABA, acetylcholine and LiCl on neurons in the primary visual cortex of the rat. The main responses to dopamine (DA) or to the D1 agonist (+/- )SKF38393 on spontaneously-active (SA) or visually-driven (VD) units was a prolonged decrease in firing and a reduction in the responsiveness to pulses of acetylcholine. The D1 antagonist SCH23390, applied iontophoretically or intravenously, blocked or attenuated the inhibitory responses to both DA and (+/- )SKF38393. The D2 agonist quinpirole (LY171555) either produced only slight excitations or had no effects on both VD and SA units. The concomitant application of lithium blocked the inhibitory responses to DA and to (+/- )SKF38393 but did not modify the responsiveness to LY171555. In addition, the DA- and (+/- )SKF38393-induced decreases in responsiveness to acetylcholine were also suppressed by lithium. These effects were on dopaminergic mechanisms, since the excitatory responses to acetylcholine alone as well as the inhibitions caused by GABA were unchanged by the application of lithium. These results imply that the modifications in sensitivity to dopaminergic agents induced by lithium are mediated by dopamine D1 receptors and are discussed in relation to adenylate-cyclase.     

Penicillin effects on iontophoretic responses in Aplysia californica

The effect of penicillin on neurons of Aplysia californica was studied using drug concentrations which would be convulsant in mammalian nervous systems. Iontophoretic responses were elicited by the application of acetylcholine, dopamine, gamma-aminobutyric acid and serotonin. Low concentrations of penicillin (2 mM) consistently and reversibly reduced the chloride-dependent hyperpolarizing responses by approximately 70%, regardless of the transmitter required to evoke them. The short depolarizing responses which are sodium sensitive are slightly reduced by a much higher (10 mM) concentration. The extent of the reduction of the excitatory response varied with the transmitter. The slow sodium-dependent depolarizations and the slow potassium-dependent hyperpolarizations were unaffected by the concentrations of penicillin used. The possibility that the convulsant effect of penicillin is due to interference with membrane conductance to chloride is discussed.     

Influence of noncholinergic drugs on rat striatal acetylcholine levels

Rat striatal acetylcholine levels were measured in rats following the administration of drugs that primarily affect dopamine metabolism. Reserpine, clozapine and chlorpromazine caused decreases in striatal acetylcholine levels of 33%, 38% and 54% respectively. Apomorphine, L-DOPA, methamphetamine and amantadine caused increases of 27%, 14%, 17% and 45% respectively. The decrease in acetylcholine levels following drugs that deplete or block dopamine, and the increase following drugs that enhance dopamine or stimulate its receptors, is consistent with a direct inhibitory action of dopamine on cholinergic striatal interneurons. Increased levels of acetylcholine would accompany decreased firing of cholinergic neurons due to dopaminergic inhibitory action. Decreased levels would accompany increased firing due to reduced dopaminergic inhibitory action.     

Central effects of systemic lidocaine mediated by glycine spinal receptors: an iontophoretic study in the rat spinal cord

The objective of this investigation was to demonstrate the possible interactions of systemic lidocaine (lido) with inhibitory receptors in the spinal cord. In the lumbar dorsal horn of anesthetized and curarized rats, 60 physiologically identified, wide dynamic range (WDR) neurons, were recorded extracellularly. Glutamate, glycine and its selective antagonist, strychnine, were iontophoretically applied onto the neurons either singularly or concurrently. The effects of systemic lido on the drug-induced frequency changes and the interaction with the glycine receptors, using strychnine as a probe, were studied. It was consistently found that (i) lido (3–4 mg/kg) inhibited the excitatory responses to iontophoretic glutamate, (ii) this inhibition was significantly antagonized by concurrent iontophoretic strychnine, (iii) iontophoretic glycine induced comparable glutamate inhibition that was reversed by strychnine. In contrast, no effect on glutamate-induced excitations was observed when lido was applied by micropressure or a different local anesthetic was systemically administered. The results suggest that central inhibitory effects of lido could by mediated by spinal strychnine-sensitive glycine receptors, activated by lido itself or possibly by its glycine residue-bearing metabolites.     

Studies on the pharmacology of neurones in the nucleus accumbens of the rat.

A study was made of the effects of iontophoretically applied drugs on single neurones in the nucleus accumgens and caudate nucleus of rats anaesthetized with urethane. Neurones in the caudate nucleus were inhibited by dopamine, dibutyryl cyclic AMP, ADTN and ergometrine. Acetylcholine and homocysteic acid caused excitation of striatal neurones. In the nucleus accumbens neurones were inhibited by dopamine, ADTN, ergometrine, dibutyryl cyclic AMP, glycine and gamma-aminobutyric acid. The responses of glycine and gamma-aminobutyric acid were antagonised by strychnine and picrotoxin, respectively. Acetylcholine and homocysteic acid caused excitation of neurones in the nucleus acumbens; the effects of acetylcholine were blocked by atropine. The results are consistent with the suggestion that dopamine is an inhibitory transmitter in the nucleus accumbens and in the caudate nucleus and support the hypothesis that the effects of dopamine are mediated by cyclic AMP. The locomotor stimulants ADTN and ergometrine mimicked the inhibitory actions of dopamine in both the striatum and in the nucleus accumbens. These results support the suggestion that dopamine receptors in the nucleus accumbens are involved in the actions of locomotor stimulant drugs.     

GABA-activated Chloride Currents of Postnatal Mouse Retinal Ganglion Cells are Blocked by Acetylcholine and Acetylcarnitine: How Specific are Ion Channels in Immature Neurons?

The goal of this study was to clarify pharmacological properties of GABA_A receptors in cells of the mouse retinal ganglion cell layer in situ. Spontaneous synaptic currents and responses to exogenous GABA were recorded from individual neurons in retinal whole mounts (postnatal days 1–3) or retinal stripe preparations (postnatal days 4–6). Drugs were applied by a fast local superfusion system. Current responses were measured with the patch-clamp technique in the whole-cell configuration. All cells responded to exogenous GABA (average EC₅₀ and Hill coefficient: 16.7 μM and 0.95 respectively) and generated GABAergic synaptic currents in response to elevated KCI. GABA-induced currents of retinal ganglion cells were blocked by bicuculline, picrotoxin and Zn²⁺, as well as strychnine, and increased by pentobarbital, clonazepam and 3α-hydroxy-5α-pregnan-20-one. In some retinal ganglion cells GABA caused an increase in the frequency of spontaneous synaptic currents, which points to a partially depolarizing action of this traditionally inhibitory neurotransmitter in the neural retina. Our major observation is that acetylcholine and acetylcarnitine blocked or reduced GABAergic inhibitory postsynaptic currents and responses to exogenous GABA. This effect was seen in only a fraction of retinal ganglion cells and occurred in both the undesensitized and the desensitized state of the GABA_A receptor. The block was voltage-independent and persisted during coapplication with the nicotinic and muscarinic acetylcholine receptor antagonists D-tubocurarine and atropine. In contrast to GABA-activated Cl⁻ currents, glycine-activated Ch currents remained unaffected by acetylcholine and acetylcarnitine. Acetylcarnitine had no effect on voltage-activated Ca²⁺ channel currents and glutamate-activated currents. Similar results were obtained in a dissociated cell culture preparation from the neonatal rat superior colliculus. In these cells acetylcholine induced a rightward shift in the dose - response curve for GABA. Taken together, these results indicate that acetylcholine and acetylcarnitine can act directly at the GABA_A binding site and thereby reduce the action of GABA in the immature retina.     

Postsynaptic pharmacology of psychoactive substances in Aplysia californica

The effects of antidepressants and neuroleptics on postsynaptic iontophoretic responses to acetylcholine and dopamine have been examined in identified neurons of the central nervous system of Aplysia californica. The results demonstrate an antidopaminergic action of neuroleptics in accordance with in vivo and in vitro results in higher animals. Some of the antidepressant drugs tested also caused a blockade of dopamine hyperpolarizing responses. The incidence of anticholinergic activity of both antidepressants and neuroleptics also correlates well with other systems. The results furthermore suggest that the psychoactive drugs tested behave as antagonists for the neurotransmitter postsynaptic receptor sites.     

Modulatory action of acetylcholine on striatal neurons: microiontophoretic study in awake,unrestrained rats

Cholinergic interneurons innervate virtually all medium spiny striatal cells, but the relevance of this input in regulating the activity and afferent responsiveness of these cells remains unclear. Studies in anaesthetized animals and slice preparations have shown that iontophoretic acetylcholine (ACh) either weakly excites or inhibits striatal neurons. These differential responses may reflect cholinergic receptor heterogeneity but may be also related to the different activity states of recorded units and different afferent inputs specific in each preparation. Single-unit recording was combined with iontophoresis in awake, unrestrained rats to examine the effects of ACh and selective muscarinic (oxotremorine M or Oxo-M) and nicotinic agonists (nicotine or NIC) on dorsal and ventral striatal neurons. These effects were tested on naturally silent, spontaneously active and glutamate-stimulated units. We found that iontophoretic ACh primarily inhibited spontaneously active and glutamate-stimulated units; the direction of the ACh response, however, was dependent on the firing rate. The effects of ACh were generally mimicked by Oxo-M and, surprisingly, by NIC, which is known to excite units in most central structures, including striatal neurons in anaesthetized preparation. Given that NIC receptors are absent on striatal cells but located primarily on dopamine terminals, we assessed the effects of NIC after complete blockade of dopamine receptors induced by systemic administration of a mixture of D1 and D2 antagonists. During dopamine receptor blockade the number of NIC-induced inhibitions dramatically decreased and NIC had mainly excitatory effects on striatal neurons. Thus, our data suggest that under physiologically relevant conditions ACh acts as a state-dependent neuromodulator, and its action involves not only postsynaptic but also presynaptic cholinoreceptors located on dopamine- and glutamate-containing terminals.     

Trimethadione effects on cholinergic responses in Aplysia neurons

The anticonvulsant, trimethadione (TMO), was tested for effects on cholinergic responses to iontophoretic application of ACh on identified neurons in Aplysia. TMO (1–10 mM) depressed the amplitudes of depolarizing responses mediated by Na⁺ and hyperpolarizing responses mediated by Cl⁻ but did not affect hyperpolarizing responses mediated by K⁺. Such a combination of effects on cholinergic responses distinguishes the action of this anti-absence drug from agents effective against tonic-clonic seizures and from convulsant drugs.     

Two pharmacologically distinct histamine receptors mediating membrane hyperpolarization on identified neurons of Aplysia californica.

Two distinct hyperpolarizing responses are produced when histamine is iontophoretically applied onto the somal membranes of identified neurons within the cerebral ganglion of Aplysia: a biphasic response consisting of a rapid component (less than 5 sec) usually superimposed upon a slowly developing component; or a monophasic slowly developing response 5-20 sec in duration. The reversal potential values for the fast (typically -65 mV) and the slow (typically -89 mV) responses, and their shift to new values when the external potassium or chloride concentrations were altered, revealed that the fast and slow potentials are produced predominantly by conductance increases to chloride and potassium ions, respectively. The effects of histamine H1- and H2-receptor agonists and antagonists were studied to characterize the pharmacological properties of histamine receptors mediating these two ionically dissimilar hyperpolarizing responses. The slow potassium-dependent hyperpolarization could be mimicked by several histamine analogues; the most potent tested were the H1-receptor agonist, 2-methylhistamine, and the H2-receptor agonist, 4-methylhistamine. Neither of these agents mimicked the fast chloride-dependent histamine response. The slow potassium-dependent responses induced by histamine or histamine agonists were completely and reversibly blocked by the H2-receptor antagonist, cimetidine. By contrast, the slow potassium-dependent hyperpolarizations produced by iontophoretically applied acetylcholine or by dopamine to the same neurons were unaffected by cimetidine. Other H1 and H2 antagonists tested were either ineffective, or only partially blocked the slow hyperpolarizations in a non-selective manner. The fast chloride-dependent hyperpolarizations were not selectively antagonized by any of the H1 or H2 reagents tested, although they were effectively suppressed by tubocurarine and strychnine. These data indicate that two pharmacologically distinct histamine receptors mediate potassium- and chloride-dependent hyperpolarizations in Aplysia neurons. Neither of these receptors, however, could be classified as strictly H1 or H2 according to criteria presently used in non-neuronal tissues. The selectivity and reversibility of cimetidine indicate that this particular antihistaminic could be a valuable pharmacological tool for defining putative histaminergic synapses in Aplysia and perhaps other nervous systems.     

Norepinephrine modulates reactivity of hippocampal cells to chemical stimulation in vitro

Intracellular activity was recorded from CA1 neurons in rat hippocampal slices. Iontophoretic application of glutamate produced a fast and reversible depolarization associated with an increase in action potential discharge rate and only minimal conductance changes. Concurrent application of norepinephrine (NE) or isoproterenol with moderate iontophoretic currents or in a microdrop potentiated the depolarizing responses to glutamate. This effect was not reproduced by serotonin. It is suggested that NE acts postsynaptically to increase the efficacy of the depolarizing action of excitatory neurotransmitters.     

Picrotoxin but not bicuculline antagonizes 5-hydroxytryptamine-induced inhibition of cerebellar Purkinje neurons

The inhibitory effects of iontophoretically applied serotonin (5-hydroxytryptamine) on Purkinje cells were examined in the presence of the GABA antagonists picrotoxin and bicuculline in in vivo and in vitro cerebellar preparations. Continuous application of the GABA-mediated chloride ionophore antagonist, picrotoxin, at currents that induced significant antagonism of GABA-elicited inhibition of Purkinje cells decreased the inhibitory effects of serotonin significantly in the same neurons. Bicuculline, an antagonist of GABA receptors, exerted different actions on serotonin-mediated inhibition of Purkinje cells. Continuous iontophoretic applications of bicuculline antagonized the inhibitory effects of GABA on Purkinje cells significantly, but failed to induce significant attenuation of serotonin-mediated inhibition. The inhibitory effects of serotonin on Purkinje cells were examined also in the presence of nipecotic acid, an inhibitor of GABA reuptake, and a low Ca2-high Mg2+ medium, which blocks synaptic transmission. Under these circumstances, serotonin-mediated inhibitions were not influenced significantly, indicating that its inhibitory effects do not involve release or reuptake of GABA. These results indicate that there might be a linkage between the actions of serotonin and GABA.     

Clozapine and haloperidol in the amygdaloid complex: differential effects on dopamine transmission with long-term treatment

Single-unit recording techniques and liquid chromatography with electrochemical detection were used to measure the effects of neuroleptic pretreatment on the efficacy of dopamine transmission in the amygdaloid complex. Rats received twice-daily injections of clozapine (10.0 mg/kg), haloperidol (1.0 mg/kg), or vehicle for 6 days. During withdrawal from haloperidol, but not clozapine, amygdaloid neurons were significantly more responsive to iontophoretic application of dopamine. Neither drug altered the neuronal response to norepinephrine or acetylcholine. Tolerance developed to the ability of haloperidol to increase the amygdaloid level of 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, but such an effect did not occur with clozapine. In fact, clozapine pretreatment led to a significant increase in both DOPAC and dopamine levels in the amygdaloid complex. These results suggest that a differential action of these drugs on dopamine transmission may explain their differential effects on postsynaptic dopamine receptors.     

Excitatory and inhibitory effects of dopamine on neuronal activity of the caudate nucleus neurons in vitro

Effects of dopamine on the rat caudate nucleus neurons were examined in a slice preparation using an intracellular recording technique. Perfusion of the bath with a low concentration (1 μM) of dopamine produced a depolarization concomitant with an increase in the spontaneous firing and the number of action potentials evoked by a depolarizing pulse applied into the cells. In contrast, higher concentrations (100–500 μM) of dopamine inhibited the spontaneous and current-induced firings without apparent effects on the resting membrane potential. In addition, during application of a high concentration (100 μM) of dopamine there was a marked elevation of the threshold potential of the action potential elicited by a higher depolarizing current. Simultaneous application of haloperidol (0.5–5 μM) antagonized both excitatory and inhibitory effects induced by the low and high concentrations of dopamine, respectively. In addition, the excitatory effect induced by a low concentration (1 μM) of dopamine was antagonized by domperidone (0.5 μM), a selective D₂ receptor antagonist, while the inhibitory effect by a high concentration (100 μM) was blocked by SCH 23390, a selective D₁ receptor antagonist. These results strongly suggest that the postsynaptic sites of caudate nucleus neurons have at least two subtypes of dopamine receptors (D₁ and D₂ receptors) that mediate inhibitory and excitatory responses of the neuron to dopamine, respectively.     

Cytochemical and pharmacological studies on polysensory neurons in the primate frontal cortex

Neurons of the squirrel monkey postarcuate cortex respond to a number of different sensory modalities. The pharmacological responses of these neurons to putative neurotransmitters, administered by microiontophoresis, was studied. Norepinephrine and serotonin had a powerful inhibitory action on the vast majority of cells at relatively low iontophoretic currents. The inhibitory action of norepinephrine was potentiated by low doses of desmethylimipramine; higher doses produced a direct slowing of discharge.Acetylcholine had mixed effects, exciting and inhibiting approximately equal numbers of neurons. The threshold current for acetylcholine responses was considerably higher than that for norepinephrine or serotonin.Treatment of freeze-dried slices of postarcuate cortex with paraformaldehyde vapor, after uptake of alpha-methyl norepinephrine or 6-hydroxytryptamine, showed an extensive plexus of fine fluorescent axons with characteristic varicosities, throughout the cortex. Many of the varicosities surrounded the soma of neurons.Light microscopic autoradiography of polysensory cortex, after subdural injection or incubation with tritiated norepinephrine or serotonin, revealed many grains along neuronal soma and major dendrites. With electron microscopy after this same treatment, autoradiographic grains were localized largely to fine unmyelinated axons and to small terminals making synaptic contact with the soma and large dendrites.After chronic treatment with 6-hydroxydopamine, fibers within the polysensory cortex which take up alpha-methyl norepinephrine or tritiated norepinephrine are greatly reduced in number, whereas fibers which take up 6-hydroxytryptamine or tritiated serotonin are still plentiful. These electrophysiological and cytochemical studies support the existence of norepinephrine and serotonin-containing inhibitory pathways to neurons of the postarcuate cortex of squirrel monkey.     

Low sensitivity of the neuroblastoma cell cholinergic receptors to erabutoxins and α-bungarotoxin

The fast depolarizing nicotinic response evoked by iontophoretic application of acetylcholine in neuroblastoma cell line N1E-115 was completely blocked by 10 μM d-tubocurarine but only partially suppressed by 7.2 μM erabutoxin a or b, or 1 μM α-bungarotoxin. The hyperpolarizing and slow depolarizing muscarinic responses were not affected by these snake toxins.     

The effects of iontophoretic application of putative neurotransmitters on the electrical activity of rat mediobasal hypothalamic neurons in relation to their steroid sensitivity

In order to characterize the neurons responsible for ACTH release and the suppression produced by adrenocortical steroids, we have studied the pharmacological sensitivity of neurons in the region containing the highest concentration of corticotropin releasing factor (CRF) in the brain and probably responsible for most of these activities. The effects of 8 putative neurotransmitters applied iontophoretically to more than 400 mediobasal hypothalamic (MBH) neurons in the rat were examined, and compared with cortisol sensitivity of the same neurons. Glutamate was the only agent that produced excitation exclusively, while GABA, serotonin, glycine and dopamine were inhibitory in action. Mixed excitation and inhibition were produced by histamine, acetylcholine (ACh) and norepinephrine (NE). All 8 cells excited by ACh were inhibited by cortisol and constitute only about 10% of neurons sampled; these neurons are considered to be strong candidates for CRF-releasing cells. On 7 of these neurons histamine was strongly excitatory and its action greatly outlasted the iontophoretic application. Histamine, however, excited many other neurons, including those neither excited by ACh nor steroids.     

Involvement of amino acids in periodic inhibitions of bulbar respiratory neurones

As previously demonstrated⁷, spontaneously firing bulbar inspiratory neurones are periodically inhibited either at the beginning of, or throughout expiration, while bulbar expiratory neurones are inhibited during inspiration. The aim of the present study was to test the hypothesis that amino acids act as transmitters of these periodic inhibitions.The study was performed using iontophoretic applications of drugs on bulbar respiratory neurones. On these neurones GABA and glycine-sensitive sites were identified and differentiated on the basis of the actions of agonist (muscimol) or antagonists (bicuculline, picrotoxin and strychnine). Using competitive antagonists (nipecotic acid, β-alanine) mechanisms responsible for GABA uptake were found in the close vicinity of respiratory-related neurones.Some but not all types of periodic inhibition were found to be reduced following application of GABA or glycine antagonists. Strychnine was found to reduce periodic inhibitions occurring at the beginning of expiration in inspiratory neurones. GABA antagonists had an effect on those periodic depressions which were prolonged throughout expiration. A different and complementary role of glycine-like and GABA-like systems in central respiratory mechanisms is proposed.     

Modulatory Action of serotonin on glutamate-induced excitation of cerebellar purkinje cells

The effects of serotonin (5-hydroxytryptamine, 5-HT) on glutamate-induced excitation of Purkinje cells were examined. Pulsatile iontophoretic applications of glutamate (1-25 nA) induced consistent increases in the spontaneous activity of Purkinje cells. When serotonin was applied continuously with currents that elicited minimum changes in the spontaneous rate, it inhibited or blocked glutamate-induced excitations significantly in most Purkinje cells. We also examined the effects of high currents of serotonin on spontaneous activity of Purkinje cells. High currents of serotonin induced 3 different effects: inhibitions, biphasic effects comprising transient inhibition followed by excitation, and excitations. Nonetheless, whether it inhibited or excited the activity of Purkinje cells, serotonin inhibited glutamate-induced excitations consistently. The effect of a putative 5-HT antagonist methysergide (UML) was also examined. Methysergide consistently attenuated or antagonized the inhibitory effects of serotonin on glutamate-induced excitations. This finding suggests strongly that inhibitory effects of 5-HT on glutamate excitations observed in the present study is the specific action of serotonin.     

Interactions of Dopamine with Glutamate- and GABA-mediated Synaptic Transmission in the Rat Entorhinal Cortex In Vitro

We have studied the interactions between dopamine and glutamate-mediated transmission in the entorhinal cortex using intracellular recording in a slice preparation from the rat brain. High concentrations (0.1 – 1 mM) of dopamine had weak, direct effects on the membrane potential with predominantly hyperpolarizing responses in layer II neurons and depolarizing responses in layer V. Studies with the dopamine antagonists sulpiride (D2 antagonist, 10 – 50 μM) and SCH-23390 (D1 antagonist, 50 μM) indicated that the hyperpolarization by dopamine could be mediated by D2 receptors, although the pharmacology was not clear-cut. The depolarizing response was not affected by either D1 or D2 antagonists. Synaptic responses of layer II and layer V cells were complex, consisting of both inhibitory and excitatory potentials. In untreated slices, dopamine reduced all components of the synaptic responses. However, when components of the responses were pharmacologically isolated, only the excitatory, glutamate-mediated potentials were consistently affected and the GABAergic inhibitory potentials were more resistant to reduction by dopamine. Excitatory potentials mediated by both N -methyl- d -aspartate and α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid receptors were reduced by dopamine, but the former were more strongly affected. Studies with antagonists suggested that the D1 receptor is more likely to be involved in the decrement of glutamate transmission. Thus, dopamine appears to modulate glutamate-mediated synaptic transmission in the entorhinal cortex, and it is conceivable that a disturbance in this interaction could be involved in the aetiology of schizophrenia.