Dopaminergic neuronal responses to a non-amphetamine CNS stimulant

Summary The present study compares the effects of d-amphetamine (d-AMP) and the potent non-amphetamine CNS stimulant, amfonelic acid (AFA), on the firing rate of single midbrain dopaminergic (DA) neurons and on neostriatal DA metabolism (dihydroxyphenylacetic acid—DOPAC). The results indicate that AFA, like d-AMP, reduces the firing rate of DA neurons, although unlike d-AMP, AFA does not cause a decrease in neostriatal DOPAC content and, in fact, enhances that produced by haloperidol (HALO). The AFA-induced decrease in firing rate, like d-AMP, is reversed by the DA receptor blocker HALO, but again unlike d-AMP, the decrease in firing rate is not prevented by catecholamine synthesis inhibition with-methyl-para-tyrosine. Thus, both amphetamine and amfonelic acid have identical electrophysiological effects on DA neurons but act by different mechanisms.     

Electrophysiological properties of spinally-projecting A5 noradrenergic neurons

Spinally-projecting A5 neurons were studied with anatomical and electrophysiological techniques in the rat. A detailed study of the number and distribution of spinally-projecting catecholaminergic (CA) and non-catecholaminergic neurons present in a defined area of ventrolateral pontine reticular formation was performed using a sequential technique for the detection of CA fluorescence and retrogradely transported HRP. Using control animals and rats with 6-hydroxydopamine-induced lesions of spinal CA axons, it was concluded that up to 93% of all noradrenergic (NE) neurons present in the area investigated send an axonal process to the thoracic spinal cord and that NE neurons constitute at least 90% of all spinally-projecting neurons present in the same area. Single unit recordings of spinally-projecting neurons were obtained in the same area of the reticular formation in urethane-anesthetized, paralyzed and respirated rats. Based on the above-mentioned anatomical data, antidromic activation from thoracic spinal cord provided a necessary and sufficient criterion for the identification of A5 NE cells. These neurons had a conduction velocity of 2.5 m/s, a discharge rate of up to 4 spikes/s and all were inhibited by i.v. clonidine or desmethylimipramine (DMI). The inhibition produced by the latter drugs was always reversed by the alpha-2 adrenergic antagonists piperoxan or yohimbine. Antidromic (AD)-activation was followed by a period of inhibition whose duration was increased by raising the intensity of the stimulus or by administration of the NE-uptake inhibitor DMI. The effect of the latter was reversed by administration of the alpha-2 antagonist piperoxan.     

Electrophysiological and biochemical sequelae of the destruction of hippocampal noradrenergic afferents by DSP4

The effects of DSP4 lesions were examined 20—53 days postlesion in the rat hippocampus. A single treatment with DSP4 produced decreases of 42–94% in the norepinephrine (NE) content of this brain region. There was, however, no effect of DSP4 treatment on either the number of affinity of beta-adrenergic receptor sites as determined by radioligand binding studies with (−)-[¹²⁵I]pindolol; furthermore, there was no relationship between the concentrations of NE and the number of receptor sites in individual hippocampi.The DSP4-induced depletion of functionally releaseable NE was confirmed by the loss of electrophysiological responsiveness to amphetamine in the in vitro hippocampus following such lesions. In contrast, electrophysiological responses to direct acting beta-adrenergic or alpha-adrenergic agonists were unchanged following DSP4 treatment. This finding again suggests tha lack of any change in postsynaptic sensitivity. The results of this study demonstrate that while the potent noradrenergic neurotoxin DSP4 is able to reduce NE concentrations significantly in noradrenergic target regions in brain, these lesions are not necessarily associated with postsynaptic changes in adrenergic systems.     

Electrophysiological responses of rat olfactory tubercle neurons to biologically relevant odours

Biologically relevant odours were used to stimulate olfactory tubercle neurons in anaesthetized male rats. Among 120 recorded neurons, 118 showed spontaneous activity (mean firing rate, 15.0 ± 1.4 spikes/s). Ninety-eight neurons were exposed to at least one of the four following odour sources: an empty vial, or a vial containing food pellets (familiar odour), a sample of oestrous rat faeces (conspecific sexual odour), or a sample of male fox faeces (predator odour). The proportion of neurons responding with a change in activity was significantly linked to the odour applied. Repetition of the stimulation with the same odour elicited the same activity change. Between 50 and 70% of neuronal activity changes were not accompanied by respiration changes. Fifty-six neurons were exposed successively to all four odours, and 38 of them showed an activity change in response to at least one. The response of a neuron to an odour was not affected by its response to the previous one, and no neuron responded in the same manner to all odours. Conversely, no odour elicited a unique response in this population of neurons. However, the proportions of excited, inhibited and insensitive neurons depended significantly on the odour applied, suggesting that the recruitment of olfactory tubercle neurons is directly dependent on the biological significance of the odour.     

Electrophysiological responses to muscarinic receptor stimulation in cultured hippocampal neurons.

Electrophysiological recordings of responses to muscarinic receptor stimulation in cultured embryonic hippocampal neurons have been largely unsuccessful to date. In this study muscarinic receptor binding was demonstrated in 2-week-old embryonic rat hippocampal cultures. Using whole-cell patch-clamp recording we found that 1-5 microM carbachol produced multiple effects including depolarization, increased action potential firing rate, increased synaptic activity and a reduction in the amplitude of medium-duration afterhyperpolarizations. Voltage-clamp analysis revealed a time-dependent current relaxation with hyperpolarizing steps from a holding potential of about -40 mV which was inhibited by 10 microM muscarine or 50 microM carbachol and had characteristics similar to those of the m-current. Both atropine and pirenzepine inhibited all of these effects indicating that these cholinergic actions were mediated by muscarinic receptors. This study shows that muscarinic responses obtained classically in hippocampal brain slices can also be produced in cultured hippocampus.     

Electrophysiological evidence that noradrenergic afferents selectively facilitate the activity of supraoptic vasopressin neurons

The functional role of the ascending projection from A1 noradrenergic neurons of the caudal ventrolateral medulla to the supraoptic nucleus of the hypothalamus was investigated by examining the effects of electrical stimulation of the A1 region on the activity of supraoptic neurons deemed to be vasopressinergic or oxytocinergic on the basis of basal firing patterns and responsivity to baroreceptor activation. A1 stimulation enhanced the activity of all putative vasopressin-secreting supraoptic neurons tested. This effect appeared to be selective in that no putative oxytocin-secreting neurons were excited by A1 stimulation. Destruction of the supraoptic noradrenergic terminal plexus by local application of the neurotoxin 6-hydroxydopamine abolished the facilitatory effects of A1 stimulation but did not noticeably alter basal activity patterns, nor the influence of baroreceptor inhibitory pathways. These findings suggest a facilitatory role for noradrenergic afferents in regulating the activity of neurohypophysially-projecting vasopressin neurons of the supraoptic nucleus.     

Electrophysiological effects of locally applied noradrenergic agents at cerebellar Purkinje neurons: receptor specificity

We have investigated the receptor subtype(s) mediating the noradrenergic inhibition of cerebellar Purkinje cell spontaneous firing rate using local application of specific agonists and antagonists, in situ, via pressure microejection. Extracellular action potentials were recorded from Purkinje neurons in anesthetized Fischer 344 rats. Timolol, a beta-receptor antagonist, did not affect norepinephrine (NE)-induced inhibition in 9 of 12 cells studied. Phentolamine, an alpha-receptor antagonist, blocked the effect of NE in 8 of 11 cells. To further determine the subtype of alpha-receptor involved, the effects of the alpha 1-antagonist prazosin and alpha 2-antagonists idazoxan and yohimbine were examined. While prazosin had no effect on NE-mediated inhibition, both idazoxan and yohimbine blocked NE effects. Idazoxan was also successful in blocking phencyclidine (PCP), an indirect noradrenergic agonist. The inhibitory action of NE upon Purkinje cell firing rate was mimicked by the selective alpha 2-agonist clonidine; this action of clonidine was blocked by idazoxan but not by timolol or prazosin. In addition, the alpha 1-adrenergic agonist phenylephrine and the beta-adrenergic agonist isoproterenol inhibited Purkinje cell firing rate. Phenylephrine effects were blocked by prazosin but not by timolol or idazoxan. Isoproterenol-induced inhibition was blocked by timolol but not phentolamine. Taken together, these studies suggest that both alpha- and beta-receptors alter Purkinje cell firing rate; the depressant action of locally applied NE, however, seems to be mediated primarily via an alpha 2-adrenergic receptor.     

Electrophysiological study of neurotropin-induced responses in guinea pig hypothalamic neurons

To investigate the direct actions of neurotropin (NSP, a nonproteinaceous extract from inflamed skin of rabbits which is in therapeutic use), intracellular recordings were made from neurons of the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LHA) in slices of guinea pig brain. In the VMH, NSP, applied by perfusion (0.1-3.0 NU/ml), caused dose-dependent depolarization in 29 of 48 neurons (60%) tested. No change in membrane resistance was observed during the depolarization, which hypothesized that the NSP-induced depolarization might be mediated through the inactivation of the Na-K pump. The NSP-induced depolarization persisted even after the elimination of synaptic activity by perfusion with Ca(2+)-free and high Mg2+ Ringer solution. NSP hyperpolarized the cell membrane of three neurons (6%) while two neurons (4%) showed biphasic responses; transient depolarization followed by long-lasting hyperpolarization. Membrane potential of the remaining 14 neurons was not changed by application of NSP. Of 14 LHA neurons tested for NSP effects, eight (57%) were depolarized, three (21%) were hyperpolarized, and one showed a biphasic response. The present results suggest that NSP significantly modulates hypothalamic neuron activity, and the central modulation of autonomic functions by NSP might be mediated through hypothalamic neurons.     

Treatment of hypersomnolence in myotonic dystrophy with a CNS stimulant

A 42-year-old man with myotonic dystrophy developed severe hypersomnolence during the year prior to admission for acute respiratory insufficiency. However, his hypersomnolence could be attributed to a central dysfunction, and during a follow-up period of 3 years, was successfully treated with methylphenidate.     

Local application of bicuculline potentiates NMDA-receptor-mediated sensory responses of brain noradrenergic neurons.

Direct application of bicuculline methiodide (BIC) to noradrenergic locus coeruleus (LC) neurons potently enhanced their sensory responsiveness. This increased responsiveness was due to the long-lasting expression of a new, N-methyl-D-aspartate (NMDA) receptor-mediated component of the synaptic response. This enhancement only occurred when a high stimulus intensity was used to induce the sensory response. A similar increase in responsiveness was observed with stimulation of the nucleus paragigantocellularis (PGi), one of the major direct afferents to LC. This action of BIC was neither mimicked by picrotoxin, penicillin, or the GABA-B antagonist, 2-hydroxy-baclofen, nor by agents that directly depolarize LC neurons. In addition, the inverse agonist of the benzodiazepine receptor, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), did not mimic this effect of BIC. The BIC-potentiated response component was eliminated by direct application of the neurotransmitter gamma-aminobutyric acid (GABA). These results indicate that BIC, acting at a possibly novel site, unmasks NMDA receptors that can be activated by sensory stimuli. This may reflect a mechanism whereby interactions between two major neurotransmitter systems, excitatory amino acids (EAAs) and GABA, potently modulate signal transmission in the brain.     

Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep

It is well known that noradrenergic locus coeruleus (LC) neurons decrease their activity during slow wave sleep (SWS) and are virtually quiescent during paradoxical sleep (PS). It has been proposed that a GABAergic input could be directly responsible for this sleep-dependent neuronal inactivation. To test this hypothesis, we used a new method combining polygraphic recordings, microiontophoresis and single-unit extracellular recordings in unanaesthetized head-restrained rats. We found that iontophoretic application of bicuculline, a specific GABA_A-receptor antagonist, during PS and SWS restore a tonic firing in the LC noradrenergic neurons. We further observed that the application of bicuculline during wakefulness (W) induced an increase of the discharge rate. Of particular importance for the interpretation of these results, using the microdialysis technique, Nitz and Siegel (Neuroscience 1997; 78 : 795) recently found an increase of the GABA release in the cat LC during SWS and PS as compared with waking values. Based on these and our results, we therefore propose that during W, the LC cells are under a GABAergic inhibitory tone which progressively increases at the entrance and during SWS and PS and is responsible for the inactivation of these neurons during these states.     

Conditioned and sensitized responses to stimulant drugs in humans

In animal models considerable evidence suggests that increased motivation to seek and ingest drugs of abuse are related to conditioned and sensitized activations of the mesolimbic dopamine (DA) system. Direct evidence for these phenomena in humans, though, is sparse. However, recent studies support the following. First, the acute administration of drugs of abuse across pharmacological classes increases extracellular DA levels within the human ventral striatum. Second, individual differences in the magnitude of this response correlate with rewarding effects of the drugs and the personality trait of novelty seeking. Third, transiently diminishing DA transmission in humans decreases drug craving, the propensity to preferentially respond to reward-paired stimuli, and the ability to sustain responding for future drug reward. Finally, very recent studies suggest that repeated exposure to stimulant drugs, either on the street or in the laboratory, can lead to conditioned and sensitized behavioral responses and DA release. In contrast to these findings, though, in individuals with a long history of substance abuse, drug-induced DA release is decreased. This diminished DA release could reflect two different phenomena. First, it is possible that drug withdrawal related decrements in DA cell function persist longer than previously suspected. Second, drug-paired stimuli may gain marked conditioned control over the release of DA and the expression of sensitization leading to reduced DA release when drug-related cues are absent. Based on these observations a two-factor hypothesis of the role of DA in drug abuse is proposed. In the presence of drug cues, conditioned and sensitized DA release would occur leading to focused drug-seeking behavior. In comparison, in the absence of drug-related stimuli DA function would be reduced, diminishing the ability of individuals to sustain goal-directed behavior and long-term objectives. This conditioned control of the expression of sensitized DA release could aggravate susceptibility to relapse, narrow the range of interests and perturb decision-making, accounting for a wide range of addiction related phenomena.     

Gonadotropin responses to naloxone may depend upon spontaneous activity in noradrenergic neurons at the time of treatment

The opiate system is thought to modulate gonadotropin secretion by its effect on catecholamine secretion. This action may be produced by opiates regulating the amount of catecholamine released from presynaptic terminals at a given frequency of depolarization and/or by increasing the rate of impulse traffic within catecholamine neurons. We examined the effects of naloxone, an opiate receptor antagonist, on luteinizing hormone (LH) and prolactin (Prl) secretion in 3 sex steroid-treated, gonadectomized rat models in which we have considerable information on the rates of turnover of norepinephrine (NE) and dopamine (DA) in the hypothalamus. In 7 day ovariectomized rats treated for 2 days with estradiol (E2), the injection of naloxone (10 mg/kg) at 09.15 h produced a small 3-fold rise in LH and a short-lived decline in Prl. In contrast, naloxone, given at 12.15 h, markedly amplified (10-fold) and advanced the time of the LH surge but did not affect afternoon Prl surges. Hypothalamic NE turnovers are low in the morning and high in the afternoon for such animals. Other ovariectomized (OVX) rats received E2 for 2 days and progesterone (P4) on day 2. Such treatment extinguishes the LH surges which normally occur the next day (day 3) but does not affect phasic Prl secretion. Naloxone, given at 09.15 h to E2P4-treated rats on day 3, did not affect basal LH levels but serum Prl declined for about 1 h. When given at 12.15 h, naloxone produced a small 3-fold rise in LH but did not affect phasic Prl release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological properties and cholinergic responses of rat ventral oral pontine reticular neurons in vitro

In order to characterize the electrophysiological properties of morphologically identified neurons of the ventral part of the oral pontine reticular (vRPO) nucleus and the effects of cholinergic agonists on them, intracellular recordings were obtained from 45 cells in a rat brain-slice preparation. Intracellular staining was performed with 2% biocytin in potassium acetate (1 M)-filled micropipettes. Results demonstrated the presence of two types of vRPO neurons. Type I cells (n=12, 24%) were characterized by a break with a decrease of the depolarizing slope following hyperpolarizing pulses which delayed the return to the resting V_m and subsequent spike-firing. The delay was antagonized by 4-AP (200–500 μM) which specifically blocks the transient outward K⁺-mediated current I_A. Type II neurons (n=38, 76%) displayed a typical depolarizing sag during hyperpolarizing current pulses which was blocked by Cs⁺. This behavior is characteristic of the hyperpolarization-activated current I_Q. These two neuronal types displayed different morphological features. Most type I and II cells (100 and 73.7%, respectively) were depolarized by acetylcholine (1–15 μM), carbachol (0.5–1 μM) and muscarine (1–10 μM) through the activation of post-synaptic muscarinic receptors. The remaining type II cells (26.3%) were hyperpolarized (1–10 min, 3–15 mV) through the activation of post-synaptic muscarinic receptors. Results are consistent with the hypothesis that the vRPO could be a neuronal target of Cch in eliciting paradoxical sleep because most of its neurons are activated by muscarinic agonists. © 1979 Elsevier Science B.V. All rights reserved.     

Electrophysiological responses of nucleus accumbens neurons to novelty stimuli and exploratory behavior in the awake, unrestrained rat

To establish a physiological correlate of goal-directed and reward-seeking non-operant behaviors, we studied the electrophysiological activity of NAcc neurons in unrestrained, naive Sprague–Dawley rats. Our results showed an inhibitory response in 21% (7/34) of the recorded NAcc neurons during spontaneous nosepoking behavior and in 16% (4/25) of the NAcc neurons when rats were fed with a favorite novel food morsel (popcorn). These data suggest that a subgroup of NAcc neurons is actively modulated during motivated behavior and during consummatory events resulting in a suppression of neuronal activity.     

Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists

A direct comparison was made of the effects of serotonin 5-HT1A and 5-HT1B selective compounds on the spontaneous firing rate of dorsal raphe serotoninergic neurons in chloral-hydrate-anesthetized rats. Following intravenous administration, the 5-HT1A selective compounds ipsapirone (TVX Q 7821) and LY 165163 potently inhibited single-unit activity in a dose-dependent manner whereas the 5-HT1B selective compounds, m-chlorophenylpiperazine (mCPP) and trifluoromethylphenylpiperazine (TFMPP), displayed only weak or irregular actions. Low microiontophoretic currents of ipsapirone and LY 165163 were also effective in suppressing spontaneous firing; dose-response relationships for the 5-HT1A compounds were indistinguishable from that of 5-HT itself. In contrast, dorsal raphe neurons were only weakly responsive to microiontophoretic application of mCPP and TFMPP; dose-response relationships for the 5-HT1B compounds were significantly displaced from that of 5-HT. In intracellular studies, ipsapirone and LY 165163, when added to the media bathing brain slices, mimicked the actions of 5-HT in hyperpolarizing dorsal raphe cell membranes and decreasing input resistance; however, the maximal effects of the 5-HT1A compounds on these membrane properties exceeded those of 5-HT. In summary, dorsal raphe 5-HT neurons appear highly responsive to 5-HT1A, but not to 5-HT1B compounds; these findings are discussed with regard to the 5-HT receptor subtypes as candidates for the somatodendritic autoreceptor of dorsal raphe neurons.     

Electrophysiological properties and cholinergic responses in guinea-pig celiac ganglion neurons in primary culture.

Prevertebral neurons enzymatically dissociated from celiac ganglia of adult guinea-pigs were maintained in long-term primary culture. Cells were plated at a density of 95 +/- 15 cm-2, and intracellular electrical activity was measured between 2 and 7 weeks after dissociation. Neurite outgrowth began within 24 h of enzymatic dissociation. Cell survival dropped below 50% after more than two weeks in culture. The resting potential (-53 mV +/- 0.8), time constant (12 ms +/- 1.3), input resistance (47 M omega +/- 8.6), rheobase (0.33 nA +/- 0.02), degree of accommodation, spike amplitude (70 mV +/- 3.0), after hyperpolarization amplitude (-9.5 mV +/- 0.55), and after hyperpolarization duration (88 ms +/- 7.6) in these cells were not different from those recorded from neurons in intact celiac ganglia. A larger proportion (greater than 90%) of cells exhibited fast accommodation (phasic) in response to depolarizing current pulses. Unevoked (spontaneous) depolarizations and action potentials were observed. The cells responded to pressure ejected acetylcholine. Two types of responses consisted of an early rapid depolarization which was attenuated by hexamethonium and a later slow depolarization which was attenuated by atropine. We conclude that prevertebral neurons from guinea-pigs can be maintained in long-term primary culture, that they retain electrophysiological properties similar to intact ganglia and exhibit complex responsivity to acetylcholine.     

Electrophysiological properties of pedunculopontine neurons and their postsynaptic responses following stimulation of substantia nigra reticulata

Membrane properties and postsynaptic responses to stimulation of the substantia nigra reticulata (SNr) of the neurons in rat pedunculopontine nucleus (PPN) were studied in an in vitro parasagittal slice preparation using intracellular recording techniques. Based on electrical membrane properties, PPN neurons were classified into 3 types (types I, II and II). The unique feature of the type I neuron was the low threshold calcium spike while the type II neuron had various inward and outward rectifications. The type III neuron showed no such features as those observed in type I or II neurons. Some recorded neurons were intracellularly labeled with biocytin to study their morphology, and their transmitter phenotype was investigated by immunocytochemistry for choline acetyltransferase (ChAT). The type I and III neurons were found to be non-cholinergic, but 50% of the labeled type II neurons were immunopositive for ChAT. Morphological features of type II neurons were also different from type I or III neurons. The soma of the type II neuron was almost always more than twice as large as that of type I and III neurons. Inhibitory postsynaptic potentials (IPSPs) were induced in all 3 types of PPN neurons following stimulation of SNr. SNr-induced IPSPs were usually followed by a slow depolarizing potential from which rebound spikes were triggered. These rebound excitations were found only in type I and II neurons. These data indicate that heterogeneous groups of neurons exist in the PPN in terms of morphology, transmitter phenotypes and electrical membrane properties.