Brain benzodiazepine levels following intravenous administration of [34]diazepam: relationship to the potentiation of purinergic depression of central nervous system neurons

Levels of [3H]benzodiazepine were measured in rat cerebral cortex following intravenous injection of [3H]diazepam using a dose and time schedule reported to elicit a marked potentiation of the depressant effects of iontophoretically applied 5'-AMP to rat cerebral cortical neurons. The levels of [3H]benzodiazepine obtained strongly suggest (i) that blockade of adenosine uptake as a mechanism for this potentiation is not consistent with the potency of diazepam as an inhibitor of adenosine uptake in vitro, and (ii) that a potentiative interaction of adenosine and diazepam may reflect the binding of these compounds to benzodiazepine receptors.     

Electrophysiological and microiontophoretic studies with buspirone: influence on the firing rate of central monoaminergic neurons and their responsiveness to dopamine, clonidine or GABA

The influence of an i.v. perfusion of buspirone on the firing rate of central monoaminergic neurons was studied in rats anaesthetized with chloral hydrate. Buspirone increased the firing rate of A10 dopaminergic neurons and blocked the inhibitory effect of iontophoretically applied dopamine on these neurons. A slight attenuation of the inhibitory effect of iontophoretically applied GABA was also observed. Buspirone increased the firing rate of locus coeruleus (LC) noradrenergic neurons and induced an attenuation of the inhibitory effect of iontophoretically applied clonidine. A slight attenuation of the inhibitory effect of iontophoretically applied GABA was also observed. Furthermore buspirone was a very potent inhibitor of the firing rate of dorsal raphe (DR) serotonergic neurons. It is concluded that activation of A10 neurons by buspirone is due to blockade of dopaminergic autoreceptors and that activation of LC neurons is related to blockade of alpha-2 autoreceptors. The significance of the interaction with gabaergic inhibition is unclear. The mechanisms involved in the inhibition of DR neurons remain to be investigated.     

A presynaptic component of the action of iontophoretically applied flurazepam on feline cortical neurones.

The effect of iontophoretically applied flurazepam on the spike activity of pericruciate cortical neurones of the cat was studied. Flurazepam increased cortical inhibition produced either by local electrical stimulation (which is known to release gamma-aminobutyric acid (GABA) or by iontophoretically applied GABA. Following intravenous treatment with thiosemicarbazide (a GABA-synthesis inhibitor), flurazepam still augmented the action of GABA but was much less effective on electrically evoked cortical inhibition. These findings suggest that part of the action of flurazepam on inhibitory cortical transmission might be at the presynaptic level.     

Sensitivity of the motor endplate to carbachol in the presence of sulfhydryl reagents

The effect that bath application of sulphydryl reagents (SR) exerts on frog sartorius motor endplate sensitivity to iontophoretically applied carbachol (CCh) has been studied. Sensitivity to CCh is expressed as the ratio of the CCh potential (mV) to the nanocoulombs delivered by the iontophoretic pulse and has been determined before and after addition of SR to the bath. Two groups of SR have been tested: oxidizing reagents, o-iodosobenzoate and reducing agents, dithiothreitol (DTT). CCh was applied iontophoretically by means of a microelectrophoretic programmer with constant current source. Exposure of the muscle to 1 mM DTT in a bath pH range of 7-8 for 2 to 85 min showed no significant differences in endplate sensitivity to CCh before and after addition of the reducing agent. o-Iodosobenzoate at a 1 mM bath concentration (pH 7) for 2 to 19 min strongly decreases endplate sensitivity to CCh. The statistical methods used were Wilcoxon rank tests and linear regression. Since previous studies have shown that oxidizing and reducing SR evoke depolarizations when applied iontophoretically at the endplate region, these results suggest that activation of the receptor is achieved only when SR are delivered iontophoretically, and that discrepancies observed can be attributed mainly to the different techniques of drug application.     

Anticonvulsant drug mechanisms of action

The effects of clinically used anticonvulsant drugs on high-frequency sustained repetitive firing (SRF) of action potentials and on postsynaptic responses to iontophoretically applied gamma-aminobutyric acid (GABA) have been compared to establish a classification of anticonvulsant drugs based on cellular mechanisms of action. By using concentrations in the range of therapeutic cerebrospinal fluid values in humans, drugs have been separated into three categories: Phenytoin, carbamazepine, and valproic acid limited SRF, but did not alter GABA responses. Phenobarbital, clonazepam, and diazepam augmented GABA responses and limited SRF only at concentrations above the therapeutic range in ambulatory patients but that are achieved in the acute treatment of status epilepticus. Ethosuximide failed to affect SRF or GABA responses even at supratherapeutic concentrations. Ability of an anticonvulsant to limit SRF correlated well with efficacy against generalized tonic-clonic seizures clinically and against maximal electroshock seizures in experimental animals. Augmentation of GABA responses and lack of limitation of SRF correlated with efficacy against generalized absence seizures in humans and against pentylenetetrazol-induced seizures in animals. However, ethosuximide must act against generalized absence seizures and against pentylenetetrazol-induced seizures by a third, as yet unknown, mechanism. Other actions occurring at supratherapeutic concentrations correlated with clinical toxicity.     

Effect of bradykinin and morphine on rat sensomotor cortex neurons]

As revealed in acute experiments on rats bradykinin applied microiontophoretically produced neuronal activation in the sensory-motor region of the rat brain cortex. Morphine applied iontophoretically prevented bradykinin effect. It is suggested that bradykinin interacts with opiate receptors in the cortical neurones.     

Calcitonin and calcitonin gene-related peptide alter the excitability of neurons in rat forebrain

Individual neurons in the hypothalamus, thalamus, cortex, and other forebrain areas of urethane-anesthetized, male rats were iontophoretically tested for their membrane sensitivity to salmon calcitonin (CT), human CT, and CT gene-related peptide (CGRP). Extracellular recording of unit activity revealed that depression of neuronal firing was the predominant effect of iontophoretically applied salmon CT (35 of 74 cells tested). Few neurons responded to salmon CT with an increase in firing rate (N = 3). When CGRP was iontophoretically applied a pattern of response resembling that of salmon CT was observed. CGRP was predominantly inhibitory and excited those neurons whose firing rate was increased by salmon CT. Inhibition was also the predominant effect of human CT. However, no neurons were excited by human CT. The results clearly demonstrate that a subpopulation of neurons with membrane sensitivity to salmon CT, human CT, and CGRP are present in the rat forebrain. This finding suggests that modulation of neuronal activity may underlie the behavioral and biochemical effects of these peptides when administered centrally. Endogenous CGRP and CT-like peptides in rat brain may be capable of regulating these events as neurotransmitters or neuromodulators.     

Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex

In this study we characterized the responses of neocortical neurons to iontophoretically applied gamma-aminobutyric acid (GABA) and examined how these GABA responses as well as the inhibitory postsynaptic potentials (IPSPs) were affected by the presence of penicillin or pentobarbital. Intracellular recordings were obtained from slices of rat neocortex maintained in vitro; injection of the dye Lucifer yellow indicated that recordings were primarily from pyramidal neurons. Orthodromically evoked responses were always depolarizing at the cell's resting membrane potential. Such depolarizing responses could easily be reversed in polarity by depolarizing the cell 10-15 mV, suggesting that the response consisted partly of an IPSP. In some cases, depolarization unmasked a small, short-latency excitatory postsynaptic potential (EPSP). Responses to iontophoretically applied GABA were also depolarizing at rest. Biphasic hyperpolarizing-depolarizing responses were occasionally observed upon depolarization of the neuron. Bath application of penicillin (1.7-3.4 mM) decreased the amplitude of the IPSPs and increased their time to peak, an effect associated with the development of epileptiform activity. Penicillin also reduced the maximum response to iontophoretically applied GABA without affecting the dose required to obtain a half-maximal response, suggesting a noncompetitive antagonism. Pentobarbital (100-200 microM) prolonged the time course and increased the amplitude of the IPSPs while producing a leftward shift in the GABA charge-response relation. These results suggest that the convulsant penicillin and the anticonvulsant pentobarbital have opposing actions on GABAergic inhibition in the neocortex.     

Electrophysiological actions of neurotensin in rat cerebellum

The electrophysiological actions of neurotensin (NT) and its analog d-Arg⁹NT were studied in rat cerebellar Purkinje neurons. NT applied by pressure ejection was a potent depressant of Purkinje (P) neuron firing. In contrast, iontophoretically applied NT was a weak depressant. Pressure-ejected d-Arg⁹NT, which is largely inactive in peripheral systems, had little effect on P neurons. The depressant effects of pressure-ejected NT were blocked by intraperitoneally administered haloperidol, iontophoretically applied magnesium or 6-OHDA pretreatment. After such treatments, locally applied NT evoked only excitations.The results of this study suggest that NT, when applied by pressure ejection, produces two effects on the Purkinje neuron. The potent inhibitory effects of locally applied NT appear to result from release of the inhibitory transmitter, norepinephrine from locus coeruleus-derived afferents. We postulate that the excitations, which appear when postsynaptic effects of norepinephrine are antagonized or release is reduced, may be the direct result of NT action at the postsynaptic P neuron membrane.     

Selective reduction by serotonin, of neuronal excitation in the locus coeruleus evoked by glutamate

In the rat, iontophoretically applied serotonin substantially attenuated the excitation of locus coeruleus neurons evoked by iontophoretic glutamate but not that elicited by acetylcholine. These results occurred independently of serotonin's variable effects on spontaneous discharge, and indicate a neuromodulatory role of serotonin in locus coeruleus.     

Evidence for an ascending inhibitory histaminergic pathway to the cerebral cortex.

Previous observations from our laboratory indicate that metiamide is a specific histamine antagonist in rat cerebral cortex. In view of the recent finding that histamine levels and L-histidine decarboxylase (EC 4.1.1.22) activity in cerebral cortex decrease following disruption of the ipsilateral medial forebrain bundle (MFB), the present investigation was undertaken to examine whether iontophoretically applied metiamide antagonizes the inhibition of deep cerebral cortical neurones produced by stimulation of the MFB. In rats anaesthetized with a mixture of methoxyflurane, nitrous oxide and oxygen, stimulation of the ipsilateral MFB or the cortical surface with iontophoretically applied histamine depressed the firing of cortical neurones. Metiamide antagonized the histamine-induced depression and reduced the duration of inhibition produced by MFB stimulation. However, it did not alter the inhibition induced by the cortical surface stimulation. These results indicate that a histaminergic pathway ascending through the MFB may inhibit rat cerebral cortical neurones.     

Increase in the sensitivity of rabbit sensomotor cortex neurons to gamma-aminobutyric acid under the influence of diazepam (microiontophoretic study)]

A study was made of the action of diazepam on the effects of the gamma-aminobutyric acid (GABA) applied electrophoretically to the neurons of the sensory-motor rabbit cortex. It was shown that diazepam intensified the depressive action of GABA on the spontaneous neuronal activity and the prolonging action of GABA on the duration of the inhibitory phase in the neuron responses to the afferent and direct stimulation of the cortex. Diazepam failed to alter the neuron response to glycine, glutamate and acetylcholine applied microelectrophoretically. It is supposed that diazepam increased the sensitivity of the receptors of the post-synaptic membrane of the neuron to GABA.     

An iontophoretic survey of opioid peptide actions in the rat limbic system: in search of opiate epileptogenic mechanisms

Iontophoretic and micropressure drug application and lesion techniques were used to investigate the cellular source of rat limbic system epileptiform responses to opioid peptides [19]. Iontophoretically applied morphine, methionine enkephalin or beta-endorphin inhibited the spontaneous or glutamate-activated firing of the great majority of single neurons in medial and lateral septum, amygdala and cingulate cortex. These inhibitions in firing were antagonized by iontophoresis of naloxone. In contrast to inhibitory effects in other limbic areas, morphine and the opioid peptides predominantly excited CA1 and CA3 pyramidal neurons in a naloxone-sensitive manner, as previously reported [36]. On rare occasions, iontophoretically applied beta-endorphin evoked repetitive waveforms similar to interictal population EPSPs or spikes. Micropressure application of opiates and peptides also excited hippocampal neurons indicating such responses were not current-induced artefacts. The possible role of the excitatory cholinergic septal hippocampal pathway in the facilitatory response of hippocampal units to the opiates was tested with iontophoretically applied atropine and scopolamine, or lesions of septal nuclei. None of these manipulations reduced the opioid-induced excitations; rather, septal lesions enhanced excitatory and epileptiform responses to the opiates. These results support the hypothesis that opiate-evoked epileptiform activity in the limbic system arises from enhanced pyramidal cell activity in the hippocampal formation, probably by a non-cholinergic mechanism.     

Responses of septal nuclei neurons to microiontophoretically administered putative neurotransmitters

In halothane anesthetized rats, neurons of the medial and lateral septal nuclei were tested with iontophoretically applied putative neurotransmitters. GABA, norepinephrine, serotonin, and acelycholine in roughly this order of potency were inhibitory with respect to spontaneous and evoked activity of both medial and lateral septal nuclei cells. No specific effects of any of the compounds were observed on septal unit responses to fornix or fimbria stimulation.     

Vasoactive intestinal polypeptide excitation of cerebral cortical neurons: lack of synergism with adenosine and noradrenaline

Vasoactive intestinal polypeptide (VIP), applied iontophoretically, excited 40% of the spontaneously firing rat cortical neurons tested. No neurons were depressed by VIP. When applied simultaneously with adenosine or noradrenaline, VIP depressed the firing of cortical neurons, but this depression could be reproduced by the passage of similar positive currents through a 50 mM NaCl-containing barrel of the multiple barrelled micropipette. VIP, therefore, excited rat cortical neurons and no depressant actions were apparent when VIP was applied together with adenosine or noradrenaline. Leakage of adenosine or noradrenaline during iontophoretic applications of the peptide may account for the reported inhibitory actions of VIP.     

Dopamine-glutamate interaction and cholinesterase activity in the corpus striatum of the adult rat

Electrophysiological, microiontophoretic and neuroanatomical techniques have been employed to investigate the relationships between intrastriatal sites of dopamine/glutamate (DA/GLU) interaction and inhomogeneities for acetylcholinesterase. The sites where iontophoretically applied DA antagonized the excitatory effects of iontophoretic GLU or cortical stimulation showed no topographic arrangement in the dorsolateral parts of the striatum. The data suggest that DA/GLU interaction in the striatum of the adult rat may occur independently from distribution of acetylcholinesterase.     

Intraseptal injection of scopolamine increases the effect of systemic diazepam on passive avoidance learning and emotionality in rats.

This experiment was designed to assess the role of the septo-hippocampal cholinergic (ACh) system in the deleterious effects produced by systemic benzodiazepine injection on learning processes in rats. Retention of a step through passive avoidance task was analysed after systemic injection of increasing doses of either scopolamine or diazepam applied alone 30 min before the acquisition phase. Results indicated a dose related impairment of retention by each drug: in addition, sub-threshold doses of scopolamine and diazepam applied in combination (diazepam: 2mg/kg plus scopolamine: 0.3mg/kg) produced a decrease of retention latencies, thus showing an additive effect of the combined treatment. Secondly, a sub-threshold dose of scopolamine (15microg/0.5microl) was also administered into the medial septal area, together with an i.p. injection of 2mg/kg of diazepam. This combined treatment produced a severe impairment of retention, in parallel with a large reduction in emotionality (number of faeces). The data are consistent with the hypothesis that peripheral administration of behaviorally effective doses of diazepam on passive avoidance learning might act partially via a septal ACh-GABA/benzodiazepine mechanism. It is also suggested that this mechanism subserves both anxiety and the memorisation of contextual stimuli associated with passive avoidance acquisition, through the modification of the septo-hippocampal activity.     

Neuronal responsiveness to gonadotropin-releasing hormone and its correlation with sexual receptivity in the rat

In the present study, an attempt was made to correlate the neuronal responsiveness of individual preoptic-septal (POA/S) units to iontophoretically applied GnRH with the onset of sexual receptivity. In both behavioral and electrophysiological studies, ovariectomized, estrogen-primed rats were used. In behaviorally tested rats, lordosis quotients (LQ) were determined at varying times following progesterone (P) injection. For electrophysiological studies, P was given 1 hr after the start of recording. GnRH was iontophoretically applied for 30 sec at 16 nA on spontaneously discharging cells. A unit was deemed excited or inhibited if a repeatable 30% change in discharge rate was observed. From 2-10 hours as the LQ increased from 17 to 90 the total number of GnRH sensitive cells did also. The majority of responsive cells were excited by the peptide. As receptivity displayed a sharp increase from 2 to 6 hours the mean responsiveness of cells excited by GnRH was significantly elevated over inhibitory responses. These findings confirm the E/P biasing effect on POA/S unit responses to GnRH. Moreover, they suggest that a dynamic relationship exists between GnRH responses at the cellular level and sexual behavior throughout the course of steroid-induced receptivity.     

Presynaptic depression of synaptic response of Renshaw cells by adenosine 5'-monophosphate

The action of AMP (adenosine 5'-monophosphate) on synaptic transmission of Renshaw cells has been studied in cats under Dial anaesthesia. AMP applied iontophoretically reversibly reduced synaptic responses of Renshaw cells evoked by stimulation of ventral roots, as well as their spontaneous firing; however, there were no marked effects on discharges of these cells caused by iontophoretic application of acetylcholine, asparatate, and glutamate. On the other hand, AMP had no comparable effect on synaptic responses of dorsal horn interneurones evoked by stimulation of dorsal roots or their spontaneous discharges.     

Residence time distribution of diazepam in the isolated perfused rat liver. Analysis with the axial dispersion model.

The application of the axial dispersion model to diazepam hepatic elimination was evaluated using data obtained for impulse-response experiments with diazepam in the single-pass isolated perfused rat liver preparation. The transient form of the two-compartment dispersion model was applied to the output concentration versus time profile of diazepam after bolus input of a radiolabelled tracer into the hepatic portal vein (n = 4), providing DN and CLint estimates of 0.251 +/- 0.093 and 135 +/- 59 ml min-1, respectively. In contrast, the one-compartment form of the axial dispersion model, which assumes instantaneous transversal distribution of substance to the accessible spaces within the liver, could not adequately describe the residence time distribution (RTD) of diazepam. Furthermore, the magnitude of DN, a stochastic parameter which characterizes the axial spreading of solutes during transit through the liver, is similar to that determined for non-eliminated substances such as erythrocytes, albumin, sucrose and water. These findings suggest that the dispersion of diazepam in the perfused rat liver is determined primarily by the architecture of the hepatic microvasculature.