Modulation of ion channels in rat neurons by the constituents of Hypericum perforatum

Despite almost forty years of widespread use of antidepressant drugs, their mode of action is still unknown. Hyperforin, a phloroglucinol derivative, is a major pharmacologically and therapeutically active constituent of Hypericum perforatum extract that is widely used as an herbal antidepressant drug. However, the mechanism or mechanisms of action of these naturally abundant, non-toxic extracts remain unclear. Enzymatically isolated patch-clamped rat central and peripheral neurons exposed to rapid changes in the composition of external medium (concentration clamp) were used in our experiments to investigate the modulation of the various voltage- and ligand-gated channels by hyperforin, as well as by other constituents of Hypericum perforatum. At nanomolar concentrations, hyperforin induced significant inhibition of various ion channels. In the case of P-type Ca2+ channels, we established that hyperforin acts via interaction with calmodulin or through calmodulin-activated pathways involving at least one second messenger. The results presented here indicate that multiple mechanisms and extract constituents may be involved in the antidepressant action of Hypericum extracts, and that they could also possess neuroprotective and analgesic effects.     

Effects of high-frequency stimulation on subthalamic neuronal activity in parkinsonian patients

BACKGROUND: High-frequency stimulation of the subthalamic nucleus (STN) is a neurosurgical alternative to medical treatment in levodopa-responsive forms of Parkinson disease. The mechanism of action of STN stimulation remains controversial, although an inhibition of overactive STN neurons has been postulated. OBJECTIVE: To determine the effects of high-frequency STN stimulation on the neuronal activity of STN neurons in Parkinson disease patients. PATIENTS: Single-unit recordings of the neuronal activity of the STN were obtained before, during, and after the application of intra-STN electrical stimulation in 15 Parkinson disease patients. Changes in firing frequency and pattern were analyzed using various combinations of stimulus frequency (range, 14-140 Hz). RESULTS: Stimulation at a frequency greater than 40 Hz applied within the STN significantly decreased the firing frequency and increased the burst-like activity in the firing pattern of STN neurons. An aftereffect was observed in cells that had been totally inhibited during high-frequency stimulation. CONCLUSION: The beneficial effects of high-frequency stimulation result from a change in the firing pattern of cellular discharge and a blockade of the spontaneous overactivity of STN neurons.     

5-HT(1A) agonist potential of pindolol: electrophysiologic studies in the dorsal raphe nucleus and hippocampus.

BACKGROUND: The ability of pindolol to block 5-HT(1A) autoreceptors on serotonin-containing neurons in the raphe nuclei is thought to underlie the clinical reports of enhanced efficacy and rate of improvement in depressed patients treated with pindolol/selective serotonin reuptake inhibitor (SSRI) combinations. Selectivity for somatodendritic 5-HT(1A) autoreceptors is a crucial requirement, as blockade of postsynaptic 5-HT(1A) sites may jeopardize the therapeutic response. Previous investigators have probed the effects of pindolol on serotonergic dorsal raphe cell firing in animal species; here we confirm their findings and extend them to include observations on postsynaptic 5-HT(1A) receptors in the hippocampus. METHODS: Extracellular single-unit recordings were made in rats using standard electrophysiologic techniques. Firing rates of serotonin-containing neurons in the dorsal raphe nucleus and CA3 hippocampal pyramidal neurons were monitored and the effects of pindolol given alone or in combination with an SSRI (fluoxetine) or a 5-HT(1A) antagonist (WAY-100,635) were determined. RESULTS: Pindolol inhibited the firing rates of serotonergic dorsal raphe neurons in a dose-dependent manner. Recovery to baseline firing rates was gradual, but this inhibition could be acutely reversed by WAY-100,635. A range of pindolol doses failed to block the inhibitory effects of fluoxetine on dorsal raphe cell firing. In the hippocampus, pindolol also inhibited cell firing as a function of dose, although these effects were insensitive to WAY-100,635 treatment. CONCLUSIONS: The ability of pindolol to inhibit serotonergic dorsal raphe cell firing is indicative of its agonist potential and is consistent with previous studies. The lack of observable antagonism of the SSRI-induced slowing of raphe unit activity casts doubt on the suitability of this mechanism of action to account for the positive findings in clinical studies utilizing pindolol/SSRI combinations. The 5-HT(1A)-independent inhibition of hippocampal CA3 cell firing by pindolol suggests that this compound invokes multiple pharmacologic actions, all of which need to be assimilated into any proposed mechanism of action.     

Pharmacological and endocrine effects of Hypericum perforatum and hypericin after repeated treatment

Clinical studies have demonstrated the antidepressant efficacy of Hypericum (St. John's wort) extracts comparable to tricyclic antidepressants such as imipramine. We examined the effects of Hypericum extract and hypericin, one active constituent, in the forced swimming test (FST) after treatment repeated for 14 days. It has recently been shown that hypericin was inactive in the FST after acute treatment, but remarkably active when solubilized by subfraction IIIc1 containing mainly procyanidin B2. Therefore, we investigated the cooperative effects of hypericin and procyanidin B2 after repeated treatment. Imipramine (15 mg/kg), Hypericum extract (500 mg/kg) and hypericin (0.1 mg/kg) given daily for 2 weeks significantly reduced immobility time in the FST. No differences were observed between animals receiving pure hypericin and those receiving hypericin in combination with procyanidin B2. As several antidepressants act on the neuroendocrine axis resulting in altered hormone concentrations, selected endocrine parameters were investigated after repeated treatment. Daily treatment with either imipramine, Hypericum extract or hypericin alone or in combination with procyanidin B2 for 14 days significantly decreased plasma ACTH and corticosterone levels. None of the substances had pronounced effects on plasma prolactin or LH levels. From our present data, we propose that cooperative effects of hypericin and procyanidin B2 are of important relevance for the acute, but not for the chronic effects of this polycylic quinone.     

The midbrain slice preparation. An in vitro model to select potential anti-parkinsonian drugs?

Most anti-parkinsonian drugs produce a dopamine-like effect on single dopaminergic neurons. Using electrophysiological recordings, this effect is observed as an inhibition of the spontaneous firing activity. The firing activity of dopaminergic cells throughout dopamine-mediated mechanisms is inhibited not only by dopamine direct agonists but also by substances that increase the release or synthesis, or block the uptake or reduce degradation of dopamine in the brain. Here we propose the electrophysiological approach on dopaminergic neurons in in vitro slice preparation as a preliminary tool for selecting new potential anti-parkinsonian agents, before they are tested in more complex animal models.     

Modulation of auditory evoked magnetic fields by benzodiazepines

The N1 and P2 components of the auditory evoked magnetic field were shown to be modified by the benzodiazepines diazepam and triazolam. Previous studies indicate that the electrical sources of these components are located in the auditory cortex, implying that benzodiazepines have a direct or indirect effect on neuronal activity at this level. The recorded changes were comparable to those previously reported using auditory evoked potential measurements. These results suggest that magnetic recordings may eventually be used as a sensitive, supplementary and location-specific measure of the central action of psychoactive substances.     

GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro

To elucidate the role of GABA_B receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABA_B agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the γ-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABA_B agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABA_B antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca²⁺ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABA_B receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABA_B receptors inhibits electrical activity of SON neurons of both male and female rats and that GABA_B receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA.     

Electrophysiological and behavioural evidence for an antagonistic modulatory role of adenosine A2A receptors in dopamine D2 receptor regulation in the rat dopamine-denervated striatum

It has been shown that striatal adenosine A2A receptors can antagonistically interact with dopamine D2 receptors at the membrane level leading to a decrease in the affinity and efficacy of D2 receptors. Extracellular recordings and rotational behaviour were employed to obtain a correlate to these findings in an animal model of Parkinson's disease (PD). The recordings were performed in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced catecholamine depletion. While recording in the dopamine-depleted striatum, local applications of the dopamine D2 agonist quinpirole reduced neuronal activity. However, when the adenosine A2A antagonist MSX-3 was applied simultaneously with quinpirole, the inhibition of neuronal firing seen after quinpirole alone was significantly potentiated (P< 0.001, n = 11). In contrast, local application of CGS 21680 attenuated the effect of quinpirole. The doses of MSX-3 and CGS 21680 used to achieve the modulation of quinpirole action had no effect per se on striatal neuronal firing. Furthermore, rotational behaviour revealed that MSX-3 dose-dependently increased the number of turns when administrated together with a threshold dose of quinpirole while no enhancement was achieved when MSX-3 was combined with SKF 38393. MSX-3 alone did not induce rotational behaviour. In conclusion, this study shows that low ineffective doses of MSX-3 enhance the effect of quinpirole on striatal firing rate, while the A2A agonist exerts the opposite action. This mechanism gives a therapeutic potential to A2A antagonists in the treatment of PD by enhancing D2 receptor function.     

Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.     

An intrinsic neuronal-like network in the rat pineal gland

Recent studies have shown that in rat pineal glands kept in vitro action potential-producing cell clusters are demonstrable. To test whether the clusters interact, multiple-unit recordings were carried out simultaneously from different clusters, with or without electrical stimulation. Clusters with rhythmic burst activity exhibit highly synchronized firing and electrical stimulation of one cluster elicits an immediate response in another one, apparently involving synapses but not gap junctions. It is hypothesized that the interacting clusters form a network. As the firing is affected by norepinephrine, acetylcholine and Ca2+, the network may monitor the interstitial concentrations of these substances and spread this information in the gland, to modulate melatonin secretion.     

Comparative evaluation of Melissa officinalis L., Tilia europaea L., Passiflora edulis Sims. and Hypericum perforatum L. in the elevated plus maze anxiety test

There are numerous plants that have been used for their 'tranquillising' properties in Portuguese folk medicine. This report will describe a comparative analysis of the effects of Melissa officinalis L., Tilia europaea L., Passiflora edulis Sims. and Hypericum perforatum L. on the performance of mice in the elevated plus maze, open-field, and horizontal-wire tests. We have tested lyophilised aqueous extracts with doses ranging from 5-100 mg/kg prepared according to traditional folk medicine. The results indicate that Hypericum perforatum L. and Tilia europaea L. induced significant raise in immobility time, diminution of rearing and other parameters, suggesting a clear sedative effect at doses ranging from 10-100 mg/kg. Below these doses, Tilia europaea L. did not induce any significant change in the tests mentioned, while Hypericum perforatum L. (5 mg/kg) increased the time spent in the open areas of the elevated plus maze and the percentage of unprotected head-dips and stretch-approach postures, thus indicating an anxiolytic effect. For this dose, there were no significant changes in motor activity as measured by classical parameters for the tests used. As the infused H. perforatum L. tested was devoid of hyperforin, it can be stated that the observed effects cannot be attributed to this substance.     

Hypericum LI 160 inhibits uptake of serotonin and norepinephrine in astrocytes

Extracts of Hypericum perforatum, commonly known as St. John's wort, are frequently used in Germany and other European countries to treat mild to moderately severe depression, but the mechanism of antidepressant activity of Hypericum is not understood. Because known mechanisms of antidepressant activity include inhibition of serotonin and/or norepinephrine uptake, we investigated the effects of standardized extracts of Hypericum LI 160 on the transport of these monoamine neurotransmitters into astrocytes, cells which surround synaptic terminals and regulate neurotransmission by means of their uptake systems. We found that LI 160 inhibited both serotonin and norepinephrine uptake in a dose-dependent manner. The two monoamine transport systems were affected differently by LI 160: for serotonin, the main effect was a 50% decrease in the rate of maximal transport, whereas for norepinephrine, the main effect was a 4.5 fold reduction in the apparent affinity of norepinephrine for its uptake sites. Upon removal of LI 160, uptake was restored, thereby indicating that the inhibition was not due to a toxic effect of Hypericum on the cells. These findings suggest that the ability of LI 160 to inhibit serotonin and norepinephrine uptake may underlie the antidepressant activity of this Hypericum extract.     

Inhibition of cathecol-O-methyltransferase (COMT) in the brain does not affect the action of dopamine and levodopa: an in vitro electrophysiological evidence from rat mesencephalic dopamine neurons

In order to study whether the membrane hyperpolarization and firing inhibition caused by dopamine and levodopa on rat midbrain dopamine cells are affected by the inhibition of brain catechol-O-methyl-transferase (COMT), intracellular electrophysiological recordings were made from these neurons maintained in vitro. Here we report that a treatment of the cerebral tissue with tolcapone, a central and peripheral inhibitor of COMT, does not change the membrane responses of midbrain dopamine neurons to dopamine and levodopa. The lack of modification of the dopaminergic effects by tolcapone suggests that the pharmacological inhibition of intracerebral COMT does not have detectable action on dopamine neurotransmission. Therefore, the therapeutic action of tolcapone in Parkinson's disease, might be dependent on the reduction of COMT activity in the extracerebral tissue.     

Effect of the Hypericum perforatum extract on serotonin turnover in the mouse brain

St. John's Wort, a traditional herbal medicine obtained from the extract of Hypericum perforatum, has been used in the treatment of mild depression. Its mechanism of action remains to be established. The present study confirmed that Hypericum extract exhibited very weak inhibitory activities towards MAO. Mouse brain MAO activities was unchanged following either acute or chronic treatment with Hypericum extract. 5-HIAA levels were found to be significantly increased in the cerebral cortex, hypothalamus, hippocampus and caudate 3 h after treatment with the Hypericum extract at a dose as low as 10 mg/Kg. An increase of 5-HT levels was also observed in hypothalamus and hippocampus. The increase of brain 5-HIAA was not further enhanced following chronic administration of the herb. The Hypericum extract significantly reduced the plasma tryptophan levels, the precursor of 5-HT. The action of Hypericum extract is consistent with the notion that serotonergic system is involved. The effect of Hypericum extract on the brain 5-HIAA and 5-FIT levels appeared to be quite different from the effect of classical 5-HT re-uptake blockers.     

Commercially available Hypericum perforatum extracts do not decrease immobility of rats in the forced swimming test

There are controversial results of clinical trials on the antidepressant effects of Hypericum perforatum, while several preclinical studies describe antidepressant properties for Hypericum extracts. This study evaluates the antidepressant effect of two commercially available hydroalcoholic extracts of H. perforatum standardized to contain 0.3% hypericin in comparison to imipramine (IMI), in the forced swimming test (FST). Wistar rats were treated with different doses of two Hypericum extracts, of hypericin or of IMI and submitted to the FST. The experiments were videotape recorded to detail immobile and active behaviors of rats during the procedures. The imported extract tested and hypericin did not modify rats' behaviors in the test, while IMI, a classical antidepressant, significantly shortened immobility and prolonged climbing behavior during forced swimming. The locally produced Hypericum extract significantly increased immobility duration as compared to the controls at the same time as climbing efforts were decreased. Therefore, the two different commercially available Brazilian hydroalcoholic H. perforatum extracts did not show the expected effects in a screening test for antidepressant agents, on the contrary, one of the extracts promoted a depressant-like effect in rats. Therefore, these extracts available to the population differ from other Hypericum extracts. At which step of the production or commercialization chain these extracts probably lost their therapeutic potential remains to be evaluated.     

Biochemical mechanisms of action of Hypericum LI 160 in glial and neuronal cells: inhibition of neurotransmitter uptake and stimulation of extracellular signal regulated protein kinase

We have investigated biochemical mechanisms that may underlie the antidepressant effects of Hypericum LI 160. We found that LI 160 inhibits uptake of serotonin and norepinephrine in cultures of rat cortical astrocytes. Observed differences in the kinetic parameters Km and Vmax as well as in the recovery of uptake after removal of Hypericum indicates that LI 160 does not affect serotonin and norepinephrine transport in the same manner. This suggests that multiple components of the extract can mediate inhibition of these neurotransmitter transporters. Hypericum LI 160 also inhibits serotonin uptake in neuronal cultures from serotonergic-rich raphe nuclei; concentration-response studies indicate LI 160 is 25 times more potent in terms of inhibition of serotonin uptake in neurons than in astrocytes. In addition, Hypericum LI 160 inhibits norepinephrine uptake in SK-N-SH cells, a human neuroblastoma cell line enriched in norepinephrine transporters. A chemical constituent of LI 160, hyperforin, is about 10 to 20 times more potent than LI 160 in inhibiting neurotransmitter uptake in astrocytes and neuronal cells; this finding is consistent with the observation that hyperforin comprises 5% of LI 160 extracts. As several weeks are needed to achieve a clinical response with antidepressants, we have also investigated whether Hypericum LI 160 affects biochemical mechanisms that underlie long-term changes such as gene expression. We found that LI 160 stimulates a sustained activation of extracellular signal regulated protein kinase (ERK), a key component of a signal transduction pathway involved in gene expression. Taken together, our findings suggest that Hypericum LI 160 can affect rapidly-acting as well as slower-developing, long-term biochemical mechanisms related to depressive disorders.     

Locus coeruleus modulation of the motor thalamus: Inhibition in nuclei ventralis lateralis and ventralis anterior

Single-cell recordings were made from 693 cells in thalamic nuclei ventralis lateralis and ventralis anterior (VL-VA). Cells were identified as thalamocortical projection cells by antidromic firing from motor cortex or classified according to responsiveness to stimulation of the brachium conjunctivum (BC), entopeduncular nucleus, and motor cortx. Only 14% of the cells tested responded to entopeduncular nucleus stimulation, whereas BC and motor cortex (orthodromic) stimulation each evoked responses in 31% of the VL-VA cells tested. The most common sources of convergent input to VL-VA cells were motor cortex and BC. In 30% of the VL-VA population tested, spontaneous firing was inhibited by stimulation of the locus coeruleus (LC). This inhibition had a long latency to onset which varied from cell to cell (100 to 1000 ms or more) and a long duration (mean = 1183 ms). The inhibition of spontaneous firing by LC was associated with a variable effect upon BC-evoked excitatory responses in VL-VA cells. In some cases, BC evoked responses were suppressed, but not abolished. In other cells, the excitatory response to BC was unaffected despite complete cessation of VL-VA cell spontaneous firing after LC stimulation. The inhibitory action of LC was not limited to any class of VL-VA cells, but occurred most frequently in neurons receiving an input from the BC. The LC inhibition of VL-VA is not related to changes in systemic blood pressure or an action at the level of the cerebellar cortex. However, LC also produces inhibitory and excitatory effects in centrum medianum neurons, which could account for some of the long-latency responses observed in VL-VA. This electrophysiological study of the action of locus coeruleus upon cellular activity in the motor thalamus argues against involvement in phasic movement and associated postural adjustments. Rather, the locus coeruleus projection to thalamus has properties which suggest a role in longer-term tonic regulation of motor activity.     

Effects of tobacco and cigarette smoke extracts on serotonergic raphe neurons in the rat

Tobacco components other than nicotine might participate in the behavioural effects of smoking. In this study, in-vivo recordings of serotonergic dorsal raphe neurons were performed in the anesthetized rat, whereas tobacco extracts, cigarette smoke extracts, nicotine, nornicotine or anabasine were intravenously injected. All substances inhibited the neurons, and all inhibitions were completely blocked by the nicotine receptor antagonist mecamylamine. The effects of the extracts were much more potent than those of individual substances. These results support the hypothesis that the acute inhibition of serotonin neurons by tobacco compounds is completely related to an effect on nicotine receptors. Tobacco extracts and tobacco smoke extracts may be useful tools for the study of the effects of central effects of smoking.     

Balanced inhibition and excitation underlies spike firing regularity in ventral cochlear nucleus chopper neurons

Ventral cochlear nucleus stellate cells respond to characteristic frequency (CF) tones with sustained (C(S)), transient (C(T)) or onset chopping (O(C)) activity. The mechanisms underlying these different response patterns are not fully understood, and the present study used in vivo intracellular recordings (n = 42) in urethane-anaesthetized rats to examine the possible influence of inhibition on action potential regularity. Hyperpolarization following the offset of a CF tone burst was used as a measure of on-CF inhibition. A cluster analysis based on several membrane potential features, including on-CF inhibition, discriminated three groups in addition to the C(S) response type - two types of C(T) responses and the O(C) type. The different patterns of firing regularity exhibited by C(S/T) neurons reflected different thresholds or degrees of overlap between these cells' narrowly tuned excitatory and inhibitory inputs. C(T) cells with closely matched inhibitory and excitatory response areas showed substantial on-CF inhibition and the greatest decline in firing regularity during a CF tone, whereas those with a mismatch between their response areas showed lateral inhibition and a less marked decline in firing regularity. The presence of inhibition in C(S) neurons did not alter their firing regularity, possibly because of the lower threshold for excitation compared with inhibition. The latency, duration and frequency extent of sustained hyperpolarization in C(S/T) cells is inconsistent with the response properties of O(C) neurons, suggesting that another source(s) of inhibition influences firing regularity, and presumably response magnitude, in these neurons.