Insulin decreases epileptiform activity in rat layer 5/6 prefrontal cortex in vitro.

Accumulating evidence indicates that insulin-mediated signaling in the brain may play important roles in regulating neuronal function. Alterations to insulin signaling are associated with the development of neurological disorders including Alzheimer's disease and Parkinson's disease. Also, hyperglycemia and insulin resistance have been associated with seizure activity and brain injury. In recent work, we found that insulin increased inhibitory GABAA -mediated tonic currents in the prefrontal cortex (PFC). In this work, we used local field potential recordings and calcium imaging to investigate the effect of insulin on seizure-like activity in PFC slices. Seizure-like events (SLEs) were induced by perfusing the slices with magnesium-free artificial cerebrospinal fluid (ACSF) containing the proconvulsive compound 4-aminopyridine (4-AP). We found that insulin decreased the frequency, amplitude, and duration of SLEs as well as the synchronic activity of PFC neurons evoked by 4-AP. These insulin effects were mediated by the PI3K/Akt signaling pathway and mimicked by gaboxadol (THIP), a δ GABAA receptor agonist. The effect of insulin on the number of SLEs was partially blocked by L-655,708, an inverse agonist with high selectivity for GABAA receptors containing the α5 subunit. Our results suggest that insulin reduces neuronal excitability by an increase of GABAergic tonic currents. The physiological relevance of these findings is discussed.

Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 µM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4ß2 nAChR agonist RJR-2403 and was not influenced by TTX (1 µM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.

Spontaneous voltage oscillations in striatal projection neurons in a rat corticostriatal slice.

In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked long-lasting plateau potentials in neostriatal neurons. Plateau potentials were often followed by spontaneous voltage transitions between two preferred membrane potentials. While the induction of plateau potentials was disrupted by non-NMDA and NMDA glutamate receptor antagonists, the maintenance of spontaneous voltage transitions was only blocked by NMDA receptor and L-type Ca2+ channel antagonists. The frequency and duration of depolarized events, resembling up-states described in vivo, were increased by NMDA and L-type Ca2+ channel agonists as well as by GABAA receptor and K+ channel antagonists. NMDA created a region of negative slope conductance and a positive slope crossing indicative of membrane bistability in the current-voltage relationship. NMDA-induced bistability was partially blocked by L-type Ca2+ channel antagonists. Although evoked by synaptic stimulation, plateau potentials and voltage oscillations could not be evoked by somatic current injection--suggesting a dendritic origin. These data show that NMDA and L-type Ca2+ conductances of spiny neurons are capable of rendering them bistable. This may help to support prolonged depolarizations and voltage oscillations under certain conditions.

D2 dopamine receptors in striatal medium spiny neurons reduce L-type Ca2+ currents and excitability via a novel PLC[beta]1-IP3-calcineurin-signaling cascade.

In spite of the recognition that striatal D(2) receptors are critical determinants in a variety of psychomotor disorders, the cellular mechanisms by which these receptors shape neuronal activity have remained a mystery. The studies presented here reveal that D(2) receptor stimulation in enkephalin-expressing medium spiny neurons suppresses transmembrane Ca(2+) currents through L-type Ca(2+) channels, resulting in diminished excitability. This modulation is mediated by G(beta)(gamma) activation of phospholipase C, mobilization of intracellular Ca(2+) stores, and activation of the calcium-dependent phosphatase calcineurin. In addition to providing a unifying mechanism to explain the apparently divergent effects of D(2) receptors in striatal medium spiny neurons, this novel signaling linkage provides a foundation for understanding how this pivotal receptor shapes striatal excitability and gene expression.

Action of substance P (neurokinin-1) receptor activation on rat neostriatal projection neurons.

Substance P (SP) acts as a neurotransmitter in the neostriatum through the axon collaterals of spiny projection neurons. However, possible direct or indirect actions of SP on the neostriatal output neurons have not been described. Targets of SP terminals within the neostriatum include interneurons, spiny neurons, afferent fibers and boutons. SP induces the release of both dopamine (DA) and acetylcholine (ACh). Since some postsynaptic actions of both DA and ACh on spiny neurons are known, we asked if activation of neostriatal NK1-class receptors is able to reproduce them. The SP NK1-receptor agonist, GR73632 (1 microM), had both excitatory and inhibitory actions on virtually all spiny neurons tested at resting potential. The excitatory action was blocked by atropine and coursed with an increase in firing rate and input resistance (R(N)). The inhibitory action was blocked by haloperidol and coursed with a reduction in firing rate and R(N). Therefore, the release of both DA and ACh induced by NK1-receptor activation modulates indirectly the excitability of the projection neurons. SP facilitates the actions of these transmitters on the spiny neuron. A residual excitatory response to the NK1-receptor agonist was observed in 30% of a sample of neurons tested in the presence of both haloperidol and atropine. The increase in R(N) that accompanied this response could be observed in the presence of 1 microM TTX or 100 microM Cd2+, suggesting a direct effect. Double labeling showed that only SP-immunoreactive neurons were facilitated by NK1-receptor activation in these conditions.

Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.

It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.

Dopamine facilitates striatal EPSPs through an L-type Ca2+ conductance.

When synaptic activity is evoked from relatively depolarized membrane potentials, D1 receptor agonists enhance the depolarization level and slow the decay of synaptic responses recorded from neostriatal spiny neurons. The population spikes' amplitude is also increased. These D1 actions facilitate firing and are evident in the presence of both NMDA and GABA selective blockers. Thus, dopaminergic D1 receptor activation facilitates the AMPA-mediated EPSP in these conditions. This facilitatory effect could be suppressed by L-type Ca2+ channel antagonists (200 nM calciseptine and 5 microM nicardipine), suggesting that it is mediated by an increase in L-current. D1-receptor activation thus mediates orthodromic facilitation of neostriatal neurons when evoked from depolarized membrane potentials. This reinforces the dopamine facilitation mediated through NMDA responses.

D1 receptor activation enhances evoked discharge in neostriatal medium spiny neurons by modulating an L-type Ca2+ conductance.

Most in vitro studies of D1 dopaminergic modulation of excitability in neostriatal medium spiny neurons have revealed inhibitory effects. Yet studies made in more intact preparations have shown that D1 receptors can enhance or inhibit the responses to excitatory stimuli. One explanation for these differences is that the effects of D1 receptors on excitability are dependent on changes in the membrane potential occurring in response to cortical inputs that are seen only in intact preparations. To test this hypothesis, we obtained voltage recordings from medium spiny neurons in slices and examined the impact of D1 receptor stimulation at depolarized and hyperpolarized membrane potentials. As previously reported, evoked discharge was inhibited by D1 agonists when holding at negative membrane potentials (approximately -80 mV). However, at more depolarized potentials (approximately -55 mV), D1 agonists enhanced evoked activity. At these potentials, D1 agonists or cAMP analogs prolonged or induced slow subthreshold depolarizations and increased the duration of barium- or TEA-induced Ca2+-dependent action potentials. Both effects were blocked by L-type Ca2+ channel antagonists (nicardipine, calciseptine) and were occluded by the L-type channel agonist BayK 8644-arguing that the D1 receptor-mediated effects on evoked activity at depolarized membrane potential were mediated by enhancement of L-type Ca2+ currents. These results reconcile previous in vitro and in vivo studies by showing that D1 dopamine receptor activation can either inhibit or enhance evoked activity, depending on the level of membrane depolarization.

Firing frequency modulation of substantia nigra reticulata neurons by 5-hydroxytryptamine.

Unitary extracellular recordings were made in in vitro rat brain slices to explore the effects of serotoninergic analogues on the spontaneous activity of substantia nigra reticulata (SNr) neurons. Most SNr neurons exhibited regular spontaneous firing (23.4 +/- 8.9 Hz, mean +/- S.E.M., n = 30) similar to that found in vivo. The most reproducible effect of serotonin (5-HT) was an increase in firing frequency found in 53% of the cells. The effect was concentration dependent and blocked by the 5-HT1/2 antagonist methysergide (1-10 microM) but unaffected by the 5-HT4- and 5-HT1-preferring antagonists DAU 6285 (5 microM) and metiothepin (5 microM), respectively. However, 5-HT also decreased the firing frequency in several neurons. In 19% of the neurons an inhibition was found alone but a biphasic response (inhibition and excitation) was found in another 28% of the neurons. Interestingly, the effect of the 5-HT-uptake inhibitor, duloxetine (100-400 nM), was frequency inhibition. Agonists that mimicked the 5-HT-induced inhibition were of the 5-HT1B-class (25 microM CP 93129 and 25 microM TFMPP). Neither the 5-HT2-antagonist ritanserin (5 microM) nor the GABA(A)-antagonist, bicuculline (30 microM) were able to block the inhibition suggesting that some SNr neurons may be directly inhibited by 5-HT.

Muscarinic antagonists microinjected into the subthalamic nucleus decrease muscular rigidity in reserpinized rats.

The ability of anticholinergic agents microinjected into the subthalamic nucleus to reduce reserpine-induced muscular rigidity was assessed in rats. The electromyographical activity of the gastrocnemius-soleus muscle was used as a parameter of muscular rigidity. Reserpine (5 mg/kg i.p.) produced the appearance of electromyographical activity. The muscarinic antagonists M3 (1.27 nmol of 4-DAMP) and M1 (2.36 nmol of pirenzepine) markedly reduced the reserpine-induced electromyographical activity, whereas the M2 antagonist AFDX-116 (2.37 nmol) had no effect. These results suggest that a high cholinergic tone in the subthalamic nucleus is associated with the reserpine-induced muscular rigidity. Moreover, the M3 muscarinic antagonist is more effective than the M1 muscarinic antagonist in reducing the muscular rigidity in reserpinized rats, a model of Parkinson's disease, by blocking the high cholinergic tone in the subthalamic nucleus.

Inhibitory action of dopamine involves a subthreshold Cs(+)-sensitive conductance in neostriatal neurons.

Intracellular recordings in in vitro slice preparations of rat brain were used to compare the actions of dopamine and dopamine receptor agonists on the subthreshold membrane properties of neostriatal neurons. A reproducible response for dopaminergic agonists was evoked after firing produced by current ramp injections that induced a subthreshold voltage displacement. Dopamine (10-100 microM) decreased both firing rate and membrane slope input resistance in virtually all cells tested. Input resistance change appeared as an increase in inward rectification. Approximate reversal potential was around -87 mV. The D1 receptor agonists SKF 38393 and Cl-APB (1-10 microM) mimicked both dopamine effects with a reversal potential around -89 mV. The effects were blocked by the presence of 5-10 mM caesium (Cs+) but not by 1 microM tetrodotoxin, suggesting that main D1 effects on input resistance are due to subthreshold Cs(+)-sensitive conductances. cAMP analogues mimicked the actions of D1 receptor agonists. The D2 agonist, quinpirole (1-10 microM), did not produce any input resistance change, nonetheless, it still produced a decrease in firing rate. This suggests that the main D2 effect on firing is due to actions on suprathreshold ion conductances. All effects were blocked by D1 and D2 antagonists, respectively. D1 or D2 effects were found in the majority of cells tested.

Circling behavior elicited by cholinergic transmission in the substantia nigra pars compacta: involvement of nicotinic and muscarinic receptors.

The influence of cholinergic transmission within the substantia nigra pars compacta on circling behavior was assessed in male rats. Microinjection of physostigmine (6-37 nmol) into the caudal part of the substantia nigra pars compacta elicited a dose-dependent contralateral circling. The circling was inhibited 93 +/- 3% by the dopamine antagonist haloperidol (53 nmol) injected into the neostriatum 90 min before the injection of physostigmine (37 nmol) into the ipsilateral substantia nigra pars compacta. The effect of haloperidol was reversible, since the circling behavior was fully restored when physostigmine was applied to the same animals 24 h later. The circling was completely blocked when physostigmine (37 nmol) was applied simultaneously with the muscarinic M1 antagonist pirenzepine (2 nmol). The M2 antagonist AF-DX 116 (2 nmol) only partially blocked the circling induced by a lower dose of physostigmine (12 nmol). The nicotinic antagonist mecamylamine (5 nmol) also inhibited the circling, but only during the 5 min following co-injection of the drugs. These results indicate that endogenous acetylcholine stimulates muscarinic and nicotinic receptors of nigrostriatal dopaminergic neurons which, in turn, increase their firing rate and cause the circling behavior. We conclude that the pedunculopontine cholinergic neurons, which innervate the substantia nigra pars compacta, modulate the motor behavior by increasing the activity of dopaminergic nigrostriatal pathway.

Dopamine modulates the afterhyperpolarization in neostriatal neurones.

Intracellular techniques were used to study the actions of dopaminergic D1 agonists on the afterhyperpolarization (AHP) that follows action potentials in rat neostriatal neurones. Dopamine or Cl-APB (10 microM), or 1-10 microM 6-Cl-PB all increased AHP amplitude. This effect was blocked by 1 microM SCH-23390, a D1 antagonist, but not by 1 microM sulpiride, a D2 antagonist. Both 500 microM dibutyryl cAMP and 5 microM BayK 8644 induced a similar AHP increase. BayK 8644 occluded the effect of agonists. The results suggest that the action of dopamine is mediated via the recently described protein kinase A enhancement of L-type Ca2+ channels. The results partially explain the decrease in firing frequency induced by dopamine and a possible site of antagonism with cholinergic modulation.

Cholinergic stimulation of rostral and caudal substantia nigra pars compacta produces opposite effects on circling behavior and striatal dopamine release measured by brain microdialysis.

Turning in circles is among the behaviors elicited by unilateral cholinergic stimulation of the substantia nigra. Recent studies have shown that microinjection of cholinergic agonists into the substantia nigra pars compacta increases dopamine release and turnover in the striatum of anesthetized rats [Hernández-López et al. (1992) Brain. Res. 598, 114-120; Blaha and Winn (1993) J. Neurosci, 13, 1035-1044]. In this study, the relationship between circling behavior and striatal dopamine release following cholinergic stimulation of the substantia nigra pars compacta neurons was assessed by brain microdialysis in awake rats. The results indicate that cholinergic stimulation of the substantia nigra pars compacta with the mixed nicotinic-muscarinic cholinergic agonist carbachol modulates striatal dopamine release, and this effect is accompanied by circling behavior and stereotypies. Microinjection of carbachol (109 nmol) in the caudal portions of the substantia nigra pars compacta induced contralateral circling associated with an increase of dopamine release in neostriatum. On the contrary, ipsilateral circling and reduction of striatal dopamine release was elicited when the same dose of the drug was applied in the rostral portions of the substantia nigra pars compacta. The above findings are in accordance with recent electrophysiological studies suggesting the existence of sub-populations of nigrostriatal dopaminergic neurons, and indicate that the substantia nigra pars compacta is functionally compartmentalized. We conclude that the cholinergic input to the substantia nigra pars compacta could modulate the motor behavior through regulating the firing rate of nigrostriatal dopaminergic neurons and dopamine release in the neostriatum.

A cholinergic input to the substantia nigra pars compacta increases striatal dopamine metabolism measured by in vivo voltammetry.

3,4-Dihydroxyphenylacetic acid (DOPAC) and ascorbic acid (AA) were measured by differential pulse voltammetry in the neostriatum of anesthetized rats. Physostigmine (2.3 nmol) applied into the substantia nigra pars compacta (SNc), increased DOPAC concentration in the ipsilateral neostriatum, but did not modify AA levels. The largest increase of striatal DOPAC (37 +/- 8% above basal) was observed when physostigmine was applied at less than 0.5 mm from SNc, and decreased with increasing distance of the injection site from the pars compacta region. Chemical stimulation of the pedunculopontine tegmental nucleus (PPN) with kainic acid (2.3 nmol) increased both DOPAC and AA concentration in the ipsilateral neostriatum. Pretreatment with the muscarinic antagonist scopolamine (5 mg/kg, i.p.) inhibited the increase of striatal DOPAC from 20 to 70 min after kainic acid injection into the PPN, whereas the increase of AA was reduced from 90 to 160 min. By contrast, the nicotinic antagonist mecamylamine (4 mg/kg, i.p.) did not inhibit neither DOPAC nor AA increase elicited by the chemical stimulation of PPN. These results support the existence of cholinergic neurotransmission within the SNc that increases the firing rate of nigrostriatal dopaminergic neurons, enhancing dopamine turnover in neostriatum without changes in AA release. They also suggest that the PPN could be the origin of cholinergic afferents to the SNc that modulate the activity of dopaminergic neurons, through activation of muscarinic cholinergic receptors. Finally, the activation of a multisynaptic loop involving a cholinergic pathway which modulates the activity of the glutamatergic corticostriatal neurons is postulated to explain the increase of AA in neostriatum observed after PPN stimulation.

Activation of nigral M1 and M2 muscarinic receptors produces opposing effects on striatal 3,4-dihydroxyphenylacetic acid measured by in vivo voltammetry.

3,4-dihydroxyphenylacetic acid (DOPAC) was measured by differential pulse voltammetry in the neostriatum of anesthetized rats. DL-Muscarine (2.9 nmol) applied into the substantia nigra pars compacta, increased DOPAC concentration in the ipsilateral neostriatum. This effect was blocked by pirenzepine (2.8 nmol), and potentiated by AF-DX 116 (2.8 nmol). These results indicate the existence of two types of muscarinic receptors on dopaminergic neurons, whose activation produces opposing effects on dopamine metabolism in neostriatum.