Rat Brain Slice Preparation Research Articles

Lamotrigine inhibits postsynaptic AMPA receptor and glutamate release in the dentate gyrus

The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory synaptic transmission on DG. Evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (eEPSCampa and eEPSCnmda) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60-120 g). Exogenously applied AMPA and NMDA-induced currents and AMPA receptor-mediated miniature EPSC (mEPSCampa) were recorded in the presence of specific antagonists. LTG inhibited both eEPSCampa and eEPSCnmda, and had no effect on exogenously applied NMDA-induced current indicating LTG inhibited glutamate release. Previous studies demonstrated that alteration in glutamate concentration in synaptic cleft causes parallel changes of eEPSCampa and eEPSCnmda. Our results showed that LTG inhibited eEPSCampa significantly more than eEPSCnmda (p < 0.05), suggesting that LTG may also have blocked the postsynaptic AMPA receptor. The hypothesis is further supported by the facts that; (1) LTG (30-100 microM) inhibited direct exogenously applied AMPA-induced currents (to 90%), (2) LTG significantly reduced the amplitude, but not the frequency of mEPSCampa and asynchronous (EPSC), and (3) LTG-induced reduction of eEPSCampa was not associated with a modification of the paired-pulse ratio. To sum up, LTG exerts a postsynaptic inhibitory mechanism on the AMPA receptor. Our results demonstrate that LTG suppresses postsynaptic AMPA receptors and reduces glutamate release in granule cells of DG. The postsynaptic effect can be one of the underlying mechanisms of LTG's anticonvulsant action.

Serotonin Activates Presynaptic and Postsynaptic Receptors in Rat Globus Pallidus

Although recent histological, behavioral, and clinical studies suggest that serotonin (5-HT) plays significant roles in the control of pallidal activity, only little is known about the physiological action of 5-HT in the pallidum. Our recent unit recording study in monkeys suggested that 5-HT provides both presynaptic and postsynaptic modulations of pallidal neurons. The present study using rat brain slice preparations further explored these presynaptic and postsynaptic actions of 5-HT. Bath application of 5-HT or the 5-HT(1A/1B/1D/5/7) receptor (R) agonist 5-carboxamidotryptamine maleate (5-CT) depolarized some and hyperpolarized other pallidal neurons. Pretreatments of slices with blockers of the hyperpolarization-cyclic nucleotide-activated current or with the 5-HT(2/7)R-selective antagonist mesulergine occluded 5-CT-induced depolarization. The 5-HT(1A)R-selective blocker N-[2[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohex-anecarboxamide maleate occluded the 5-CT-induced hyperpolarization. These results suggested involvement of 5-HT(7)R and 5-HT(1A)R in the postsynaptic depolarization and hyperpolarization, respectively. 5-CT presynaptically suppressed both internal capsule stimulation-induced excitatory postsynaptic currents (EPSCs) and striatal stimulation-induced inhibitory postsynaptic currents (IPSCs). The potencies of 5-CT on the presynaptic effects were 20- to 25-fold higher than on postsynaptic effects, suggesting that 5-HT mainly modulates presynaptic sites in the globus pallidus. Experiments with several antagonists suggested involvement of 5-HT(1B/D)R in the presynaptic suppression of EPSCs. However, the receptor type involved in the presynaptic suppression of IPSCs was inconclusive. The present results provided evidence that 5-HT exerts significant control over the synaptic inputs and the autonomous activity of pallidal neurons.

Neurokinins robustly activate the majority of septohippocampal cholinergic neurons

In the brain, tachykinins acting via the three cloned neurokinin (NK) receptors are implicated in stress-related affective disorders. Hemokinin-1 is a novel tachykinin that reportedly prefers NK1 to NK2 or NK3 receptors. Although NK1 and NK3 receptors are abundantly expressed in the brain, NK2-receptor-mediated electrophysiological effects have rarely been described as NK2 receptors are expressed only in a few brain regions such as the nucleus of the medial septum/diagonal band. Medial septal/diagonal band neurons that control hippocampal mnemonic functions also colocalize NK1 and NK3 receptors. Functionally, intraseptal activation of all three NK receptors increases hippocampal acetylcholine release and NK2 receptors have specifically been implicated in stress-induced hippocampal acetylcholine release. Electrophysiological studies on the effects of NKs on septohippocampal cholinergic neurons are lacking and electrophysiological effects of hemokinin-1 have thus far not been reported in brain neurons. In the present study we examined the electrophysiological and pharmacological effects of multiple NKs on fluorescently tagged septohippocampal cholinergic neurons using whole-cell patch-clamp recordings in a rat brain slice preparation. We demonstrate that a vast majority of septohippocampal cholinergic cells are activated by NK1, NK2 and NK3 receptor agonists as well as by hemokinin-1 via direct post-synaptic mechanisms. Pharmacologically, hemokinin-1 recruits not only NK1 but also NK2 and NK3 receptors to activate septohippocampal cholinergic neurons that are the primary source of acetylcholine for the hippocampus.

Activation of a G Protein–coupled Inwardly Rectifying K+Current and Suppression of I hContribute to Dexmedetomidine-induced Inhibition of Rat Hypothalamic Paraventricular Nucleus Neurons

Alpha2-adrenoceptor agonist has been reported to produce inhibition of arginine vasopressin release, diuresis, and sympatholytic effects. However, its mechanisms of central action remain incompletely understood. Hypothalamic paraventricular nucleus (PVN) neurons, which are in direct contact with noradrenergic synapses and are controlled by the hyperpolarization-activated currents, are called Ih (H current). The effect of dexmedetomidine, a highly selective and potent agonist, at alpha2 adrenoceptors on Ih is unknown. The purpose of this study was to examine the effects of dexmedetomidine on the PVN neuron, which is involved in the arginine vasopressin release and autonomic regulation. The authors investigated the effects of dexmedetomidine on the membrane properties in PVN magnocellular neurons and an Ih in PVN parvocellular neurons with a whole cell patch clamp technique using a rat brain slice preparation. Dexmedetomidine dose-dependently hyperpolarized PVN magnocellular neurons. In the voltage clamp mode, dexmedetomidine induced an outward current, with a reversal potential of -94 mV, and this was shown to depend on the external concentration of K. Pretreatment with Ba or peptide toxin tertiapin blocked hyperpolarization induced by dexmedetomidine. The effect of dexmedetomidine was blocked by an alpha2-adrenoceptor antagonist, yohimbine. Ih was suppressed dose dependently by dexmedetomidine in PVN parvocellular neurons. Pretreatment with Cs occluded the Ih suppression by dexmedetomidine. Yohimbine blocked the Ih suppression by dexmedetomidine. The Ih sensitive to dexmedetomidine was weakly modulated by intracellular cyclic adenosine monophosphate. Dexmedetomidine inhibited PVN magnocellular neurons by activation of the G protein-coupled inwardly rectifying K current and inhibited PVN parvocellular neurons by suppression of Ih.

Exogenous Ethyl Pyruvate versus Pyruvate during Metabolic Recovery after Oxidative Stress in Neonatal Rat Cerebrocortical Slices

Exogenous pyruvate and ethyl pyruvate (EP), the key ingredient in a new Ringer's solution in clinical trials, are antioxidants as well as metabolic substrates. In vivo studies show both to be protective in oxidative stress, with EP being better. The authors used an acute rat brain slice preparation to compare EP and pyruvate rescue after H(2)O(2) oxidative stress, asking whether EP was again better and whether its actions were exclusively metabolic. Oxygenated neonatal P7 cerebrocortical slices were exposed for 1 h to 2 mM H(2)O(2), and recovered for 4 h with artificial cerebrospinal fluid having 2 mM glucose and (1) 20 mM EP, (2) 20 mM pyruvate, or (3) 1 mM of the nonmetabolizable radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN). Perchloric acid extracts were studied with 31P/1H nuclear magnetic resonance at 14.1 T. Acute cell injury was assessed by counting terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL)-stained cells. At the end of recovery, preservation of adenosine triphosphate and N-acetylaspartate was better with EP than with pyruvate. Adenosine triphosphate preservation was best when PBN and EP were coadministered. 1H nuclear magnetic resonance revealed changes in lactate, alanine, gamma-aminobutyric acid, glutamate, glutamine, succinate, taurine, and myoinositol. Two-dimensional [1H-13C] heteronuclear single quantum coherence spectroscopy found that 13C-EP administration produced the same tricarboxylic acid metabolites as C-pyruvate. TUNEL-positive cell percentages with EP were less than half of those for PBN or pyruvate rescue (P < 0.05). EP enters cells, provides pyruvate as a tricarboxylic acid substrate, and is more protective. Although EP provides metabolic protection of adenosine triphosphate levels, it does not maximize antioxidant protection.

Cholecystokinin enhances GABAergic inhibitory transmission in basolateral amygdala

The neuropeptide cholecystokinin (CCK) is anxiogenic in studies of human and animal behavior. As the amygdala formation has been implicated in generation of emotional states such as anxiety, we tested the effect of CCK on spontaneous synaptic events in the basolateral amygdala (BLA) using whole cell patch recordings in rat brain slice preparation. We found that CCK increased the frequency of spontaneous inhibitory postsynaptic potentials (sIPSPs) and currents (sIPSCs). This effect was blocked by the fast sodium channel blocker tetrodotoxin (TTX), indicating that the CCK effect is likely mediated by direct excitation of GABAergic interneurons. The CCK(B) receptor subtype antagonist, CR2945, blocked the CCK effect, while CCK4, a specific CCK(B) agonist, increased sIPSC frequency. We hypothesize that these actions may underlie the anxiogenic effects of CCK observed in behavioral studies.

Modulation of postsynaptic potentials in rat cortical neurons by valerian extracts macerated with different alcohols: involvement of adenosine A1‐ and GABAA‐receptors

Valeriana officinalis (valerian) is used traditionally as a mild sedative. Research into valerian is sparse, and studies differ greatly with respect to design, measures and preparations used. This study compares the action of a methanol (M-E), ethanol (E-E) and an extract macerated with ethylacetate (EA-E) from roots of valerian (Valeriana officinalis L., Valerianaceae) on postsynaptic potentials (PSPs) in cortical neurons. Intracellular recordings were performed in rat brain slice preparations containing pyramidal cells of the cingulate cortex. PSPs were induced by electrical field stimulation. The M-E induced strong inhibition in the concentration range 0.1-15 mg/mL, whereas the E-E (1-10 mg/mL) did not influence significantly the PSPs. The maximum inhibition induced by the M-E was completely antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microm), an antagonist on the adenosine A(1) receptor. Contrary to the M-E, the EA-E (10 mg/mL) induced an increase of the PSPs, which was completely blocked by the GABA(A) receptor antagonist picrotoxin (100 microm). The data suggest that activation of adenosine A(1) and GABA(A) receptors is mediated by different components within the valerian extract. The two mechanisms may contribute independently to the sleep-inducing effect of valerian.

Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation

Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.

Bensultap decreases neuronal excitability in molluscan and mammalian central nervous system

Electrophysiological experiments were performed on in vitro neuronal preparations from terrestrial snail and rat brain slices, to determine the effect of the insecticide bensultap. Although bensultap has low toxicity in mammals, our results showed that bensultap altered the synaptic transmission in the vertebrate as well as in the invertebrate central nervous system. Bensultap caused a significant decrease of the ACh-induced current. The effect depended on the preapplication time and the concentration of the chemical. Bensultap also had an effect on the kinetic parameters of the ACh-induced current; the desensitization time was altered in a concentration-dependent manner. In the rat brain slice preparations, we observed an increase in the amplitude of the evoked responses after a 30 min treatment. There was no effect on paired-pulse depression, but LTP-induction was significantly inhibited by bensultap. The efficacy of synaptic transmission was modified by bensultap through effects on both input integration and output organization.

The β‐amyloid protein of Alzheimer’s disease binds to membrane lipids but does not bind to the α7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Abeta exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In this study, we examined the binding of Abeta1-42 to endogenous and recombinantly expressed alpha7nAChRs. Abeta1-42 did neither inhibit the specific binding of alpha7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of alpha7nAChRs expressed in Xenopus oocytes. Similarly, Abeta1-42 did not compete for alpha-bungarotoxin-binding sites on SH-SY5Y cells stably expressing alpha7nAChRs. The effect of the Abeta1-42 on tau phosphorylation was also examined. Although Abeta1-42 altered tau phosphorylation in alpha7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to alpha7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Abeta bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Abeta may disrupt membrane lipid structure or fluidity. We conclude that the effects of Abeta are unlikely to be mediated by direct binding to the alpha7nAChR. Instead, we speculate that Abeta may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.

Presynaptic α-adrenoceptors in median preoptic nucleus modulate inhibitory neurotransmission from subfornical organ and organum vasculosum lamina terminalis

The median preoptic nucleus (MnPO) in the lamina terminalis receives a prominent catecholaminergic innervation from the dorsomedial and ventrolateral medulla. The present investigation used whole cell patch-clamp recordings in rat brain slice preparations to evaluate the hypothesis that presynaptic adrenoceptors could modulate GABAergic inputs to MnPO neurons. Bath applications of norepinephrine (NE; 20-50 microM) induced a prolonged and reversible suppression of inhibitory postsynaptic currents (IPSCs) and reduced paired-pulse depression evoked by stimulation in the subfornical organ and organum vasculosum lamina terminalis. These events were not correlated with any observed changes in membrane conductance arising from NE activity at postsynaptic alpha(1)- or alpha(2)-adrenoceptors. Consistent with a role for presynaptic alpha(2)-adrenoceptors, responses were selectively mimicked by an alpha(2)-adrenoceptor agonist (UK-14304) and blockable with an alpha(2)-adrenoceptor antagonist (idazoxan). Although the alpha(1)-adrenoceptor agonist cirazoline and the alpha(1)-adrenoceptor antagonist prazosin were without effect on these evoked IPSCs, NE was noted to increase (via alpha(1)-adrenoceptors) or decrease (via alpha(2)-adrenoceptors) the frequency of spontaneous and tetrodotoxin-resistant miniature IPSCs. Collectively, these observations imply that both presynaptic and postsynaptic alpha(1)- and alpha(2)-adrenoceptors in MnPO are capable of selective modulation of rapid GABA(A) receptor-mediated inhibitory synaptic transmission along the lamina terminalis and therefore likely to exert a prominent influence in regulating cell excitability within the MnPO.

Repetitive Activation of Glutamatergic Inputs Evokes a Long-Lasting Excitation in Rat Globus Pallidus Neurons In Vitro

External globus pallidus (GPe) neurons express abundant metabotropic glutamate receptor 1 (mGluR1) in their somata and dendrites and receive glutamatergic inputs mainly from the subthalamic nucleus. We investigated whether synaptically released glutamate could activate mGluR1s using whole cell and cell-attached recordings in rat brain slice preparations. Repetitive internal capsule stimulation evoked EPSPs followed by a slow depolarizing response (sDEPO) lasting 10-20 s. Bath application of both GABA(A) and GABA(B) receptor antagonists increased the amplitude of sDEPOs. A mixture of AMPA/kainate and N-methyl-d-aspartate receptor antagonists did not alter sDEPOs. The induction of sDEPOs was only partially mediated by mGluR1 because mGluR1 antagonists reduced but failed to completely block the responses. Voltage-clamp recordings revealed that slow inward currents sensitive to mGluR1 antagonist were larger at -60 than at -100 mV, whereas the currents insensitive to mGluR1 antagonist were larger at -100 than at -60 mV. In cell-attached recordings, repetitive internal capsule stimulation evoked long-lasting excitations in GPe neurons, which were also partially suppressed by mGluR1 antagonists. Application of a glutamate uptake inhibitor or an mGluR1 agonist significantly increased the spontaneous firing rate but decreased the excitations to repetitive stimulation. These results suggest that synaptically released glutamate can activate mGluR1, contributing to the induction of long-lasting excitation in GPe neurons and that background mGluR1 activation suppresses the slow mGluR1 responses. Thus mGluR1 may play important roles in the control of GPe neuronal activity.

A rat brain slice preparation for characterizing both thalamostriatal and corticostriatal afferents

The striatum, the primary input nucleus of the basal ganglia, is crucially involved in motor and cognitive function and receives significant glutamate input from the cortex and thalamus. Increasing evidence suggests fundamental differences between these afferents, yet direct comparisons have been lacking. We describe a slice preparation that allows for direct comparison of the pharmacology and biophysics of these two pathways. Visualization of slices from animals previously injected with BDA into the parafascicular nucleus revealed the presence of axons of thalamic origin in the slice. These axons were especially well-preserved after traversing the reticular nucleus, the location chosen for stimulation of thalamostriatal afferents. Initial characterization of the two pathways revealed both non-NMDA and NMDA receptor-mediated currents at synapses from both afferents and convergence of the afferents in 51% of striatal efferent neurons. Annihilation of action potentials was not observed in collision experiments, nor was current spread from the site of stimulation to striatum found. Differences in short-term plasticity suggest that the probability of release differs for the two inputs. The present work thus provides a novel rat brain slice preparation in which the effects of selective stimulation of cortical versus thalamic afferents to striatum can be studied in the same preparation.

Systemic effect of cannabinoids on the spontaneous firing rate of locus coeruleus neurons in rats

Previous reports have described modulation of noradrenergic activity by cannabinoid receptors. The aim of the present research was to examine the effect of two synthetic cannabinoid CB 1/CB 2 receptor agonists, R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolol-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone (WIN 55212-2) and (−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl) cyclohexanol (CP 55940), on the spontaneous activity of locus coeruleus noradrenergic neurons by single-unit extracellular recordings in vivo and in vitro. In anaesthetized rats, intravenous administrations of WIN 55212-2 (31.3–500 μg/kg) or CP 55940 (31.3–500 μg/kg) increased the firing rate of locus coeruleus neurons in a dose-dependent manner. The stimulatory effect of WIN 55212-2 was blocked by pretreatment with the cannabinoid CB 1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1 H-pyrazole-3-carboxamide (SR 141716A; 2 mg/kg). Paradoxically, local administration of WIN 55212-2 (8.3–31.3 pmol) into the locus coeruleus and intracerebroventricular injections of WIN 55212–2 (10–20 μg) or CP 55940 (20–40 μg) failed to change the spontaneous firing rate of locus coeruleus neurons. Likewise, in rat brain slice preparations perfusion with WIN 55212-2 (10 μM) or CP 55940 (10–30 μM) did not specifically affect the spontaneous firing rate of locus coeruleus cells. Therefore, we conclude that synthetic cannabinoids increase the spontaneous firing activity of noradrenergic neurons in the rat locus coeruleus through cannabinoid CB 1 receptors. This stimulation appears to be indirectly induced via a receptor mechanism probably located at the peripheral level.

Input-specific long-term potentiation in the rat lateral amygdala of horizontal slices

Long-term potentiation (LTP) at input synapses to the lateral nucleus of the amygdala (LA) is a candidate mechanism for memory storage during fear learning. Cellular mechanisms of LTP have been nearly exclusively investigated in coronal brain slices. In our experiments, we used a horizontal brain slice preparation of rats that preserved most of the connections to cortical areas and the hippocampus. The stimulation electrodes were located either within the external capsule (EC) or the LA. The aim of the present study was to investigate the mechanisms of LTP induced either by weak theta burst stimulation (TBS) or strong high frequency stimulation (HFS) using the two different stimulation sites. Whereas both TBS and HFS of afferences running through the LA induced stable LTP, TBS failed to induce LTP of EC-inputs to the LA. The present findings also show that LTP in the LA exhibits vulnerability at different time windows after induction. The time window was dependent on the kind of stimulated afferences. Later LTP becomes resistant to disruption by low frequency stimulation. We could show that both used inputs depended on NMDA receptors for LTP-induction. LTP induced by stimulation of fibers within the LA was not altered by nifedipine (10 μM). In contrast, EC-induced LTP was dependent on L-type voltage-gated calcium channels (VGCC). Finally, we found a higher magnitude of LTP in females using TBS, whereas HFS did not cause gender-specific differences. Our study supports the conclusion that the form of LA-LTP depend on which afferences are activated and what pattern of stimulation is used to induce LTP.

Suprachiasmatic nucleus communicates with anterior thalamic paraventricular nucleus neurons via rapid glutamatergic and gabaergic neurotransmission: State-dependent response patterns observed in vitro

The hypothalamic suprachiasmatic nucleus uniquely projects to the midline thalamic paraventricular nucleus. To characterize this projection, patch clamp techniques applied in acute rat brain slice preparations examined responses of anterior thalamic paraventricular nucleus neurons to focal suprachiasmatic nucleus stimulation. Whole cell recordings from slices obtained during daytime (n=40) revealed neurons with a mean membrane potential of −66±1.2 mV, input conductance of 1.5±0.1 nS and state-dependent tonic or burst firing patterns. Electrical stimulation (one or four pulses) in suprachiasmatic nucleus elicited monosynaptic excitatory postsynaptic potentials (mean latency of 12.6±0.6 ms; n=12), featuring both AMPA and N-methyl-d-aspartate–glutamate receptor-mediated components, and monosynaptic bicuculline-sensitive inhibitory postsynaptic potentials (mean latency of 16.6±0.6 ms; n=7) reversing polarity at −72±2.6 mV, close to the chloride equilibrium potential. Glutamate microstimulation of suprachiasmatic nucleus also elicited transient increases in spontaneous excitatory or inhibitory postsynaptic currents in anterior thalamic paraventricular neurons. Recordings from rats under reverse light/dark conditions (n=22) yielded essentially similar responses to electrical stimulation. At depolarized membrane potentials, suprachiasmatic nucleus-evoked excitatory postsynaptic potentials triggered single action potentials, while evoked inhibitory postsynaptic potentials elicited a silent period in ongoing tonic firing. By contrast, after manual adjustment of membrane potentials to hyperpolarized levels, neuronal response to the same ‘excitatory’ stimulus was a low threshold spike and superimposed burst firing, while responses to ‘inhibitory’ stimuli paradoxically elicited excitatory rebound low threshold spikes and burst firing. These data support the existence of glutamatergic and GABAergic efferents from the suprachiasmatic nucleus to its target neurons. Additionally, in thalamic paraventricular nucleus neurons, responses to activation of their suprachiasmatic afferents may vary in accordance with their membrane potential–dependent intrinsic properties, a characteristic typical of thalamocortical neurons.

Intrinsic subthreshold oscillations of the membrane potential in pyramidal neurons of the olfactory amygdala

The amygdala complex is a heterogeneous group of temporal lobe brain structures involved in the processing of biologically significant sensory stimuli and in the generation of appropriate responses to them. The amygdala has also been implicated in certain forms of emotional learning and memory. While much progress has been made in understanding neural processing in the basolateral subgroup of the amygdala, physiological studies in the cortical regions of the complex, also known as olfactory amygdala, are missing. Using a rat brain slice preparation, we conducted whole-cell recordings on pyramidal neurons of the periamygdaloid cortex and the anterior cortical nucleus, two structures receiving direct connections from the olfactory bulb. Upon depolarization by current injection through the recording electrode, a fraction of periamygdaloid cortex and most anterior cortical nucleus layer II pyramidal neurons displayed an intermittent discharge pattern, where clusters of action potentials were interspersed by periods of membrane potential subthreshold oscillations. Oscillations frequency increased with membrane potential and correlated linearly with the cluster spiking frequency. Frequency ranged from 3 to 20 Hz, considering different cells and membrane potential values (up to approximately 30 mV above resting potentials of typically approximately -70 mV). Subthreshold oscillations were preserved after pharmacological inhibition of fast excitatory and inhibitory synaptic transmission, but were abolished by application of the sodium channel blocker tetrodotoxin. We conclude that pyramidal neurons of the olfactory cortical amygdala display intrinsically generated voltage-dependent membrane potential rhythmic fluctuations in the theta-low beta range, requiring the activation of a sodium conductance.

Excitatory effect of histamine on neuronal activity of rat globus pallidus by activation of H 2 receptors in vitro

Previous studies have revealed distribution of histaminergic fibers and presence of histamine receptors in globus pallidus (GP). In this study, the brain slice preparation of adult rats was used to examine the effect of histamine on the spontaneous unitary discharge of GP neurons and the underlying receptor mechanism. Ninety-five GP neurons were extracellularly recorded from 42 slices containing the GP, of which 87 (91.6%) were excited by the stimulation of histamine. The histamine-induced excitation was concentration-dependent and persisted in low Ca 2+/high Mg 2+ medium ( n = 9), demonstrating that the action of histamine on the GP neurons was postsynaptic. The excitatory effect of histamine on the GP neurons was not blocked by selective histamine H 1 receptor antagonist triprolidine ( n = 16) or chlorpheniramine ( n = 6), but was effectively suppressed by ranitidine, a highly selective histamine H 2 receptor antagonist ( n = 21). On the other hand, highly selective histamine H 2 receptor agonist dimaprit mimicked the excitatory effect of histamine on the GP neurons ( n = 23), while histamine H 1 receptor agonists, including 2-pyridylethylamine ( n = 22), 2-thiazolyethylamine ( n = 9) and betahistine ( n = 9), did not cause GP neurons any response. The dimaprit-induced GP neuronal excitation was effectively antagonized by selective histamine H 2 receptor antagonist ranitidine ( n = 14) but not influenced by selective histamine H 1 receptor antagonist triprolidine ( n = 12). Moreover, adenylate cyclase (AC) activator forskolin ( n = 7) was observed to evoke GP neurons an excitatory response, whereas the histamine-induced excitation was effectively reduced by H-89 ( n = 9), a selective and potent inhibitor of protein kinase A (PK A). Finally, it was noted that neurons of both subdivisions of the GP, the internal (GPi, n = 35) and external (GPe, n = 60) segment, showed no differences in their responses to stimulations of the tested histaminergic reagents. These results demonstrated that histamine excited GP (including GPi and GPe) neurons via histamine H 2 receptors and H 2 receptors linked intracellular G-protein-AC-PK A signaling pathway, suggesting that the hypothalamic histaminergic afferent fibers innervating GP may play an important modulatory role in motor control through its excitatory effect on GP neurons.

Synaptically Released GABA Activates Both Pre- and Postsynaptic GABAB Receptors in the Rat Globus Pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.

Metabotropic glutamate receptor activity induces a novel oscillatory pattern in neonatal rat hypoglossal motoneurones

Tongue muscles innervated by the hypoglossal nerves play a crucial role to ensure airway patency and milk suckling in the neonate. Using a slice preparation of the neonatal rat brain, we investigated the electrophysiological characteristics of hypoglossal motoneurones in the attempt to identify certain properties potentially capable of synchronizing motor commands to the tongue. Bath-applied DHPG, a selective agonist of group I metabotropic glutamate receptors (mGluRs), generated persistent, regular electrical oscillations (4-8 Hz) recorded from patch-clamped motoneurones. Under voltage clamp, oscillations were biphasic events, comprising large outward slow currents alternated with fast, repeated inward currents. Electrical oscillations had amplitude and period insensitive to cell membrane potential, and required intact glutamatergic transmission via AMPA receptors. Oscillations were mediated by subtype 1 receptors of group I mGluRs (mGluR1s), and were routinely observed during pharmacological block of glycinergic and GABAergic inhibition, although they could also be recorded in standard saline. Simultaneous recordings from pairs of motoneurones within the same hypoglossal nucleus demonstrated that oscillations were due to their strong electrical coupling and were blocked by the gap junction blocker carbenoxolone. Pacing of slow oscillations apparently depended on the operation of K(ATP) channels in view of the block by tolbutamide or glibenclamide. Under current clamp, oscillations generated more regular spike firing of motoneurones and facilitated glutamatergic excitatory inputs. These data suggest that neonatal motoneurones of the nucleus hypoglossus possess a formerly undisclosed ability to express synchronous electrical oscillations, unveiled by activation of mGluR1s.