Mouse Brain Slice Preparation Research Articles

Orphan receptor-GPR52 inverse agonist efficacy in ameliorating chronic stress-related deficits in reward motivation and phasic accumbal dopamine activity in mice

Reward processing dysfunctions e.g., anhedonia, apathy, are common in stress-related neuropsychiatric disorders including depression and schizophrenia, and there are currently no established therapies. One potential therapeutic approach is restoration of reward anticipation during appetitive behavior, deficits in which co-occur with attenuated nucleus accumbens (NAc) activity, possibly due to NAc inhibition of mesolimbic dopamine (DA) signaling. Targeting NAc regulation of ventral tegmental area (VTA) DA neuron responsiveness to reward cues could involve either the direct or indirect—via ventral pallidium (VP)—pathways. One candidate is the orphan G protein-coupled receptor GPR52, expressed by DA receptor 2 NAc neurons that project to VP. In mouse brain-slice preparations, GPR52 inverse agonist (GPR52-IA) attenuated evoked inhibitory postsynaptic currents at NAc-VP neurons, which could disinhibit VTA DA neurons. A mouse model in which chronic social stress leads to reduced reward learning and effortful motivation was applied to investigate GPR52-IA behavioral effects. Control and chronically stressed mice underwent a discriminative learning test of tone-appetitive behavior-sucrose reinforcement: stress reduced appetitive responding and discriminative learning, and these anticipatory behaviors were dose-dependently reinstated by GPR52-IA. The same mice then underwent an effortful motivation test of operant behavior-tone-sucrose reinforcement: stress reduced effortful motivation and GPR52-IA dose-dependently restored it. In a new cohort, GRABDA-sensor fibre photometry was used to measure NAc DA activity during the motivation test: in stressed mice, reduced motivation co-occurred with attenuated NAc DA activity specifically to the tone that signaled reinforcement of effortful behavior, and GPR52-IA ameliorated both deficits. These findings: (1) Demonstrate preclinical efficacy of GPR52 inverse agonism for stress-related deficits in reward anticipation during appetitive behavior. (2) Suggest that GPR52-dependent disinhibition of the NAc-VP-VTA-NAc circuit, leading to increased phasic NAc DA signaling of earned incentive stimuli, could account for these clinically relevant effects.

DIRECT, a low-cost system for high-speed, low-noise imaging of fluorescent bio-samples.

A targeted imaging system has been developed for applications requiring recording from stationary samples at high spatiotemporal resolutions. It works by illuminating regions of interest in rapid sequence, and recording the signal from the whole field of view onto a single photodetector. It can be implemented at low cost on an existing microscope without compromising existing functionality. The system is characterized in terms of speed, spatial resolution, and tissue penetration depth, before being used to record individual action potentials from ASAP-3 expressing neurons in an ex vivo mouse brain slice preparation.

Tachykinins amplify the action of capsaicin on central histaminergic neurons

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

Role of seizure activity within the parafascicular nucleus in cessation of respiratory rhythm and potentially SUDEP

Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in patients with uncontrolled epilepsy, yet the mechanisms underlying SUDEP are unknown. Recent data suggests that as seizures propagate in the brain, they can cause respiratory and/or cardiac arrest, leading to death (e.g. SUDEP). Although the precise mechanisms are unknown, seizure-induced respiratory arrest (S-IRA) is the most likely cause of SUDEP. The neural pathways propagating seizure (SZR) activity from areas in the brain where seizures initiate (cortex, hippocampus and thalamus) to autonomic respiratory centers in the brainstem, resulting in respiratory failure and SUDEP are unknown. To identify SZR propagation pathways, I am using a new mouse brainslice preparation that includes cortex, hippocampus and thalamus as well as autonomic medullary areas controlling breathing. Preliminary electrophysiological data suggest that seizure-like activity resulting in repeated activation of the parafascicular nucleus (PFn) of the thalamus, disrupts and then stops rhythmic respiratory activity generated by the medullary respiratory neural network, the pre-Bötzinger Complex (preBötC). I am testing my hypothesis that SZR propagation from the cortex to the PFn can interrupt respiratory activity, which may underlie or significantly contribute to respiratory arrest and/or SUDEP. The centromedial/parafascicular (CMn/PFn) thalamic nuclear complex is a deep brain stimulation target for control of epilepsy. However, little is known about the role the PFn may play in seizure propagation, control of breathing and/or SUDEP. The data support the hypothesis that the PFn may play a role in SUDEP, as it: 1) initiates and/or propagates SZRs; 2) innervates respiratory rhythm networks in the medulla, including the preBötC; and, 3) its activation inhibits breathing.

Tonic Control of Secretory Acid Sphingomyelinase Over Ventral Hippocampal Synaptic Transmission and Neuron Excitability.

The acid sphingomyelinase (ASM) converts sphingomyelin into ceramide. Recent work has advanced the ASM/ceramide system as a major player in the pathogenesis of major depressive disorder (MDD). Indeed, ASM activity is enhanced in MDD patients and antidepressant drugs like fluoxetine act as functional inhibitors of ASM. Here, we employed the specific ASM inhibitor ARC39 to explore the acute effects of the enzyme on hippocampal synaptic transmission and cell excitability in adult mouse brain slice preparations. In both field potential and whole-cell recordings, ARC39 (1–3 μM) enhanced excitatory synaptic input onto ventral hippocampal CA1 pyramidal cells. The specificity of drug action was demonstrated by its lacking effect in slices from ASM knockout mice. In control condition, ARC39 strongly reduced firing in most CA1 pyramidal cells, together with membrane hyperpolarization. Such pronounced inhibitory action of ARC39 on soma excitability was largely reversed when GABAA receptors were blocked. The idea that ARC39 recruits GABAergic inhibition to dampen cell excitability was further reinforced by the drug’s ability to enhance the inhibitory synaptic drive onto pyramidal cells. In pyramidal cells that were pharmacologically isolated from synaptic input, the overall effect of ARC39 on cell firing was inhibitory, but some neurons displayed a biphasic response with a transient increase in firing, suggesting that ARC39 might alter intrinsic firing properties in a cell-specific fashion. Because ARC39 is charged at physiological pH and exerted all its effects within minutes of application, we propose that the neurophysiological actions reported here are due to the inhibition of secretory rather than lysosomal ASM. In summary, the ASM inhibitor ARC39 reveals a tonic control of the enzyme over ventral hippocampal excitability, which involves the intrinsic excitability of CA1 pyramidal cells as well as their excitatory and inhibitory synaptic inputs.

Investigating KYNA production and kynurenergic manipulation on acute mouse brain slice preparations

Manipulation of kynurenic acid (KYNA) level through kynurenine aminotransferase-2 (KAT-2) inhibition with the aim of therapy in neuro-psychiatric diseses has been the subject of extensive recent research. Although mouse models are of particular importance, neither the basic mechanism of KYNA production and release nor the relevance of KAT-2 in the mouse brain has yet been clarified.Using acute mouse brain slice preparations, we investigated the basal and L-kynurenine (L-KYN) induced KYNA production and distribution between the extracellular and intracellular compartments. Furthermore, we evaluated the effect of specific KAT-2 inhibition with the irreversible inhibitor PF-04859989. To ascertain that the observed KYNA release is not a simple consequence of general cell degradation, we examined the structural and functional integrity of the brain tissue with biochemical, histological and electrophysiological tools.We did not find relevant change in the viability of the brain tissue after several hours incubation time. HPLC measurements proved that mouse brain slices intensively produce and liberate KYNA to the extracellular compartment, while only a small proportion retained in the tissue both in the basal and L-KYN supplemented state. Finally, specific KAT-2 inhibition significantly reduced the extracellular KYNA content.Taken together, these results provide important data about KYNA production and release, and in vitro evidence for the first time of the function of KAT-2 in the adult mouse brain. Our study extends investigations of KAT-2 manipulation to mice in a bid to fully understand the function; the final, future aim is to assign therapeutical kynurenergic manipulation strategies to humans.

Geniculohypothalamic GABAergic projections gate suprachiasmatic nucleus responses to retinal input.

Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt their physiology and behaviour in line with the solar day. In addition to direct retinal projections, the clock receives input from the visual thalamus, although the role of this geniculohypothalamic pathway in circadian photoreception is poorly understood. In the present study, we develop a novel brain slice preparation that preserves the geniculohypothalamic pathway to show that GABAergic thalamic neurons inhibit retinally-driven activity in the central clock in a circadian time-dependent manner. We also show that in vivo manipulation of thalamic signalling adjusts specific features of the hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily activated by crossed retinal inputs. Our data provide a mechanism by which geniculohypothalamic signals can adjust the magnitude of circadian and more acute hypothalamic light responses according to time-of-day and establish an important new model for future investigations of the circadian visual system. Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in allowing animals to optimize physiology and behaviour alongside daily changes in the environment. Retinal inputs encoding changes in external illumination provide the principle source of such information. The SCN also receives input from other retinorecipient brain regions, primarily via the geniculohypothalamic tract (GHT), although the contribution of these indirect projections to circadian photoreception is currently poorly understood. To address this deficit, in the present study, we established an in vitro mouse brain slice preparation that retains connectivity across the extended circadian system. Using multi-electrode recordings, we first confirm that this preparation retains intact optic projections to the SCN, thalamus and pretectum and a functional GHT. We next show that optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, and also that this effect exhibits a pronounced day/night variation and involves a GABAergic mechanism. This inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside pharmacological inactivation or optogenetic excitation to show that GHT signalling actively modulates specific features of the SCN light response, indicating that GHT cells are primarily activated by crossed retinal projections. Taken together, our data establish a new model for studying network communication in the extended circadian system and provide novel insight into the roles of GHT-signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the SCN according to time of day.

Pulse-Width Modulation of Optogenetic Photo-Stimulation Intensity for Application to Full-Implantable Light Sources.

Optogenetics allows control of neuronal activity with unprecedented spatiotemporal precision, and has enabled both significant advances in neuroscience and promising clinical prospects for some neurological, cardiac, and sensory disorders. The ability to chronically stimulate light-sensitive excitable cells is crucial for developing useful research tools and viable long-term treatment strategies. Popular optogenetic stimulation devices often rely on bench-top light-sources tethered via an optical fibre to the research animal, or significant componentry protruding externally from animal. These approaches are prone to infection, vulnerable to damage and restrict the experimental approaches that can be conducted. An ideal optogenetic stimulator would be contained entirely within the animal and provide precisely controlled optical output. However, existing prototypes of fully implantable devices rely on amplitude tuning of wireless power, which can vary strongly with environmental conditions. Here we show that pulse-width modulation (PWM) of the intensity of a light-emitting diode (LED) can enable control of photo-stimulation intensity equivalent to direct amplitude modulation. This result has significant implications for fully implantable light delivery tools, as PWM can be implemented with simple and miniaturized circuit architectures. We have modified a telemeter device previously developed by our group to include a small form-factor LED capable of generating sufficient optical power with manageable electrical power requirements and minimal heat generation. We have tested key device components in an in vitro mouse brain slice preparation and shown that pulse-width-modulation is an alternative method to modulate photo-stimulation intensity using a miniature circuit and providing easy control.

Auditory Golgi cells are interconnected predominantly by electrical synapses.

The mossy fiber-granule cell-parallel fiber system conveys proprioceptive and corollary discharge information to principal cells in cerebellum-like systems. In the dorsal cochlear nucleus (DCN), Golgi cells inhibit granule cells and thus regulate information transfer along the mossy fiber-granule cell-parallel fiber pathway. Whereas excitatory synaptic inputs to Golgi cells are well understood, inhibitory and electrical synaptic inputs to Golgi cells have not been examined. Using paired recordings in a mouse brain slice preparation, we find that Golgi cells of the cochlear nucleus reliably form electrical synapses onto one another. Golgi cells were only rarely electrically coupled to superficial stellate cells, which form a separate network of electrically coupled interneurons in the DCN. Spikelets had a biphasic effect on the excitability of postjunctional Golgi cells, with a brief excitatory phase and a prolonged inhibitory phase due to the propagation of the prejunctional afterhyperpolarization through gap junctions. Golgi cells and stellate cells made weak inhibitory chemical synapses onto Golgi cells with low probability. Electrical synapses are therefore the predominant form of synaptic communication between auditory Golgi cells. We propose that electrical synapses between Golgi cells may function to regulate the synchrony of Golgi cell firing when electrically coupled Golgi cells receive temporally correlated excitatory synaptic input.

Striatal neurones have a specific ability to respond to phasic dopamine release

The cAMP/protein kinase A (PKA) signalling cascade is ubiquitous, and each step in this cascade involves enzymes that are expressed in multiple isoforms. We investigated the effects of this diversity on the integration of the pathway in the target cell by comparing prefrontal cortical neurones with striatal neurones which express a very specific set of signalling proteins. The prefrontal cortex and striatum both receive dopaminergic inputs and we analysed the dynamics of the cAMP/PKA signal triggered by dopamine D1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D1 response between pyramidal cortical neurones and striatal medium spiny neurones: the cAMP/PKA response was much stronger, faster and longer lasting in striatal neurones than in pyramidal cortical neurones. We identified three molecular determinants underlying these differences: different activities of phosphodiesterases, particularly those of type 4, which strongly damp the cAMP signal in the cortex but not in the striatum; stronger adenylyl cyclase activity in the striatum, generating responses with a faster onset than in the cortex; and DARPP-32, a phosphatase inhibitor which prolongs PKA action in the striatum. Striatal neurones were also highly responsive in terms of gene expression since a single sub-second dopamine stimulation is sufficient to trigger c-Fos expression in the striatum, but not in the cortex. Our data show how specific molecular elements of the cAMP/PKA signalling cascade selectively enable the principal striatal neurones to respond to brief dopamine stimuli, a critical process in incentive learning.

A modulatory effect of the feedback from higher visual areas to V1 in the mouse

Using a mouse brain slice preparation, we studied the modulatory effects of a feedback projection from higher visual cortical areas, mostly or exclusively area LM (or V2), on two inputs to layer 4 cells in the first visual area (V1). The two inputs to these cells were geniculocortical and an unspecified intracortical input, possibly involving layer 6 cells. We found that activation of metabotropic glutamate receptors (mGluRs) from stimulation of the feedback projection reduced the evoked excitatory postsynaptic currents of both of these inputs to layer 4 but that this modulation acts in an input-specific way. Reducing the strength of the geniculocortical input in adults involved both presynaptic and postsynaptic group I mGluRs (although in younger animals presynaptic group II mGluRs were also involved), whereas modulation of the intracortical input acted entirely via postsynaptic group II mGluRs. These results demonstrate that one of the effects of this feedback pathway is to control the gain of geniculocortical transmission.

Alterations of corticostriatal plasticity by ammonium and rescue by green tea polyphenols

Hyperammonemia is a major pathophysiological factor in encephalopathies associated with acute and chronic liver failure. On mouse brain slice preparations we analyzed the effects of ammonium on the characteristics of corticostriatal long-term potentiation (LTP) induced by high-frequency electrical stimulation (HFS) of cortical input and the long-lasting effects of pharmacological NMDA receptor (NMDAR) activation. Ammonium chloride exposure enhanced the expression of HFS-induced LTP at the expense of LTD and promoted the generation of NMDA-induced LTD. This treatment did not affect two NMDAR-independent forms of plasticity: taurine-induced LTP and histamine-induced LTD. Alterations in NMDA-induced plasticity were prevented by treatment with green tea polyphenols suggesting the contribution of oxidative stress to the expression of abnormal corticostriatal plasticity.

Development of a micro-imaging probe for functional brain imaging

Multicellular neuronal activities should be investigated to reveal the dynamics of the neuronal circuit. Optical recording from neuronal populations is suitable for recording multicellular activities. We fabricated the prototype of the micro-imaging probe in combination with a gradient index lens and image fiber. This probe has a smaller diameter than traditional probes. We found an optimal optical configuration for maximizing the efficiency of the imaging probe. Using this optical configuration with the prototype of the imaging probe, the fluorescence images were captured from neurons expressing green fluorescent protein in a cerebellar block preparation, and the calcium-dependent images were sampled in a mouse brain slice preparation. Our optical system would facilitate the in vivo imaging studies with less invasive manners using thinner optic fiber than previously made.

Ammonia‐induced deficit in corticostriatal long‐term depression and its amelioration by zaprinast

Hyperammonemia is a major pathophysiological factor in encephalopathies associated with acute and chronic liver failure. On mouse brain slice preparations, we analyzed the effects of ammonia on the characteristics of corticostriatal long-term depression (LTD) induced by electrical stimulation of cortical input or pharmacological activation of metabotropic glutamate receptors. Long exposure of neostriatal slices to ammonium chloride impaired the induction and/or expression of all studied forms of LTD. This impairment was reversed by the phosphodiesterase inhibitor zaprinast implying lowered cGMP signaling in LTD suppression. Polyphenols from green tea rescued short-term corticostriatal plasticity, but failed to prevent the ammonia-induced deficit of LTD. Zaprinast counteracts the ammonia-induced impairment of long-term corticostriatal plasticity and may thus improve fine motor skills and procedural learning in hepatic encephalopathy.

Evaluation of the Protective Effects of Cyclosporin A and FK506 on Abnormal Cytosolic and Mitochondrial Ca2+ Dynamics During Ischemia and Exposure to High Glutamate Concentration in Mouse Brain Slice Preparations

We examined the protective effects of the immunosuppressants cyclosporin A (CsA) and FK506 on abnormal cytosolic Ca2+ ([Ca2+]c) and mitochondrial Ca2+ concentration ([Ca2+]m) dynamics induced by ischemia or high l-glutamate concentration in mouse brain slice preparations. We used fura-4F and rhod-2 as indicators for [Ca2+]c and [Ca2+]m, respectively, in their acetoxymethylester form. Slice preparations loaded with either of these two indicators were exposed to ischemic artificial cerebrospinal fluid (oxygen- and glucose-deprived medium) for 12 min or to aerobic medium with high l-glutamate concentration (isotonic 20 mM l-glutamate) for 5 min. CsA (1 – 10 μM) showed significant protective effects on the maximum increase in ischemia-induced [Ca2+]c and [Ca2+]m. FK506 (10 μM) showed significant protective effects on the [Ca2+]m increase, but not on the ischemia-induced [Ca2+]c increase. Both immunosuppressants showed almost equal protective effects on the [Ca2+]c and [Ca2+]m increases induced by high l-glutamate concentration. These results suggest that the protective effects of CsA and FK506 on Ca2+ overloading may be dependent upon the common pharmacological sites of actions relating to their effects as immunosuppressants. The small, but significant depressant effects of these drugs could give us important clues for rescuing critical brain damage induced by Ca2+ overloading.

Entorhinal theta-frequency input to the dentate gyrus trisynaptically evokes hippocampal CA1 LTP

There exists substantial evidence that some forms of explicit learning in mammals require long-term potentiation (LTP) at hippocampal CA3-CA1 synapses. While CA1 LTP has been well characterized at the monosynaptic level, it still remains unclear how the afferent systems to the hippocampus can initiate formation of this neuroplastic phenomenon. Using voltage-sensitive dye imaging (VSDI) in a mouse brain slice preparation, we show that evoked entorhinal cortical (EC) theta-frequency input to the dentate gyrus highly effectively generates waves of neuronal activity which propagate through the entire trisynaptic circuit of the hippocampus (“HTC-Waves”). This flow of activity, which we also demonstrate in vivo, critically depends on frequency facilitation of mossy fiber to CA3 synaptic transmission. The HTC-Waves are rapidly boosted by the cognitive enhancer caffeine (5 μM) and the stress hormone corticosterone (100 nM). They precisely follow the rhythm of the EC input, involve high-frequency firing (>100 Hz) of CA3 pyramidal neurons, and induce NMDA receptor-dependent CA1 LTP within a few seconds. Our study provides the first experimental evidence that synchronous theta-rhythmical spiking of EC stellate cells, as occurring during EC theta oscillations, has the capacity to drive induction of CA1 LTP via the hippocampal trisynaptic pathway. Moreover, we present data pointing to a basic filter mechanism of the hippocampus regarding EC inputs and describe a methodology to reveal alterations in the “input–output relationship” of the hippocampal trisynaptic circuit.

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

Inhibition by 5-HT of the synaptic responses evoked by callosal fibers on cortical neurons in the mouse

We have studied the modulation by 5-HT of the synaptic excitatory responses evoked by callosal fibers on cortical pyramidal neurons. We have used a mouse brain slice preparation that preserves the callosal fibers and allows their selective activation. EPSCs evoked by callosal stimulation (ccEPSCs) were recorded with patch electrodes from pyramidal neurons identified visually. We observed that 5-HT (10-40μM) inhibited the ccEPSCs peak amplitude in 64% of the neurons; 5-HT had no effect in the remaining neurons. 5-HT also increased the frequency and amplitude of spontaneous EPSCs. This inhibition was accompanied with an increase in the coefficient of variation of the fluctuations of the ccEPSCs amplitude and with an increase in the ratio of the amplitudes of paired ccEPSCs. Agonists of 5-HT receptor subtypes 5-HT(1A) (8-OH-DPAT) and 5-HT(2A) (DOI) mimicked the effect of 5-HT; also, the effect of 8-OH-DPAT and DOI was blocked in the presence of specific blockers of 5-HT(1A) (WAY 100135) and 5-HT(2A) (MDL 11,939) receptors. Application of 5-HT did not change the amplitude of currents evoked by direct application of glutamate to neurons in which 5-HT decreased the amplitude of ccEPSC. The effects of 5-HT on ccEPSCs and on the synaptic currents evoked by intracortical stimulation were not correlated; this suggests that the effect of 5-HT was specific to particular synaptic inputs to a neuron. These results demonstrate the presynaptic modulation of the callosal synaptic responses by 5-HT and the implication of 5-HT(1A) and 5-HT(2A) receptors in this effect.

Orexins/hypocretins control bistability of hippocampal long‐term synaptic plasticity through co‐activation of multiple kinases

Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep-wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTP(OX)) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTD(OX)) in the same pathway. LTP(OX) is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTD(OX) in adult animals. Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions.

Ghrelin Potentiates Miniature Excitatory Postsynaptic Currents in Supraoptic Magnocellular Neurones

Ghrelin is an orexigenic peptide discovered in the stomach as a ligand of the orphan G-protein coupled receptor, and participates in the regulation of growth hormone (GH) release. Previous studies have demonstrated that ghrelin suppressed water intake and stimulated the secretion of arginine vasopressin in rats. We examined the effect of ghrelin on the excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) using whole-cell patch-clamp recordings in in vitro rat and mouse brain slice preparations. The application of ghrelin (10(-7) approximately 10(-6) m) caused a significant increase in the frequency of the miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The increased frequency of the spontaneous EPSCs persisted in the presence of tetrodotoxin (1 microM). Des-n-octanoyl ghrelin (10(-6) m) did not have a significant effect on the mEPSCs. The ghrelin-induced potentiation of the mEPSCs was significantly suppressed by previous exposure to the transient receptor potential vanilloid (TRPV) blocker, ruthenium red (10 microM) and GH secretagougue type 1a receptor selective antagonist, BIM28163 (10 microM). The effects of ghrelin on the supraoptic MNCs in trpv1 knockout mice were significantly attenuated compared to those in wild-type mice counterparts. These results suggest that ghrelin participates in the regulation of synaptic inputs to the MNCs in the SON via interaction with the GH secretagogue type 1a receptor, and that the TRPV1 channel may be involved in ghrelin-induced potentiation of mEPSCs to the MNCs in the SON.