Arduino based intra-cerebral microinjector device for neuroscience research.

Stereotaxic surgery is a less invasive form of surgery that uses a three-dimensional coordinate system to place instruments at a specific location in the brain. Through this type of surgery, one can place needles among other tools within the structures of the brain. Therefore, injections can be given in order to deliver substances that cannot cross the blood-brain barrier. The two most important parameters of the microinjection to control are volume and speed. The volume should not be so large that it displaces the brain tissue and tears it. The injection speed must also be slow so that the liquid that comes out of the syringe can diffuse into the tissue without displacing it and damaging it. Thus, the objectives of the present work are: 1) To develop not a 3D printed prototype but an end-user device. 2) The device must be for animal research only. 3) It must have the same precision in volume and speed as commercial devices. 4) It must be adjustable for microsyringes from 0.5 µl to 1 ml. 5) It must be possible to place it directly on the stereotaxic surgery apparatus and to use it separately. 6) The price must be substantially lower than that of the commercial devices.

Nicotine Increases Spontaneous Glutamate Release in the Rostromedial Tegmental Nucleus.

The rostromedial tegmental nucleus (RMTg) is a bilateral structure localized in the brainstem and comprise of mainly GABAergic neurons. One of the main functions of the RMTg is to regulate the activity of dopamine neurons of the mesoaccumbens pathway. Therefore, the RMTg has been proposed as a modulator of the reward system and adaptive behaviors associated to reward learning. The RMTg receives an important glutamatergic input from the lateral habenula. Also, it receives cholinergic inputs from the laterodorsal and pedunculopontine tegmental nuclei. Previously, it was reported that nicotine increases glutamate release, evoked by electric stimulation, in the RMTg nucleus. However, the mechanisms by which nicotine induces this effect were not explored. In the present work, we performed electrophysiological experiments in brainstem slices to study the effect of nicotine on spontaneous excitatory postsynaptic currents recorded from immunocytochemically identified RMTg neurons. Also, we used calcium imaging techniques to explore the effects of nicotine on multiple RMTg neurons simultaneously. We found that nicotine promotes the persistent release of glutamate through the activation of α7 nicotinic acetylcholine receptors present on glutamatergic afferents and by a mechanism involving calcium release from intracellular stores. Through these mechanisms, nicotine increases the excitability and synchronizes the activity of RMTg neurons. Our results suggest that the RMTg nucleus mediates the noxious effects of the nicotine, and it could be a potential therapeutic target against tobacco addiction.

Electrophysiological characterization of glucose sensing neurons in the hypothalamic arcuate nucleus of male rats.

The arcuate nucleus (ARC), located at the base of hypothalamus, contains two main populations of neurons involved in the regulation of food intake and energy expenditure. The NPY neurons are orexigenic and their activation stimulates food intake while the activation of POMC neurons promote the opposite effect. Several works have tried to identify these neurons based on their electrophysiological and pharmacological characteristics. However, the classification of ARC neurons is still inconclusive. In this work, glucose concentrations were changed within at physiological range, and the response of rat ARC neurons to this stimulus was used to identify them. Subsequently, the cells were classified on the basis of their passive and active electrophysiological properties. Finally, calcium imaging experiments were done to study the response of ARC neurons populations changing glucose concentrations. We found that NPY and putative POMC neurons can be distinguished based on their electrophysiological properties such as input resistance and firing pattern. Calcium imaging experiments confirmed the diversity of ARC neurons.

GABAergic modulation of serotonergic neurons in the dorsal raphe nucleus.

The dorsal raphe nucleus (DRN), located in the brainstem, is involved in several functions such as sleep, temperature regulation, stress responses, and anxiety behaviors. This nucleus contains the largest population of serotonin expressing neurons in the brain. Serotonergic DRN neurons receive tonic γ-aminobutyric acid (GABA)inhibitory inputs from several brain areas, as well as from interneurons within the same nucleus. Serotonergic and GABAergic neurons in the DRN can be distinguished by their size, location, pharmacological responses, and electrophysiological properties. GABAergic neurons regulate the excitability of DRN serotonergic neurons and the serotonin release in different brain areas. Also, it has been shown that GABAergic neurons can synchronize the activity of serotonergic neurons across functions such as sleep or alertness. Moreover, dysregulation of GABA signaling in the DRN has been linked to psychiatric disorders such as anxiety and depression. This review focuses on GABAergic transmission in the DRN. The interaction between GABAergic and serotonergic neurons is discussed considering some physiological implications. Also, the main electrophysiological and morphological characteristics of serotonergic and GABAergic neurons are described.

Fast Inactivation of CaV2.2 Channels Is Prevented by the Gß1 Subunit in Rat Sympathetic Neurons.

Voltage-dependent regulation of CaV2.2 channels by G-proteins is performed by the ß (Gß) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of CaV2.2 channels in superior cervical ganglion neurons that overexpressed Gß subunits. CaV2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gß1 subunit reduced inactivation, while Gß5 subunit did not alter at all inactivation kinetics compared to control recordings. CaV2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gß1-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gß1-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gß1 subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of CaV2.2 channels resting in the inactivated state. These results support that the Gß1 subunit inhibits the fast inactivation of CaV2.2 channels in SCG neurons. They explain the long-observed sustained Ca2+ current under G-protein modulation.

PIP2 in pancreatic ß-cells regulates voltage-gated calcium channels by a voltage-independent pathway.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic ß-cells. However, whether CaV channels are regulated by PIP2, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic ß-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch-clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca2+ chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca2+-dependent mechanism in pancreatic ß- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

Gß2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline in rat sympathetic neurons.

Several neurotransmitters and hormones acting through G protein-coupled receptors elicit a voltage-dependent regulation of CaV2.2 channels, having profound effects on cell function and the organism. It has been hypothesized that protein-protein interactions define specificity in signal transduction. Yet it is unknown how the molecular interactions in an intracellular signaling cascade determine the specificity of the voltage-dependent regulation induced by a specific neurotransmitter. It has been suspected that specific effector regions on the Gß subunits of the G proteins are responsible for voltage-dependent regulation. The present study examines whether a neurotransmitter's specificity can be revealed by simple ion-current kinetic analysis likely resulting from interactions between Gß subunits and the channel-molecule. Noradrenaline is a neurotransmitter that induces voltage-dependent regulation. By using biochemical and patch-clamp methods in rat sympathetic neurons we examined calcium current modulation induced by each of the five Gß subunits and found that Gß2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline. Furthermore, overexpression of the Gß2 isoform reproduces the effect of noradrenaline in the willing-reluctant model. These results advance our understanding on the mechanisms by which signals conveying from a variety of membrane receptors are able to display precise homeostatic responses.

Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus.

Cholinergic signaling mediated by nicotinic receptors has been associated to a large number of physiological and behavioral processes such as learning, memory, attention, food-intake and mood disorders. Although it is well established that many nicotinic actions are mediated through an increase in serotonin (5-HT) release, the physiological mechanisms by which nicotine produces these effects are still unclear. The dorsal raphe nucleus (DRN) contains the major amount of 5-HT neurons projecting to different parts of the brain. DRN also contains nicotinic acetylcholine receptors (nAChRs) located at somatic and presynaptic elements. Nicotine produces both inhibitory and excitatory effects on different subpopulations of 5-HT DRN neurons. In this review, we describe the presynaptic and postsynaptic mechanisms by which nicotine increases the excitability of DRN neurons as well as the subtypes of nAChRs involved. We also describe the inhibitory effects of nicotine and the role of 5-HT1A receptors in this effect. These nicotinic actions modulate the activity of different neuronal subpopulations in the DRN, changing the 5-HT tone in the brain areas where these groups of neurons project. Some of the physiological implications of nicotine-induced 5-HT release are discussed.

Flattening plasma corticosterone levels increases the prevalence of serotonergic dorsal raphe neurons inhibitory responses to nicotine in adrenalectomised rats.

Major depression is characterized by a diminished activity of the brain serotonergic system as well as by the flattening of plasma cortisol levels. Nicotine improves mood in patients with major depression and in experimentally depressed animals by increasing brain serotonin (5-HT), noradrenaline and dopamine levels. The present study was directed to determine if flattening plasma glucocorticoid levels changes nicotine's stimulatory effects upon 5-HT DRN neurons. The experiments were performed in brain slices obtained from rats previously (14 days) adrenalectomised and implanted subcutaneously with one pellet containing 75mg of corticosterone (Adx+CSR rats). Whole cell voltage and current clamp techniques were used to study the activity of immunocitochemically identified 5-HT DRN neurons. Administration of nicotine (1µM) in sham-operated animals produced stimulatory effects in all 5-HT DRN neurons studied. In Adx+CSR rats however, nicotine inhibited 75% of 5-HT DRN neurons and increased the potassium-dependent inward rectifying current. The inhibitory effect of nicotine upon 5-HT DRN neurons was dependent on serotonin release inside the DRN, since it was converted into a stimulatory response by the selective antagonist of 5-HT1A receptors N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY100635, 25nM). Adx+CSR rats also presented an increased function of 5-HT1A autoreceptors, since, in these rats, serotonin (1-10µM) produced a higher increase in the potassium dependent inward rectifying current in comparison with sham-operated animals. Serotonin release inside DRN was mediated by α4ß2 nicotinic acetylcholine receptors since the selective antagonist of these receptors dihydro-ß-erytroidine hydrobromide (DHßE, 100nM) blocked the inhibitory effects of nicotine 5-HT DRN neurons. These data indicate that, in the experimental model of adrenalectomised rats implanted with corticosterone pellets, nicotine increases the function of 5-HT1A receptors of 5-HT DRN neurons.

Presynaptic α4ß2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4ß2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4ß2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors.

Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed.

Calcium-activated afterhyperpolarizations regulate synchronization and timing of epileptiform bursts in hippocampal CA3 pyramidal neurons.

Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca(2+)-activated K(+)-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg(2+)-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO(4) reduced burst frequency. Block of GABA(A-B) inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity.

1-Ethyl-2-benzimidazolinone (EBIO) suppresses epileptiform activity in in vitro hippocampus.

Ca2+-activated K+ currents with medium (mI(AHP)) and slow (sI(AHP)) kinetics, that mediate the post-spike medium and slow after-hyperpolarization (AHP), respectively, play critical roles in regulating neuronal excitability and the spread of epileptiform activity and could provide new therapeutic targets for the management of epileptic patients. We tested if the enhancement of the mI(AHP) by 1-ethyl-2-benzimidazolinone (EBIO) could suppress epileptiform activity in two in vitro models of epileptogenesis induced in CA3 hippocampal pyramidal neurons by superfusion with 4-AP- and kainate-Mg2+-free solutions. Both interictal- and ictal-like epileptiform activities were reversibly suppressed by EBIO concentrations between 200 microM and 1 mM. EBIO predominantly acted by a strong reduction of excitability via an increase (approximately 450%) of the mI(AHP), without changing the sI(AHP). Glutamatergic excitatory synaptic transmission was also diminished (approximately 50%) by 1 mM EBIO. In contrast, EBIO concentrations <400 microM had no effect on synaptic excitation, consistent with a lesser sensitivity to the drug than the mI(AHP). Apamine (100 nM), a toxin that specifically inhibits the mI(AHP), rapidly and reversibly antagonized the blocking effects of EBIO on epileptiform activity. Our results suggest that manipulations that enhance the mI(AHP) may prove adequate in the treatment of epilepsies; they also suggest that an abnormal down regulation of the mI(AHP) may be a key factor in the genesis of hyperexcitable states.

Expression and functional characterization of GABA transporters in crayfish neurosecretory cells.

The effect of GABA on membrane potential and ionic currents of X-organ neurons isolated from the crayfish eyestalk was investigated. Under voltage-clamp conditions, GABA elicited an inward Na+ current followed by a sustained outward chloride current. Sodium current was partially blocked in a dose-dependent manner by antagonists of GABA plasma membrane transporters such as beta-alanine, nipecotic acid, 1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711), and SKF89976-A at concentrations between 1 and 100 microm. This current was totally blocked by the combined application of NO 711 (5 microm) and beta-alanine (50 microm). We obtained an EC(50) of 5 microm and a Hill coefficient of 0.97 for the GABA transport mediated response. These results together with studies of immunolocalization using antibodies against neuronal vertebrate GABA transporters (GATs) indicate the presence of GAT-1- and GAT-3-like proteins in X-organ neurons. To isolate the sustained outward Cl- current, extracellular free sodium solution was used to minimize the contribution of GAT activity. We concluded that this current was caused by the activation of GABA(A)-like receptors with an EC50 of 10 microm and a Hill number of 1.7. To assign a functional role to the GATs in the X-organ sinus gland system, we determine the GABA concentration (0.46-0.15 microm) in hemolymph samples using HPLC. In summary, our results suggest that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the excitability of the X-organ neurons, maintaining an excitatory tone that is dependent on the circulating GABA level.