Microinjection Of AMPA Research Articles

Decreased Glutamatergic Synaptic Strength in the Periaqueductal Gray Contributes to Maintenance of Visceral Pain in Male Rats with Experimental Pancreatitis

Visceral pain originating from chronic inflammation of the pancreas is often intractable and difficult to manage clinically. However, the pathogenesis of the central nervous system underlying visceral pain is still poorly understood. The aim of the present study was to investigate the role of the midbrain ventrolateral periaqueductal gray (vlPAG) in a rat model of chronic visceral pain induced by pancreatitis. In the present study, we used a well-established rat model of chronic pancreatitis induced by tail vein injection of dibutyltin dichloride (DBTC). To assess the DBTC-induced visceral pain, we examined the abdominal withdrawal by von Frey filament test. We further studied the synaptic transmission in the vlPAG by whole-cell patch-clamp electrophysiological recordings. Rats receiving DBTC injection exhibited a significantly increased withdrawal frequency to mechanical stimulation of the abdomen compared to rats injected with vehicle. Interestingly, compared to rats injected with vehicle, we found that neurons dissected from DBTC-treated rats exhibited a significantly decreased synaptic strength, which was revealed by a diminishedα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartic acid (AMPA/NMDA) ratio in the vlPAG. Moreover, our results further demonstrated that neurons obtained from DBTC-treated rats displayed a higher paired-pulse ratio, as well as less frequent and smaller amplitudes of miniature excitatory postsynaptic currents in the vlPAG compared to rats injected with vehicle. Furthermore, intra-vlPAG microinjection of AMPA alleviated DBTC-induced abdominal hypersensitivity. Taken together, our findings suggest that diminished glutamatergic synaptic strength via both presynaptic and postsynaptic mechanisms in the midbrain vlPAG is associated with DBTC-induced abdominal hypersensitivity. In addition, activation of AMPA receptors in the vlPAG alleviates DBTC-induced abdominal hypersensitivity.

Longitudinal observations using simultaneous fMRI, multiple channel electrophysiology recording, and chemical microiontophoresis in the rat brain

BackgroundfMRI blood oxygenation level-dependent (BOLD) signal has been widely used as a surrogate for neural activity. However, interpreting differences in BOLD fMRI based on underlying neuronal activity remains a challenge. Concurrent rsMRI data collection and electrophysiological recording in combination with microiontophoretically injected modulatory chemicals allows for improved understanding of the relationship between resting state BOLD and neuronal activity. New MethodsSimultaneous fMRI, multi-channel intracortical electrophysiology and focal pharmacological manipulation data to be acquired longitudinally in rats for up to 2 months. Our artifact replacing technique is optimized for combined LFP and rsMRI data collection. ResultsIntracortical implantation of a multichannel microelectrode array resulted in minimal distortion and signal loss in fMRI images inside a 9.4T MRI scanner. rsMRI-induced electrophysiology artifacts were replaced using an in-house developed algorithm. Microinjection of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) enhanced dopaminergic neuronal activity in the ventral tegmental area (VTA) and altered LFP signal and fMRI functional connectivity in the striatum. Comparisons with Existing Method(s)Nanomanufacturing advances permit the production of MRI-compatible microelectrode arrays (with 16 or more channels), extending research beyond conventional methods limited to fewer channels. Our method permits longitudinal data collection of LFP and rsMRI and our algorithm effectively detects and replaces fMRI-induced electrophysiological noise, permitting rsMRI data collection concomitant with LFP recordings. ConclusionsOur model consists of longitudinal concurrent fMRI and multichannel intracortical electrophysiological recording during microinjection of pharmacological agents to modulate neural activity in the rat brain. We used commercial micro-electrodes and recording system and can be readily generalized to other labs.

Effects of neonatal inflammation on descending modulation from the rostroventromedial medulla

Cutaneous tissue inflammation during the first postnatal week is known to alter long-term development of spinal cord nociceptive circuitry and to alter behavioral responses to noxious stimuli in adult animals. The impact of neonatal inflammation on descending projections arising from supraspinal sites that modulate spinal nociceptive processing is unknown. In the present study, we investigated if altered behavioral responses to pain in adult animals after neonatal inflammation are associated with changes in descending modulation of nocifensive responses elicited from the rostroventromedial medulla (RVM) in lightly anesthetized rats. Compared to handled control animals, hindpaw injection of 0.25% carrageenan (CG) at postnatal day 3 produced adult basal hypoalgesia and increased hyperalgesia 24 h after reinflammation with Complete Freund's Adjuvant (CFA) in awake animals. These effects were specific to the neonatally treated hindpaw, partially replicating previous findings, but were absent in lightly anesthetized animals. However, focal electrical stimulation of the RVM in lightly anesthetized CG treated animals produced significantly greater descending inhibition of nocifensive responses to noxious thermal stimuli applied to the hindpaws and the tail. These effects were partially replicated by intra-RVM microinjection of AMPA. No differences in the efficacy of RVM stimulation between CG and control animals were observed 24 h after reinflammation with CFA. These findings indicate that neonatal tissue injury and inflammation produces lasting alterations in descending modulatory systems that modify nociceptive processing. Taken together with previous studies, these results indicate that changes in pain sensitivity following neonatal tissue injury involve long-term alterations in spinal and supraspinal circuitry.

Activation of AMPA Receptors in the Suprachiasmatic Nucleus Phase-Shifts the Mouse Circadian Clock In Vivo and In Vitro

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN.

AMPA receptor subunit GluR1 downstream of D-1 dopamine receptor stimulation in nucleus accumbens shell mediates increased drug reward magnitude in food-restricted rats

Previous findings suggest that neuroadaptations downstream of D-1 dopamine (DA) receptor stimulation in nucleus accumbens (NAc) are involved in the enhancement of drug reward by chronic food restriction (FR). Given the high co-expression of D-1 and GluR1 AMPA receptors in NAc, and the regulation of GluR1 channel conductance and trafficking by D-1-linked intracellular signaling cascades, the present study examined effects of the D-1 agonist, SKF-82958, on NAc GluR1 phosphorylation, intracranial electrical self-stimulation reward (ICSS), and reversibility of reward effects by a polyamine GluR1 antagonist, 1-NA-spermine, in ad libitum fed (AL) and FR rats. Systemically administered SKF-82958, or brief ingestion of a 10% sucrose solution, increased NAc GluR1 phosphorylation on Ser845, but not Ser831, with a greater effect in FR than AL rats. Microinjection of SKF-82958 in NAc shell produced a reward-potentiating effect that was greater in FR than AL rats, and was reversed by co-injection of 1-NA-spermine. GluR1 abundance in whole cell and synaptosomal fractions of NAc did not differ between feeding groups, and microinjection of AMPA, while affecting ICSS, did not exert greater effects in FR than AL rats. These results suggest a role of NAc GluR1 in the reward-potentiating effect of D-1 DA receptor stimulation and its enhancement by FR. Moreover, GluR1 involvement appears to occur downstream of D-1 DA receptor stimulation rather than reflecting a basal increase in GluR1 expression or function. Based on evidence that phosphorylation of GluR1 on Ser845 primes synaptic strengthening, the present results may reflect a mechanism via which FR normally facilitates reward-related learning to re-align instrumental behavior with environmental contingencies under the pressure of negative energy balance.

Central nitric oxide modulates hindquarter vasodilation elicited by AMPA receptor stimulation in the NTS of conscious rats

Microinjection of S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the nucleus of the solitary tract (NTS) of conscious rats causes hypertension, bradycardia, and vasoconstriction in the renal, mesenteric, and hindquarter vascular beds. In the hindquarter, the initial vasoconstriction is followed by vasodilation with AMPA doses >5 pmol/100 nl. To test the hypothesis that this vasodilation is caused by activation of a nitroxidergic pathway in the NTS, we examined the effect of pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 nmol/100 nl, microinjected into the NTS) on changes in mean arterial pressure, heart rate, and regional vascular conductance (VC) induced by microinjection of AMPA (10 pmol/100 nl in the NTS) in conscious rats. AMPA increased hindquarter VC by 18 +/- 4%, but after pretreatment with L-NAME, AMPA reduced hindquarter VC by 16 +/- 7% and 17 +/- 9% (5 and 15 min after pretreatment, P < 0.05 compared with before pretreatment). Pretreatment with L-NAME reduced AMPA-induced bradycardia from 122 +/- 40 to 92 +/- 32 beats/min but did not alter the hypertension induced by AMPA (35 +/- 5 mmHg before pretreatment, 43 +/- 6 mmHg after pretreatment). Control injections with D-NAME did not affect resting values or the response to AMPA. The present study shows that stimulation of AMPA receptors in the NTS activates both vasodilatatory and vasoconstrictor mechanisms and that the vasodilatatory mechanism depends on production of nitric oxide in the NTS.

Effect of excitatory amino acid receptor agonists on penile erection after administration into paraventricular nucleus of hypothalamus in the rat

ObjectivesTo investigate whether the excitatory amino acid receptor agonists can activate the paraventricular nucleus of the hypothalamus (PVN) and induce penile erections in the rat. MethodsMale adult Sprague-Dawley rats were used. A 26-gauge needle was inserted into the corpus cavernosum to monitor the intracavernous pressure (ICP) simultaneously with the systemic arterial pressure and heart rate. The study was divided into seven parts: stereotaxic delivery of N-methyl-d-aspartic acid (NMDA) (50 ng/500 nL) into the PVN; administration of the NMDA noncompetitive antagonist MK-801 (100 ng/250 nL) and NMDA (50 ng/250 nL) into the PVN; administration of the NMDA competitive antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 100 ng/250 nL) and NMDA (50 ng/250 nL) into the PVN; microinjection of (±)-α-(amino)-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 100 ng/500 nL) into the PVN; microinjection of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 100 ng/500 nL) into the PVN; saline 500 nL into the PVN; and intracavernous administration of NMDA (50 ng/100 μL). ResultsOn administration of NMDA into the PVN, a significant increase occurred in the ICP from a resting 8.3 ± 1.8 mm Hg to a peak at 59.0 ± 8.4 mm Hg. No change occurred in the resting ICP after administration of either the mixture of MK-801 and NMDA or CPP and NMDA into the PVN. Microinjection of AMPA, ACPD, or saline into the PVN and intracavernous administration of NMDA were all ineffective to induce an increase in ICP. ConclusionsThe results of this study suggest that ionotropic excitatory amino acid (NMDA) may have an effect on inducing penile erection through activation of the PVN in the rat.

Cardiovascular responses to microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats

Previous studies have shown that microinjection of NMDA or non-NMDA receptor agonists into the nucleus tractus solitarius of anesthetized rats produces bradycardia and hypotension. In the present study, we evaluated the autonomic components of the cardiovascular responses to the microinjection of α-amine-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) into the nucleus tractus solitarius of awake rats. AMPA (0.05 nmol 50 nl −1) was microinjected into the nucleus tractus solitarius before and at 2, 10, 30 and 180 min after intravenous injection of methylatropine (2 mg kg −1). The control response to the microinjection of AMPA into the lateral aspect of the commissural nucleus tractus solitarius consisted of bradycardia and hypotension, (−190±20 bpm and −49±7 mm Hg), which were effectively blocked at 2 min (−5±2 bpm and −4±5 mm Hg), 10 min (−3±2 bpm and −6±3 mm Hg) and 30 min (−9±4 bpm and −5±5 mm Hg) after methylatropine. Although the bradycardic and hypotensive responses remained significantly reduced 180 min after injection of methylatropine, the responses tended to normalize. The data show that microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats produced intense bradycardia essentially mediated by parasympathetic excitation, which was the cause of the concomitant hypotensive response.

Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys.

The role of the mesencephalic locomotor region (MLR) in initiating and controlling the power of swimming was studied in semi-intact preparations of larval and adult sea lampreys. The brain and the rostral portion of the spinal cord were exposed in vitro, while the intact caudal two-thirds of the body swam freely in the Ringer's-containing chamber. Electrical microstimulation (2-10 Hz; 0. 1-5.0 microA) within a small periventricular region in the caudal mesencephalon elicited well-coordinated and controlled swimming that began within a few seconds after the onset of stimulation and lasted throughout the stimulation period. Swimming stopped several seconds after the end of stimulation. The power of swimming, expressed by the strength of the muscle contractions and the frequency and the amplitude of the lateral displacement of the body or tail, increased as the intensity or frequency of the stimulating current were increased. Micro-injection of AMPA, an excitatory amino acid agonist, into the MLR also elicited active swimming. Electrical stimulation of the MLR elicited large EPSPs in reticulospinal neurons (RS) of the middle rhombencephalic reticular nucleus (MRRN), which also displayed rhythmic activity during swimming. The retrograde tracer cobalt-lysine was injected into the MRRN and neurons (dia. 10-20 microm) were labelled in the MLR, indicating that this region projects to the rhombencephalic reticular formation. Taken together, the present results indicate that, as higher vertebrates, lampreys possess a specific mesencephalic region that controls locomotion, and the effects onto the spinal cord are relayed by brainstem RS neurons.

Glutamate Transmission in the Nucleus Accumbens Mediates Relapse in Cocaine Addiction

Elevated dopamine transmission in the nucleus accumbens is thought to be a primary mediator of addiction to cocaine. However, repeated exposure to cocaine is associated with the recruitment of glutamate transmission. This poses the possibility that the behaviors characterizing cocaine addiction, such as craving-induced relapse, may not be preferentially mediated by dopamine transmission. An animal model of relapse was used to demonstrate that glutamate, and not dopamine transmission in the nucleus accumbens, is a primary mediator of cocaine-induced reinstatement of drug-seeking behavior. Reinstatement was produced by a systemic injection of cocaine or by the microinjection of the glutamate receptor agonist AMPA or dopamine into the nucleus accumbens. It was found that microinjection of an AMPA receptor antagonist into the nucleus accumbens blocked reinstatement by all compounds, whereas a dopamine receptor antagonist was effective only in blocking reinstatement by intra-accumbens dopamine administration. These data suggest an important role for nucleus accumbens glutamate and not dopamine transmission in cocaine-induced relapse to drug-seeking behavior.

Excitatory amino acids and neurodegeneration: a hypothetical role of calcium precipitation

Activation of excitatory amino acid (EAA) receptors can induce neurodegeneration by two major mechanisms of excitotoxicity, one related to the influx of Na+, Cl− and water, and the other to the increase in intracellular calcium concentration ([Ca2+]i). Thus, acute microinjection of EAAs in several areas of the central nervous system (CNS) has been used to produce neurodegenerative models. We studied the excitotoxic pattern associated with acute microinjection of AMPA in rat hippocampus, medial septum-diagonal band of Broca (MS-DBB), prefrontal cortex and retina. In all cases progressive neuronal loss, glial reaction and development of intra- and extracellular calcium concretions were observed. However, a CNS-area differential vulnerability was revealed, as shown by the specific atrophy of MS-DBB and its limited calcification. Whether calcium deposits are a defensive mechanism against the massive increment of free cytoplasmatic calcium is discussed on the basis of ultrastructural data and previous results.

The gain of the baroreflex bradycardia is reduced by microinjection of NMDA receptor antagonists into the nucleus tractus solitarii of awake rats

The baroreflex activation with phenylephrine infusion produces a bradycardic response. In the present study, the role of NMDA receptors in the nucleus tractus solitarii (NTS) in the processing of the parasympathetic component of the baroreflex was evaluated using acid phosphonivaleric (AP-5), a selective NMDA receptor antagonist. Baroreflex activation was performed before and after bilateral microinjection of AP-5 into the intermediate commissural NTS (0.5 mm lateral to the midline). Microinjection of the vehicle (saline, 0.9%) or a dose of 2 nmol/50 nl of AP-5 into the NTS produced no effect on the gain of the baroreflex while a dose of 10 nmol/50 nl of AP-5 produced a significant reduction in the gain of the baroreflex 2 min after microinjection [−1.43±0.22 vs. −0.43±0.03 bpm/mmHg, ( n=6)], with a return to control levels 10 min after the microinjections. The dose of 10 nmol/50 nl was selective for NMDA receptors considering that the cardiovascular responses to microinjection of AMPA (0.05 pmol/50 nl), a non-NMDA receptor agonist, were not affected by this dose of AP-5 and the responses to microinjection of NMDA (2 nmol/50 nl) were blocked. The data show that the bradycardic response to baroreflex activation was blocked by AP-5 microinjected into the NTS, indicating that the neurotransmission of the parasympathetic component of the baroreflex is mediated by NMDA receptors in the NTS.

Repeated cocaine augments excitatory amino acid transmission in the nucleus accumbens only in rats having developed behavioral sensitization

Rats were pretreated with daily cocaine or saline injections for 1 week. The rats treated with daily cocaine were separated into two groups: a sensitized group of animals demonstrating > 20% increase in motor activity on the last injection compared with the first injection of daily cocaine, and a nonsensitized group showing < 20% elevation. At 2-3 weeks after the last daily injection, four experiments were performed to assess changes in excitatory amino acid (EAA) transmission in the nucleus accumbens produced by repeated cocaine administration. (1) Rats were challenged with a microinjection of AMPA into the shell or core of the nucleus accumbens. The sensitized rats demonstrated greater motor activity than did the saline-pretreated or nonsensitized animals after AMPA injection into either subnucleus. (2) It was shown that the behavioral distinction between sensitized, nonsensitized, and control rats in behavioral responsiveness to AMPA was not mediated by differences in AMPA-induced dopamine release. (3) The extracellular content of glutamate was measured after a cocaine challenge given at 21 d of withdrawal. Cocaine elevated the levels of glutamate in the core of sensitized rats, but not of nonsensitized or control rats. (4) Microinjection of the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione into the core abolished the augmented motor response to a cocaine challenge in sensitized rats, but was without effect on cocaine-induced motor activity in nonsensitized animals. These results indicate that repeated cocaine administration increases EAA transmission in the nucleus accumbens only in rats that develop behavioral sensitization to cocaine.