Excitatory synaptic transmission in the lateral and central amygdala

The amygdala plays a major role in the acquisition and expression of fear conditioning. NMDA receptor-dependent synaptic plasticity within the basolateral amygdala has been proposed to underlie the acquisition and possible storage of fear memories. Here the properties of fast glutamatergic transmission in the lateral and central nuclei of the amygdala are presented. In the lateral amygdala, two types of neurons, interneurons and projection neurons, could be distinguished by their different firing properties. Glutamatergic inputs to interneurons activated AMPA receptors with inwardly rectifying current-voltage relations (I-Vs), whereas inputs to projection neurons activated receptors that had linear I-Vs, indicating that receptors on interneurons lack GluR2 subunits. Inputs to projection neurons formed dual component synapses with both AMPA and NMDA components, whereas at inputs to interneurons, the contribution of NMDA receptors was very small. Neurons in the central amygdala received dual component glutamatergic inputs that activated AMPA receptors with linear I-Vs. NMDA receptor-mediated EPSCs had slow decay time constants in the central nucleus. Application of NR2B selective blockers ifenprodil or CP-101,606 blocked NMDA EPSCs by 70% in the central nucleus, but only by 30% in the lateral nucleus. These data show that the distribution of glutamatergic receptors on amygdalar neurons is not uniform. In the lateral amygdala, interneurons and pyramidal neurons express AMPA receptors with different subunit compositions. Synapses in the central nucleus activate NMDA receptors that contain NR1 and NR2B subunits, whereas synapses in the lateral nucleus contain receptors with both NR2A and NR2B subunits.

Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B Synapses in the adult central amygdala

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes.

Altered inhibitory synaptic transmission in superficial dorsal horn neurones in spastic and oscillator mice

The spastic (spa) and oscillator (ot) mouse have naturally occurring mutations in the inhibitory glycine receptor (GlyR) and exhibit severe motor disturbances when exposed to unexpected sensory stimuli. We examined the effects of the spa and ot mutations on GlyR- and GABA(A)R-mediated synaptic transmission in the superficial dorsal horn (SFDH), a spinal cord region where inhibition is important for nociceptive processing. Spontaneous mIPSCs were recorded from visually identified neurones in parasagittal spinal cord slices. Neurones received exclusively GABA(A)R-mediated mIPSCs, exclusively GlyR-mediated mIPSCs or both types of mIPSCs. In control mice (wild-type and spa/+) over 40 % of neurones received both types of mIPSCs, over 30 % received solely GABA(A)R-mediated mIPSCs and the remainder received solely GlyR-mediated mIPSCs. In spa/spa animals, 97 % of the neurones received exclusively GABA(A)ergic or both types of mIPSCs. In ot/ot animals, over 80 % of the neurones received exclusively GABA(A)R-mediated mIPSCs. GlyR-mediated mIPSC amplitude and charge were reduced in spa/spa and ot/ot animals. GABA,Rmediated mIPSC amplitude and charge were elevated in spa/spa but unaltered in ot/ot animals. GlyR- and GABA(A)R-mediated mIPSC decay times were similar for all genotypes, consistent with the mutations altering receptor numbers but not kinetics. These findings suggest the spastic and oscillator mutations, traditionally considered motor disturbances, also disrupt inhibition in a sensory region associated with nociceptive transmission. Furthermore, the spastic mutation results in a compensatory increase in GABA(A)ergic transmission in SFDH neurones, a form of inhibitory synaptic plasticity absent in the oscillator mouse.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.

Long-lasting synaptic modification in the rat hippocampus resulting from NMDA receptor blockade during development

Recent reports have suggested that proper maturation of synapses in the hippocampus requires activation of NMDA receptors. We previously demonstrated that neonatal ethanol exposure results in a lasting reduction in synaptic strength in the hippocampus. To determine if this reduction was due to ethanol's effects on NMDA receptors, we investigated long-term changes in synaptic properties resulting from administration of NMDA receptor antagonists to neonatal animals. Rats were injected daily from PND 4-9 with either the noncompetitive NMDA receptor antagonist MK-801, the competitive NMDA receptor antagonist CPP, or the AMPA receptor antagonist NBQX. Control rats were either injected daily with physiological saline during the same period or left to develop normally. Hippocampal slices were prepared from nembutal-anesthetized animals between PND 35 and PND 40. The maximum pEPSP and PS values were not significantly different between controls and NMDA antagonist-treated animals. However, slices from animals injected with NMDA receptor antagonists required higher stimulus currents to attain comparable pEPSPs. The ratio of the slope of the pEPSP to the amplitude of the presynaptic volley was also reduced, as were pEPSP responses to specific stimulus currents. None of these effects were observed in slices prepared from animals treated with the AMPA receptor antagonist NBQX. Glutamate receptor antagonism did not produce lasting changes in long-term potentiation or paired-pulse facilitation. These results indicate activation of NMDA receptors during development is necessary for proper development of synapses. (C) 2001 Wiley-Liss, Inc.

Diverse synaptic mechanisms generate direction selectivity in the rabbit retina

The synaptic conductance of the On-Off direction-selective ganglion cells was measured during visual stimulation to determine whether the direction selectivity is a property of the circuitry presynaptic to the ganglion cells or is generated by postsynaptic interaction of excitatory and inhibitory inputs. Three synaptic asymmetries were identified that contribute to the generation of direction-selective responses: (1) a presynaptic mechanism producing stronger excitation in the preferred direction, (2) a presynaptic mechanism producing stronger inhibition in the opposite direction, and (3) postsynaptic interaction of excitation with spatially offset inhibition. Although the on- and off-responses showed the same directional tuning, the off-response was generated by all three mechanisms, whereas the on- response was generated primarily by the two presynaptic mechanisms. The results indicate that, within a single neuron, different strategies are used within distinct dendritic arbors to accomplish the same neural computation.

Behavioral and synaptic circuit analysis in models of neuropsychiatric disorders: Dissecting the in vivo role of the postsynaptic density proteins nArgBP2 and Shank3 using genetic engineered mice

Dissertation presented to obtain the Ph.D degree in Biology

Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission

Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.

Cerebrospinal Fluid Synaptic Proteins as Useful Biomarkers in Tyrosine Hydroxylase Deficiency

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type “B”: early onset severe encephalopathy; type “A”: later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicularmonoamine transporter (VMAT2)weremeasured in the CSF of 10 subjectswith THdeficiency byWestern blot analysis. In 3 patients, data of pre- and post-treatmentwith L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSFwas significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before LDOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

Trans-Synaptic Signaling by Activity-Dependent Cleavage of Neuroligin-1

Throughout the brain, patterns of activity in postsynaptic neurons influence the properties of synaptic inputs. Such feedback regulation is central to neural network stability that underlies proper information processing and feature representation in the central nervous system. At the cellular level, tight coupling of presynaptic and postsynaptic function is fundamental to neural computation and synaptic plasticity. The cohort of protein complexes at the pre and postsynaptic membrane allows for tight synapse-specific segregation and integration of diverse molecular and electrical signals.(...)

Stress-triggered synaptic malfunction: a gate along the path from depressionto dementia

Tese de Doutoramento em Ciências da Saúde.

Procesos de plasticidad sináptica en gyrus dentado hipocampal durante la administración crónica y el síndrome de abstinencia a cocaína. Synaptic plasticity in hippocampal dentate gyrus during chronic administration and withdrawal from cocaine.

Los procesos neuronales adaptativos que se observan como consecuencia de la administración crónica de drogas de abuso, son similares a los procesos plásticos que subyacen al aprendizaje y la memoria. Por otra parte, el hipocampo forma parte del circuito neuronal responsable de los cambios conductuales observados como consecuencia de la administración crónica de diferentes drogas de abuso. De acuerdo con esto, resultados previos de nuestro laboratorio demostraron que la plasticidad sináptica en el hipocampo y las claves contextuales relacionadas con la administración de la droga, son relevantes para el incremento de la plasticidad hipocampal por la administración crónica de diazepam. Específicamente en el gyrus dentado hipocampal se han descripto fenómenos plásticos relacionados con la exposición crónica a psicofármacos, tales como facilitación en la transmisión sináptica, disminución de la proliferación celular y el aumento del factor de transcripción ?Fos B. Debido a la correlación existente entre los mecanismos de plasticidad neuronal, los aprendizaje asociativos y formación de memorias y aquellos responsables de la adicción, el objetivo general de este trabajo es caracterizar los cambios inducidos por la exposición repetida de cocaína y durante el periodo de abstinencia, en la excitabilidad neuronal de las células del gyrus dentado hipocampal, los canales iónicos afectados y los posibles mecanismos bioquímicos involucrados en dichos cambios, que podrían explicar las alteraciones conductuales observadas después de dicho tratamiento. Con este propósito, se estudiará: 1) la plasticidad sináptica (potenciación a largo plazo, LTP y depotenciación a largo plazo, LTD) en el gyrus dentado, mediante registros electrofisiológios multiunitarios; 2)la excitabilidad de las células granulares del gyrus dentado y la actividad de los canales iónicos, utilizando la técnica de patch clamp; 3) las alteraciones en la neurotransmisión glutamatergica, midiendo los niveles del neurotransmisor in vivo, utilizando la técnica de microdiálisis; el tráfico de receptores glutamatérgicos, utilizando la técnica de western-blott, 4) la participación del óxido nítrico en los cambios adaptativos observados como consecuencia de la sensibilización a cocaína. Además, mediante la utilización de técnicas comportamentales (avoidance inhibitorio), se estudiarán las posibles alteraciones de conductas que se sabe dependen de la integridad funcional del hipocampo.En relación a los resultados del presente proyecto se espera obtener un incremento en la plasticidad sináptica, en la excitabilidad neuronal de las células granulares del gyrus dentado de la formación hipocámpica, en la liberación extracelular de glutamato in vivo, como así también en el tráfico de receptores glutamatérgicos. Además se espera obtener un aumento de las vías de señalización activadas por la acción de glutamato, como la de óxido nítrico/GMPc, como consecuencia de la administración crónica de cocaína. Con este aumento global de la plasticidad sináptica hipocampal, las conductas dependientes de esta estructura debieran estar facilitadas, demostrando así una participación activa del hipocampo en los procesos de sensibilización y posiblemente en la adicción a psicoestimulantes. La caracterización del impacto del desarrollo de sensibilización a cocaína en la excitabilidad neuronal en el hipocampo, sobre los sistemas de neurotransmisión y las vías de señalización involucradas contribuirían a dilucidar los mecanismos que contribuyen al desarrollo de sensibilización a cocaína, los cuales podrían representar potenciales blancos terapéuticos para el tratamiento de la adicción, considerando principalmente aspectos específicos de la actividad eléctrica neuronal y la plasticidad sináptica asociada con las diferentes fases del ciclo de la adicción.

Synaptic plasticity in mature cultured hippocampal-entorhinal cortex slices : activity-dependent regulation of cAMP response-element binding protein

LTP, synaptic plasticity, hippocampus, organotypic cultures, CREB

Early life stress-induced histone acetylations correlate with activation of the synaptic plasticity genes Arc and Egr1 in the mouse brain

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013