Fredy Cifuentes

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca2+ signaling

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimers disease and amyotrophic lateral sclerosis.

Role of presynaptic and postsynaptic IP3-dependent intracellular calcium release in long-term potentiation in sympathetic ganglion of the rat

In the rat superior cervical ganglion, a form of long term potentiation (LTP) can be elicited by a brief high frequency stimuli applied to the preganglionic nerve. Cumulative evidence shows that a transient increase in cytoplasmic Ca2+ concentration is essential for the generation of the ganglionic LTP. Calcium influx and calcium release from intracellular calcium stores contribute to LTP. However, the differential role of presynaptic and postsynaptic calcium signaling has not been established. Herein, by using heparin, a membrane‐impermeant inositol trisphosphate receptor (IP3R) blocker, we explored the contribution of presynaptic and postsynaptic IP3‐sensitive calcium stores to the ganglionic LTP. The LTP was produced by a conditioning train of 40 Hz for 3 s. We analyzed the effects of heparin on the posttetanic potentiation: PTP magnitude and PTP time constant, and on two parameters that describe the LTP: LTP decay time (elapsed time required by the potentiated response to fall to 20% above the basal value) and LTP extent (the integral of the potentiated response). Heparin (100 and 200 μg/ml) was loaded in the preganglionic, the postganglionic, or in both nerves. We found that in all tested conditions heparin significantly decreased LTP but practically did not affect PTP. The preganglionic and postganglionic inhibitory effects of heparin were not additive. De‐N‐sulfated heparin, an ineffective IP3R blocker, had no effect on LTP, but abolished the heparin blocking effect. Data suggest that presynaptic and postsynaptic IP3‐dependent intracellular calcium release equally contribute to ganglionic LTP, supporting our proposal of a trans‐synaptic mechanism for LTP. Synapse, 2010.

Segregation of met–enkephalin from vesicular acetylcholine transporter and choline acetyltransferase in sympathetic preganglionic varicosities mostly lacking synaptophysin and synaptotagmin

Sympathetic preganglionic neurons (SPN) coexpress the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase and different peptides in their cell bodies, but can express them independently in separate varicosities, indicating that SPN segregate transmitters to different synapses. Consequently, there are populations of preganglionic varicosities (peptidergic and noncholinergic) that store peptides but not ACh. We studied in the cell bodies and axon processes of the rat SPN the expression and the proportional coexpression of the vesicular ACh transporter-like immunoreactivity (VAChT), a specific marker of cholinergic synaptic vesicles or ChAT-like immunoreactivity (ChAT), and the peptide methionine enkephalin-like immunoreactivity (mENK), and confirmed the presence of a population of SPN peptidergic, noncholinergic varicosities. We characterized these varicosities by exploring the occurrence of synaptophysin-like immunoreactivity (Syn), a marker of small clear vesicles, and synaptotagmin-like immunoreactivity (Syt), a preferential marker of large dense core vesicles. We found that (i) VAChT and mENK, like ChAT-mENK, were coexpressed in only 59% of the mENK-containing varicosities, although they colocalized in the SPN cell bodies; and (ii) almost 60% of the population of mENK-containing varicosities did not express Syn or Syt, and over 80% of the mENK-containing varicosities negative for VAChT also lacked Syn. These data prove that SPN segregate mENK from VAChT and ChAT, and show that most of the subset of mENKergic varicosities negative for VAChT also does not express Syn, suggesting the presence of a different vesicular pattern in these sympathetic preganglionic varicosities.

Long-term potentiation in mammalian autonomic ganglia: an inclusive proposal of a calcium-dependent, trans-synaptic process.

Ganglionic synapses have the capability to express long-term potentiation (gLTP) after application of a brief high-frequency stimulus. It has been suggested a possible role of gLTP in some cardiovascular diseases. Although a number of characteristics of gLTP have been described, the precise locations and mechanisms underlying gLTP are not completely known. Current findings support two major conflicting presynaptic and postsynaptic hypotheses. The presynaptic hypothesis posits a presynaptic increase in acetylcholine (ACh) release, whereas the postsynaptic hypothesis proposes a long-lasting enhancement of the nicotinic response on the postsynaptic membrane. An alternative trans-synaptic hypothesis proposes the presynaptic release of a cotransmitter from large dense core vesicles, which postsynaptically enhances synaptic efficacy and accounts for gLTP. Here, we review the studies of LTP, with emphasis on gLTP in mammals, and we examine the findings that support the presynaptic, the postsynaptic and the trans-synaptic hypotheses. We then review our data on the contribution of calcium to gLTP as an approach to elucidate the mechanisms of gLTP. Data on the contribution of calcium to gLTP and on prolonged high-frequency stimulus-dependent fading of LTP have led us to support the trans-synaptic process as responsible for gLTP. Finally, we present a formal working model for the mechanisms of gLTP.

Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation

Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons.

Neurotrophin-dependent plasticity of neurotransmitter segregation in the rat superior cervical ganglion in vivo.

Neurons are able to segregate transmitters to different axon endings. Segregation is a plastic neuronal feature; it can be modulated by synaptic environment. We have demonstrated that neurotrophin and other cellular factors regulate segregation in sympathetic neurons in culture. Herein we tested the hypothesis that sympathetic neurons in vivo are also capable to exhibit neurotrophin‐dependent plasticity of segregation. To explore the effect of neurotrophin on segregation, we reduced ganglionic NGF content by the transection of postganglionic nerves (axotomy) of the superior cervical ganglia. By immunohistochemistry, Western blot, and PCR analyses, we explored the effect of axotomy on the NGF and BDNF content of ganglionic neurons, and on the segregation extent of vesicular acetylcholine transporter (VAChT) and methionine enkephalin (mENK) in pre‐ganglionic varicosities. We analyzed NGF‐dependence of the changes found by applying exogenous NGF. Axotomy reduced ganglionic NGF and BDNF content, increased NGF transcripts, and increased VAChT‐mENK segregation. Axotomy also increased the number of VAChT immunopositive varicosities, and caused the appearance of a population of VAChT‐, mENK‐ or SV2‐containing varicosities lacking Synaptophysin (Syn). Administration of NGF prevented changes in NGF content, kept NGF transcripts increased, and counteracted changes in segregation and in the number of cholinergic varicosities. The exogenous NGF did not preclude change in BDNF content or in the occurrence of the VAChT‐ or mENK‐containing varicosities lacking Syn. Data demonstrate that segregation of transmitters in vivo is plastic and it is modulated by environmental signals like NGF. We propose a possible functional correlate of segregation plasticity in the sympathetic ganglia.

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.