Age-Dependent Changes in the Occurrence and Segregation of GABA and Acetylcholine in the Rat Superior Cervical Ganglia.

The occurrence, inhibitory modulation, and trophic effects of GABA have been identified in the peripheral sympathetic nervous system. We have demonstrated that GABA and acetylcholine (ACh) may colocalize in the same axonal varicosities or be segregated into separate ones in the rat superior cervical ganglia (SCG). Neurotransmitter segregation varies with age and the presence of neurotrophic factors. Here, we explored age-dependent changes in the occurrence and segregation of GABA and ACh in rats ranging from 2 weeks old (wo) to 12 months old or older. Using immunohistochemistry, we characterized the expression of L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine transporter (VAChT) in the rat SCG at 2, 4, 8, 12 wo and 12 months old or older. Our findings revealed that GAD67 was greater at 2 wo compared with the other ages, whereas VAChT levels were greater at 4 wo than at 12 wo and 12 months old or older. The segregation of these neurotransmitters was more pronounced at 2 and 4 wo. We observed a caudo-rostral gradient of segregation degree at 8 and 12 wo. Data point out that the occurrence and segregation of GABA and ACh exhibit developmental adaptative changes throughout the lifetime of rats. We hypothesize that during the early postnatal period, the increase in GABA and GABA-ACh segregation promotes the release of GABA alone which might play a role in trophic actions.

Long-term potentiation and its neurotrophin-dependent modulation in the superior cervical ganglion of the rat are influenced by KCNQ channel function.

Ganglionic long-term potentiation (gLTP) in the rat superior cervical ganglion (SCG) is differentially modulated by neurotrophic factors (Nts): brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). KCNQ/M channels, key regulators of neuronal excitability, and firing pattern are modulated by Nts; therefore, they might contribute to gLTP expression and to the Nts-dependent modulation of gLTP. In the SCG of rats, we characterized the presence of the KCNQ2 isoform and the effects of opposite KCNQ/M channel modulators on gLTP in control condition and under Nts modulation. Immunohistochemical and reverse transcriptase polymerase chain reaction analyses showed the expression of the KCNQ2 isoform. We found that 1 µmol/L XE991, a channel inhibitor, significantly reduced gLTP (∼50%), whereas 5 µmol/L flupirtine, a channel activator, significantly increased gLTP (1.3- to 1.7-fold). Both modulators counterbalanced the effects of the Nts on gLTP. Data suggest that KCNQ/M channels are likely involved in gLTP expression and in the modulation exerted by BDNF and NGF.

Functional Implications of Neurotransmitter Segregation.

Here, we present and discuss the characteristics and properties of neurotransmitter segregation, a subtype of neurotransmitter cotransmission. We review early evidence of segregation and discuss its properties, such as plasticity, while placing special emphasis on its probable functional implications, either in the central nervous system (CNS) or the autonomic nervous system. Neurotransmitter segregation is a process by which neurons separately route transmitters to independent and distant or to neighboring neuronal processes; it is a plastic phenomenon that changes according to synaptic transmission requirements and is regulated by target-derived signals. Distant neurotransmitter segregation in the CNS has been shown to be related to an autocrine/paracrine function of some neurotransmitters. In retinal amacrine cells, segregation of acetylcholine (ACh) and GABA, and glycine and glutamate to neighboring terminals has been related to the regulation of the firing rate of direction-selective ganglion cells. In the rat superior cervical ganglion, segregation of ACh and GABA to neighboring varicosities shows a heterogeneous regional distribution, which is correlated to a similar regional distribution in transmission strength. We propose that greater segregation of ACh and GABA produces less GABAergic inhibition, strengthening ganglionic transmission. Segregation of ACh and GABA varies in different physiopathological conditions; specifically, segregation increases in acute sympathetic hyperactivity that occurs in cold stress, does not vary in chronic hyperactivity that occurs in hypertension, and rises in early ages of normotensive and hypertensive rats. Given this, we propose that variations in the extent of transmitter segregation may contribute to the alteration of neural activity that occurs in some physiopathological conditions and with age.

Long-term potentiation is differentially expressed in rostral and caudal neurons in the superior cervical ganglion of normal and hypertensive rats.

Neurons in the superior cervical ganglia (SCG) are classified as rostral and caudal according to their regional locations. Although diverse phenotypes have been reported for these two subpopulations, differences in neuroplasticity, like long-term potentiation (LTP), have not been characterized. Here, we explored possible regional differences of LTP expression in rostral and caudal neurons of the SCG in control rats, Wistar and Wistar Kyoto (WKy), and in the spontaneously hypertensive rats (SHR) as a model of hypertension. We characterized the expression of gLTP evoked by a tetanic train (40 Hz, 3 s) in an in vitro SCG preparation. gLTP was recorded in rostral and caudal neurons at 8-weeks-old (wo) in Wistar rats, 6-wo and 12-wo in SHR and WKy rats. We found that gLTP was differentially expressed; gLTP was larger in caudal neurons in Wistar and adult WKy rats. In adult 12-wo hypertensive SHR, gLTP was expressed in caudal but not in rostral neurons. In contrast, in 6-wo pre-hypertensive SHR, gLTP was expressed in rostral but not in caudal neurons; while in 6-wo WKy, gLTP was expressed in caudal but not in rostral neurons. The lack of gLTP expression in caudal neurons of 6-wo SHR was not due to a GABAergic modulation because several GABA-A receptor antagonists failed to unmask gLTP. Data show that neuroplasticity, particularly gLTP expression, varied according to the ganglionic region. We propose that differential regional expression of gLTP may be correlated with selective innervation on different target organs.

Ganglionic Long-Term Potentiation in Prehypertensive and Hypertensive Stages of Spontaneously Hypertensive Rats Depends on GABA Modulation.

The sympathetic nervous system (SNS) regulates body functions in normal and pathological conditions and is characterized by the presence of a neuroplastic phenomenon, termed ganglionic long-term potentiation (gLTP). In hypertension, either in spontaneously hypertensive rats (SHR) or in humans, sympathetic hyperfunction, such as elevated SNS outflow and changes in synaptic plasticity have been described. Because enhanced SNS outflow is detected in the hypertensive stage and, more importantly, in the prehypertensive phase of SHR, here we explored whether synaptic plasticity, particularly gLTP, was modified in the superior cervical ganglia (SCG) of prehypertensive SHR. Furthermore, considering that GABA modulates sympathetic synaptic transmission and gLTP in Wistar rats, we studied whether GABA might modulate gLTP expression in SHR. We characterized gLTP in the SCG of young prehypertensive 6-week-old (wo) and adult hypertensive (12 wo) SHR and in the SCG of Wistar Kyoto (WKy) normotensive control rats of the same ages. We found that gLTP was expressed in 6 wo SHR, but not in 12 wo rats. By contrast, in WKy, gLTP was expressed in 12 wo, but not in 6 wo rats. We also found that gLTP depends on GABA modulation, as blockade of GABA-A subtype receptors with its antagonist bicuculline unmasked gLTP expression in adult SHR and young WKy. We propose that (1) activity-dependent changes in synaptic efficacy are altered not only during hypertension but also before its onset and (2) GABA may play a modulatory role in the changes in synaptic plasticity in SHR, because the blockade of GABA-A receptors unmasked the expression of gLTP. These early changes in neuroplasticity and GABA modulation of gLTP could be part of the sympathetic hyperfunction observed in hypertension.

Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters.

Sympathetic neurons of the rat superior cervical ganglion (SCG) can segregate their neurotransmitters and co-transmitters to separate varicosities of single axons. We have shown that transmitter segregation is a plastic phenomenon and that it is correlated with the strength of synaptic transmission. Here, we determined whether sympathetic dysfunction occurring in stress and hypertension was correlated with plastic changes of neurotransmitter segregation. We characterized the expression of the markers, L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine (ACh) transporter (VAChT) in the SCG of cold stressed and spontaneously hypertensive rats (SHR). Considering that the SCG comprises a heterogeneous neuronal population, we explored whether the expression and segregation of neurotransmitters would also have an intraganglionic heterogeneous distribution in ganglia of stressed and hypertensive rats. Furthermore, since hypertension in SHR is detected around 8-10 weeks, we evaluated expression and segregation of ACh and GABA in adult hypertensive (12-week old (wo)) and young pre-hypertensive (6-wo) SHR. We found an increase in segregation of ACh and GABA with no change in transmitter expression in ganglia of stressed animals. In contrast, in SHR, there was an increase in GABA expression, although segregation did not vary. Segregation showed a caudo-rostral gradient in controls but not in the ganglia of stressed animals. GABA expression showed a rostro-caudal gradient in adult SHR, which was not present in young 6-wo rats. In young SHR, ACh increased and, unexpectedly, segregation of ACh and GABA was higher than in adults. Data suggest that ACh and GABA segregation increases in acute sympathetic hyperactivity like stress, but does not vary in chronic hyperactivity such as in hypertension. Changes in segregation are age-dependent and might be involved in the mechanisms underlying stress and hypertension.

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.

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.

Differential contribution of BDNF and NGF to long-term potentiation in the superior cervical ganglion of the rat.

Synaptic transmission in the sympathetic nervous system is a plastic process modulated by different factors. We characterized the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on basal transmission and ganglionic long-term potentiation (LTP) in the rat superior cervical ganglion. LTP was elicited by supramaximal tetanic stimulation (40 Hz, 3 s) of the sympathetic trunk and was quantified by measuring LTP decay time and LTP extent. Neurotrophins did not affect basal transmission, however, they differentially affected LTP. BDNF (200 ng/ml) increased LTP decay time and LTP extent 2.0-fold (p < 0.01). In contrast, NGF showed a dual effect: 200 ng/ml NGF reduced LTP decay time and LTP extent to 53% and to 32% of control value (p < 0.0001 and p < 0.02; respectively), whereas >350 ng/ml NGF significantly increased LTP decay time and LTP extent (p < 0.02). Digital analysis of compound action potentials suggests that neurotrophins could change the synchronization of unitary action potentials. Pharmacological data obtained in intact ganglia show that C2-ceramide produced a 2-fold enhancement in LTP, whereas tyrphostin AG879, an inhibitor of tyrosine kinase activity, reversed the NGF blockade and produced by itself an enhancement in LTP. In sliced ganglia we observed that an anti-TrkA antibody reversed the NGF-induced LTP blockade. Immunohistochemistry studies revealed that 83% of ganglionic neurons express TrkA, whereas 52% express p75 receptor, and 18% express TrkB receptor. We propose that p75 neurotrophin receptors and probably TrkB signaling enhance LTP, whereas TrkA signaling reduces it.

Neurotransmitter segregation: functional and plastic implications.

Synaptic cotransmission is the ability of neurons to use more than one transmitter to convey synaptic signals. Cotransmission was originally described as the presence of a classic transmitter, which conveys main signal, along one or more cotransmitters that modulate transmission, later on, it was found cotransmission of classic transmitters. It has been generally accepted that neurons store and release the same set of transmitters in all their synaptic processes. However, some findings that show axon endings of individual neurons storing and releasing different sets of transmitters, are not in accordance with this assumption, and give support to the hypothesis that neurons can segregate transmitters to different synapses. Here, we review the studies showing segregation of transmitters in invertebrate and mammalian central nervous system neurons, and correlate them with our results obtained in sympathetic neurons. Our data show that these neurons segregate even classic transmitters to separated axons. Based on our data we suggest that segregation is a plastic phenomenon and responds to functional synaptic requirements, and to 'environmental' cues such as neurotrophins. We propose that neurons have the machinery to guide the different molecules required in synaptic transmission through axons and sort them to different axon endings. We believe that transmitter segregation improves neuron interactions during cotransmission and gives them selective and better control of synaptic plasticity.