GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.
"We used triple immunohistochemical methods to label neural tracers with DAB (above) and parvalbumin (PV) or calbindin (CB) neurons with either silver-enhanced gold-conjugated or tetramethylbenzidine (TMB) labeled secondary antibodies. These methods show distinct labeling at the EM: DAB appears as a dark uniform precipitate, silver-enhanced gold particles as circular clumps of variable size, and TMB as rod-shaped crystals (e.g., Pinto et al., 2003; Gonchar and Burkhalter, 2003; Moore et al., 2004; Zikopoulos and Barbas, 2007). "
[Show abstract][Hide abstract] ABSTRACT: The anterior cingulate cortex (ACC), situated in the caudal part of the medial prefrontal cortex, is involved in monitoring on-going behavior pertaining to memory of previously learned outcomes. How ACC information interacts with the medial temporal lobe (MTL) memory system is not well understood. The present study used a multitiered approach to address two questions on the interactions between the ACC and the parahippocampal cortices in the rhesus monkey: (1) What are the presynaptic characteristics of ACC projections to the parahippocampal cortices? (2) What are the postsynaptic targets of the pathway and are there laminar differences in innervation of local excitatory and inhibitory systems? Labeled ACC terminations were quantified in parahippocampal areas TH and TF and a cluster analysis showed that boutons varied in size, with a population of small (≤0.97 μm) and large (>0.97 μm) terminations that were nearly evenly distributed in the upper and deep layers. Exhaustive sampling as well as unbiased stereological techniques independently showed that small and large boutons were about evenly distributed within cortical layers in the parahippocampal cortex. Synaptic analysis of the pathway, performed at the electron microscope (EM), showed that while most of the ACC projections formed synapses with excitatory neurons, a significant proportion (23%) targeted presumed inhibitory classes with a preference for parvalbumin (PV+) inhibitory neurons. These findings suggest synaptic mechanisms that may help integrate signals associated with attention and memory.
"In the quantitative analysis of neurons which project to the antrum we found that of all the synapses observed with these neurons, 23.0 +/− 3.6% contained GAD67-IR. This percentage is very similar to data of others (Moore et al., 2004) who reported findings on GAD-IR terminals synapsing with airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguous. From all of the synaptic contacts with retrogradely labeled AVPNs, 20.2% were GAD-IR. "
[Show abstract][Hide abstract] ABSTRACT: We reported pharmacological data suggesting that stimulation of a vago-vagal reflex activates GABAergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the antrum, but not to the fundus (Ferreira et al., 2002). The purpose of this study was to use an ultrastructural approach to test the hypothesis that GABAergic terminals form synapses with DMV antrum-projecting neurons, but not with DMV fundus-projecting neurons. A retrograde tracer, CTB-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of anesthetized rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTB-HRP, and immunocytochemically for glutamic acid decarboxylase isoenzyme 67 immunoreactivity (GAD67-IR) by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 214 synapses on 195 neurons that projected to the antrum revealed that 23.0+/-3.6% (n = 4) of synaptic contacts were with GAD67-IR terminals. The examination of 220 synapses on 203 fundus-projecting neurons revealed that only 7.9+/-3.1% (n = 4) of synaptic contacts were with GAD67-IR terminals. The difference between GAD67-IR synaptic contacts with antrum- and fundus-projecting neurons was statistically significant (p<0.05). These data suggest that brainstem circuitry controlling the antrum involves GABAergic transmission.
"The three methods show distinct labeling at the EM: DAB appears as a dark uniform precipitate, silver-enhanced gold particles as circular clumps of variable size, and TMB as rod-shaped crystals [e.g., (Pinto et al., 2003; Gonchar and Burkhalter, 2003; Moore et al., 2004; Medalla et al., 2007; Zikopoulos and Barbas, 2007); and personal observations]. "
[Show abstract][Hide abstract] ABSTRACT: Dorsolateral prefrontal areas 46 and 10 are involved in distinct aspects of cognition. Area 46 has a key role in working memory tasks, and frontopolar area 10 is recruited in complex multitask operations. Both areas are innervated by the anterior cingulate cortex (ACC), a region associated with emotions and memory but is also important for attentional control through unknown synaptic mechanisms. Here, we found that in rhesus monkeys (Macaca mulatta) most axon terminals labeled from tracers injected into ACC area 32 innervated spines of presumed excitatory neurons, but ∼20-30% formed mostly large synapses with dendritic shafts of presumed inhibitory neurons in the upper layers (I-IIIa) of dorsolateral areas 10, 46, and 9. Moreover, area 32 terminals targeted preferentially calbindin and, to a lesser extent, calretinin neurons, which are thought to be inhibitory neurons that modulate the gain of task-relevant activity during working memory tasks. Area 46 was distinguished as a recipient of more (by ∼40%) area 32 synapses on putative inhibitory neurons. Area 10 stood apart as recipient of significantly larger (by ∼40% in volume) area 32 terminals on spines of putative excitatory neurons. These synaptic specializations suggest that area 32 has complementary roles, potentially enhancing inhibition in area 46 and strengthening excitation in area 10, which may help direct attention to new tasks while temporarily holding in memory another task.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 12/2010; 30(48):16068-81. DOI:10.1523/JNEUROSCI.1773-10.2010 · 6.34 Impact Factor
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