Ultrastructural evidence for selective GABAergic innervation of CNS vagal projections to the antrum of the rat.

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.

Ultrastructural evidence for selective noradrenergic innervation of CNS vagal projections to the fundus of the rat.

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT 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 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

Genioglossal hypoglossal muscle motoneurons are contacted by nerve terminals containing delta opioid receptor but not mu opioid receptor-like immunoreactivity in the cat: a dual labeling electron microscopic study.

This study has investigated (1) the distribution of delta opioid receptor (DOR) or mu opioid receptor (MOR) containing elements in the hypoglossal nucleus of the adult cat; and (2) the association of these processes with retrogradely labeled genioglossus muscle motoneurons. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the genioglossus muscle on the right side of four isoflurane-anesthetized cats. Forty-four to 52 h later, the animals were sacrificed. Motoneurons containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. The tissues were then processed for immunocytochemistry, using an antiserum raised against DOR or MOR using diaminobenzidine (DAB) as the chromogen. At the light microscopic level, retrogradely labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed immediately caudal to obex. DOR-like immunoreactive processes were apparent at the light microscopic level in the hypoglossal nucleus, but MOR-like immunoreactive processes were not. Both DOR and MOR-like immunoreactive processes were observed in other brainstem areas such as the spinal trigeminal nucleus. At the electron microscopic level, DOR-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled genioglossus muscle motoneuronal dendrites and perikarya in the hypoglossal nucleus. Nineteen (19) percent of the DOR terminals contacted retrogradely labeled genioglossus muscle motoneurons. DOR-immunoreactive terminals also synapsed on unlabeled dendrites and somata. Few MOR-like immunoreactive terminals were found at the EM level in the hypoglossal nucleus, and none of these terminals contacted retrogradely labeled neuronal profiles from the GG muscle. These are the first ultrastructural studies demonstrating synaptic interactions between functionally identified hypoglossal motoneurons and DOR terminals, and that enkephalins most likely act presynaptically to modulate the release of other neurotransmitters that affect GG motoneuron activity. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, are modulated by terminals containing DOR, and that enkephalins acting on DOR but not MOR in the hypoglossal nucleus may play a role in the control of tongue protrusion.

Genioglossal hypoglossal motoneurons contact substance P-like immunoreactive nerve terminals in the cat: a dual labeling electron microscopic study.

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.