Sue A. Aicher

N-methyl-D-aspartate receptors are present in vagal afferents and their dendritic targets in the nucleus tractus solitarius.

N-Methyl-D-aspartate receptors are present in the nodose ganglion, which contains the cell bodies of vagal afferents, and in the nucleus tractus solitarius, where these afferent fibers terminate. This suggests that N-methyl-D-aspartate receptors are located presynaptically on visceral vagal afferents and/or their target neurons in the nucleus tractus solitarius. To test this hypothesis, we combined anterograde transport of biotinylated dextran amine, following injections into the left nodose ganglion, with electron microscopic immunogold labeling of antipeptide antiserum against the R1 subunit of the N-methyl-D-aspartate receptor in the nucleus tractus solitarius of rat brain. Within the medial nucleus tractus solitarius, the N-methyl-D-aspartate receptor R1 immunoreactivity was seen in dendrites (39% of 639 profiles), axons and axon terminals (41%), and a few neuronal perikarya and glia. Many vagal afferent axons and terminals (40% of 468 profiles) contained N-methyl-D-aspartate receptor R1 immunogold labeling. In addition, 42% of the dendrites contacted by vagal afferent terminals (n = 206) contained N-methyl-D-aspartate receptor R1 immunoreactivity. In axons and dendrites, the gold particles were occasionally seen within asymmetric postsynaptic junctions or at non-synaptic sites on the plasma membrane. More commonly, however, N-methyl-D-aspartate receptor R1 labeling was seen on membranes of vesicular cytoplasmic organelles, suggesting that there is abundant N-methyl-D-aspartate receptor protein available for activity-dependent mobilization to the plasmalemma. Since many vagal afferents are glutamatergic, our results implicate N-methyl-D-aspartate receptors in autoregulation of the presynaptic release and postsynaptic responses to glutamate at the level of the first central synapse in the nucleus tractus solitarius.

Monosynaptic projections from the nucleus tractus solitarii to C1 adrenergic neurons in the rostral ventrolateral medulla: Comparison with input from the caudal ventrolateral medulla

The rostral ventrolateral medulla (RVL) contains reticulospinal adrenergic (C1) neurons that are thought to be sympathoexcitatory and that form the medullary efferent limb of the baroreceptor reflex pathway. The RVL receives direct projections from two important autonomic regions, the caudal ventrolateral medulla (CVL) and the nucleus tractus solitarii (NTS). In the present study, we used anterograde tracing from the CVL or the NTS combined with immunocytochemical identification of C1 adrenergic neurons in the RVL to compare the morphology of afferent input from these two autonomic regions into the RVL.

Hippocampal α2A‐adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes

Alpha‐adrenergic receptor, subtype 2A (α2A‐AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, α2A‐AR immunoreactivity (α2A‐AR‐I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat α2A‐AR. By light microscopy, dense α2A‐AR‐I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular α2A‐AR‐I was found in the neuropil in all other laminae, notably stratum lacunosum‐moleculare. Ultrastructurally, α2A‐AR‐I was found in neuronal cytoplasm associated with large multivesicular‐like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of α2A‐AR‐I in the strata oriens, radiatum, and lacunosum‐moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): α2A‐AR‐I was found in axons and terminals (∼40%), glia (∼30%), dendritic spines (∼25%), and dendritic shafts (∼5%). This mixed pre‐ and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most α2A‐AR‐I was found in axons (∼60%) and glia (∼30%). Alpha‐2A—AR‐labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained α2A‐AR‐I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH‐labeled terminals were in close proximity to α2A‐AR‐labeled spines and glia. These studies demonstrate that hippocampal α2A‐AR‐I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal α2A‐ARs. J. Comp. Neurol. 395:310–327. 1998.

Nucleus tractus solitarius efferent terminals synapse on neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla

The caudal ventrolateral medulla (CVL) contains neurons that are vasodepressor and are a critical component of the baroreceptor reflex pathway. While electrophysiological studies suggest that CVL neurons are intercalated in the baroreceptor pathway between the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (RVL), there is no direct evidence for this projection. Therefore, we identified CVL neurons that project to RVL by retrogradely labelling them with wheat germ agglutinin-apo-horseradish peroxidase conjugated to colloidal gold (WAHG) injected into the RVL. Retrogradely labelled neurons were seen in previously identified vasodepressor areas of the rostral CVL that are critical for the baroreceptor reflex. Double labelling for WAHG and tyrosine hydroxylase (TH) immunocytochemistry indicated that CVL neurons that project to the RVL (CVL --> RVL neurons) are distinct from the noradrenergic neurons of the A1 cell group. To establish the presence of a direct projection from the NTS to CVL --> RVL neurons, the retrograde tracer WAHG was pressure injected into the RVL and the anterograde tracer biocytin was iontophoresed into the NTS of anesthetized rats. After 4-6 h, anesthetized rats were perfused transcardially with 3.75% acrolein in 2% paraformaldehyde and sections through the CVL were processed for both markers. By light microscopy, numerous biocytin-labelled varicose processes overlapped neurons containing WAHG in the CVL. By electron microscopy, biocytin was found in myelinated and unmyelinated axons and in axon terminals (0.9 + 0.02 microns) that contained primarily small clear vesicles. These terminals formed predominantly asymmetric synapses on large (1.5-6.0 microns in diameter) dendrites within the CVL. Some of the post-synaptic perikarya and large dendrites contained WAHG associated with lysosomes and multivesicular bodies, indicating that they belong to neurons which project to the RVL. We conclude that CVL --> RVL neurons are (a) distinct from A1 noradrenergic cells; (b) receive direct synaptic contacts from NTS efferent terminals; (c) are potently and monosynaptically excited (asymmetric synapses) by NTS efferent terminals. These data support the hypothesis that CVL neurons are intercalated between the NTS and the RVL in the baroreceptor reflex pathway.

Anatomical substrates for baroreflex sympathoinhibition in the rat.

The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS-->CVL-->RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks.

Receptor-selective analogs demonstrate NPY/PYY receptor heterogeneity in rat brain

Neuropeptide Y (NPY) receptors are heterogeneous, consisting of at least two subclasses, Y1 and Y2. We sought evidence for differential expression of NPY receptor subtypes in the rat brain. Tissue was incubated with 125I-peptide YY (PYY) which labels NPY and PYY binding sites. The Y1-selective agonist, p[Pro34]NPY, and the Y2-selective agonist, pNPY 13-36, were used as displacing ligands. Autoradiographic analyses of regional receptor binding demonstrated heterogeneity across brain regions. We conclude that Y1- and Y2-receptors may be independently expressed in the brain. While the predominate NPY/PYY receptor subtype in the brain is Y2, there are also Y1-receptors in some brain regions such as the superficial layers of the parietal cortex.

Presynaptic localization of the carboxy-terminus epitopes of the μ opioid receptor splice variants MOR-1C and MOR-1D in the superficial laminae of the rat spinal cord

Opioids inhibit nociceptive transmission at the level of the spinal cord, possibly through inhibition of neurotransmitter release by presynaptic mu opioid receptors (MORs) thus preventing the activation of ascending pathways and the perception of pain. Most nociceptive primary afferents are unmyelinated fibers containing peptides such as substance P and/or calcitonin gene-related peptide. However, few terminals contain both substance P and MOR. Recently, we identified new carboxy-terminal MOR splice variants that are localized in the superficial laminae of the dorsal horn. We now report the precise cellular distribution of two of these MOR-1 variants, MOR-1C (exon 7/8/9 epitope) and MOR-1D (exon 8/9 epitope), at the ultrastructural level. In the superficial laminae of the dorsal horn, the majority of the labeling of MOR-1C and MOR-1D was found in unmyelinated axons. This distribution contrasts with that of MOR-1 (exon 4 epitope), in which labeling is equally found in dendrites and soma, as well as in axons. The presence of dense core vesicles in many of the MOR-1C-like immunoreactive terminals implies that this splice variant might be involved in presynaptic inhibition of transmitter release from peptide-containing afferents to the dorsal horn. Consistent with this finding, confocal microscopy analyses showed that many MOR-1C profiles in laminae I-II also contained calcitonin gene-related peptide, whereas fewer MOR-1 profiles contained either substance P or calcitonin gene-related peptide in this same region. From these findings we suggest that there are differential distributions of MOR-1 splice variants as well as distinct peptide colocalizations in the dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor is postsynaptic to substance P-containing axon terminals in the rat superficial dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor is thought to mediate the postsynaptic effects of excitatory amino acids released from primary afferent terminals in the superficial layers of the dorsal horn of the spinal cord where synergistic associations with substance P (SP) have been implicated in the production of hyperalgesia. We examined the electron microscopic dual immunocytochemical localization of SP and the R1 subunit of the NMDA receptor (NMDAR1) in this region to determine the cellular basis for interactions between SP and NMDA receptor ligands. Of 971 profiles immunolabeled for NMDAR1, 40% were dendrites and the remainder were primarily unmyelinated axons and astrocytic processes. In dendrites, NMDAR1-like immunoreactivity (NMDAR1-LI) was associated with synaptic and non-synaptic portions of the plasma membrane, as well as intracellular membranes including smooth endoplasmic reticulum. These NMDAR1-labeled dendrites received synaptic input from unlabeled terminals and from terminals containing SP and/or NMDAR1-LI and they occasionally (25/389) also contained SP. In contrast, of 540 SP-immunoreactive profiles, 60% were axon terminals and the majority (252/324) of these SP-labeled terminals were presynaptic to NMDAR1-containing dendrites. These results provide anatomical evidence that the synergistic nociceptive effects of SP and NMDA ligands are attributed mainly to dual modulation of the activity of single dendritic targets in the dorsal horn of the spinal cord. They also suggest that activation of NMDA receptors may also play a role in the modulation of SP neurons, presynaptic release of SP or other neurotransmitters, and in glial function in the dorsal horn.

opioid receptors are present in vagal afferents and their dendritic targets in the medial nucleus tractus solitarius

Ligands of the μ‐opiate receptor (MOR) are known to influence many functions that involve vagal afferent input to the nucleus tractus solitarius (NTS), including cardiopulmonary responses, gastrointestinal activity, and cortical arousal. The current study sought to determine whether a cellular substrate exists for direct modulation of vagal afferents and/or their neuronal targets in the NTS by ligands of the MOR. Anterograde tracing of vagal afferents arising from the nodose ganglion was achieved with biotinylated dextran amine (BDA), and the MOR was detected by using antipeptide MOR antiserum. The medial subdivision of the intermediate NTS was examined by electron microscopy for the presence of peroxidase‐labeled, BDA‐containing vagal afferents and immunogold MOR labeling. MOR was present in both presynaptic axon terminals and at postsynaptic sites, primarily dendrites. In dendrites, MOR immunogold particles usually were located along extrasynaptic portions of the plasma membrane. Of 173 observed BDA‐labeled vagal afferent axon terminals, 33% contained immunogold labeling for MOR within the axon terminal. Many of these BDA‐labeled terminals formed asymmetric, excitatory‐type synapses with dendrites, some of which contained MOR immunogold labeling. MORs were present in 19% of the dendrites contacted by BDA‐labeled terminals but were present rarely in both the vagal afferent and its dendritic target. Together, these results suggest that MOR ligands modulate either the presynaptic release from or the postsynaptic responses to largely separate populations of vagal afferents in the intermediate NTS. These results provide a cellular substrate for direct actions of MOR ligands on primary visceral afferents and their second‐order neuronal targets in NTS. J. Comp. Neurol. 422:181–190, 2000.

Presynaptic and postsynaptic relations of μ-opioid receptors to γ- aminobutyric acid-immunoreactive and medullary-projecting periaqueductal gray neurons

The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the μ‐opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., γ‐aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary‐projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual‐labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR‐immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual‐immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA‐immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary‐projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG. J. Comp. Neurol. 419:532–542, 2000.