Efferent projections of the nucleus of the solitary tract to peri-locus coeruleus dendrites in rat brain: evidence for a monosynaptic pathway.

Locus coeruleus (LC) neurons respond to autonomic influences, are activated by physiological stressors, and discharge in parallel with peripheral sympathetic nerves. The circuitry underlying modulation of LC activity by physiological manipulations (i.e., hemodynamic stress, hypovolumia) remains unclear. Specifically, monosynaptic projections from primary baroreceptor centers to the LC have been suggested by electrophysiological studies but have not been unequivocally established. Light microscopic anterograde tract-tracing studies have previously shown that neurons originating in the nucleus of the solitary tract (NTS) project to a region of the rostrodorsal pontine tegmentum, which contains noradrenergic dendrites of the LC; however, it is not known whether these NTS efferents specifically target LC dendrites. Therefore, we combined peroxidase labeling of biotinylated dextran amine (BDA) or Phaseolus vulgaris-leucoagglutinin (PHA-L) from the NTS with gold-silver labeling for tyrosine hydroxylase (TH) in the rostrolateral peri-LC region. Injections placed into neighboring nuclei (nucleus gracilis, hypoglossal nucleus) served as controls. Only injections centered in the NTS produced anterograde labeling in peri-LC regions containing TH processes. By electron microscopy, BDA- or PHA-L-labeled axon terminals originating from the NTS contained small, clear, and some large dense-core vesicles and formed heterogeneous synaptic contacts characteristic of both excitatory- and inhibitory-type transmitters. Approximately 19% of the BDA and PHA-L axon terminals examined originating from the commissural portion of the NTS formed synaptic specializations with dendrites exhibiting TH immunoreactivity in the peri-LC. These results demonstrate that neurons projecting from the cardiovascular-related portion of the NTS target noradrenergic dendrites, indicating that barosensitive NTS neurons may directly modulate the activity of LC neurons and may serve to integrate autonomic responses in brain by influencing the widespread noradrenergic projections of the LC. In addition, these findings demonstrate that extranuclear dendrites are an important termination site for afferents to the LC.     

Selective distribution of the NMDA-R1 glutamate receptor in astrocytes and presynaptic axon terminals in the nucleus locus coeruleus of the rat brain: An immunoelectron microscopic study

The regional and cellular distribution of the different classes of excitatory amino acid receptors with respect to the noradrenergic neurons of the nucleus locus coeruleus (LC) are unknown. We therefore combined immunoperoxidase labeling for the R1 subunit of the N-methyl-D-aspartate (NMDA) receptor with immunogold-silver localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in single sections through the rat LC to determine the subcellular localization of this glutamate receptor subtype with respect to the noradrenergic neurons. At the light microscopic level, there was light to moderate labeling for the NMDA-R1-like (li) receptor in the caudal pole of the LC and dense labeling in the dorsolateral aspect of the LC adjacent to the superior cerebellar peduncle. In the rostral pole of the LC which is enriched with noradrenergic dendrites, significant overlap between both immunoreactivities could be observed. At the ultrastructural level, immunoperoxidase labeling for NMDA-R1 was selectively distributed in astrocytic processes and within presynaptic axon terminals but was rarely seen in catecholamine-containing somata or dendrites. Peroxidase labeling for NMDA-R1, however, was occasionally observed in dendrites in the rostral pole of the LC. Most of these dendrites lacked detectable levels of TH, although TH immunoreactivity was apparent in the neuropil. Dendrites containing NMDA-R1-li immunoreactivity often received asymmetric (excitatory-type) contacts from unlabeled terminals. NMDA-R1-li-immunoreactive axon terminals usually contained small clear, as well as large dense-core vesicles and were often apposed to unlabeled dendrites, axon terminals and/or glial processes. These results provide the first ultrastructural evidence that NMDA-R1-li immunoreactivity is selectively distributed within astrocytic processes and presynaptic axon terminals within the LC. © 1996 Wiley-Liss, Inc.     

Input from central nucleus of the amygdala efferents to pericoerulear dendrites,some of which contain tyrosine hydroxylase immunoreactivity

Light microscopic anterograde tracing studies indicate that neurons in the central nucleus of the amygdala (CNA) project to a region of the dorsal pontine tegmentum ventral to the superior cerebellar peduncle which contains noradrenergic dendrites of the nucleus locus coeruleus (LC). However, it has not been established whether the efferent terminals from the CNA target catecholamine-containing dendrites of the LC or dendrites of neurons from neighboring nuclei which may extend into this region. To examine this question, we combined immunoperoxidase labeling of the anterograde tracer biotinylated dextran amine (BDA) from the CNA with immunogold-silver labeling of the catecholamine-synthesizing enzyme tryrosine hydroxylase (TH) in the rostrolateral LC region of adult rats. By light microscopy, BDA-labeled processes were dense in the dorsal pons within the parabrachial nuclei as well as in the pericoerulear region immediately ventral to the superior cerebellar peduncle. Higher magnification revealed that BDA-labeled varicose fibers overlapped TH-labeled processes in this pericoerulear region. By electron microscopy, anterogradely labeled axon terminals contained small, clear as well as some large dense core vesicles and were commonly apposed to astrocytic processes along some portion of their plasmalemma. BDA-labeled terminals mainly formed symmetric type synaptic contacts characteristic of inhibitory transmitters. Of 250 BDA-labeled axon terminals examined where TH immunoreactivity was present in the neuropil, 81% contacted unlabeled and 19% contacted TH-labeled dendrites. Additionally, amygdala efferents were often apposed to unlabeled axon terminals forming asymmetric (excitatory type) synapses. These results demonstrate that amygdaloid efferents may directly alter the activity of catecholaminergic and non-catecholaminergic neurons in this pericoerulear region of the rat brain. Furthermore, our study suggests that CNA efferents may indirectly affect the activity of pericoerulear neurons through modulation of excitatory afferents. Amygdaloid projections to noradrenergic neurons may help integrate behavioral and visceral responses to threatening stimuli by influencing the widespread noradrenergic projections from the LC. © 1996 Wiley-Liss, Inc.     

A.E. Bennett Research Award. Anatomic basis for differential regulation of the rostrolateral peri-locus coeruleus region by limbic afferents.

BACKGROUND: Neurochemical and electrophysiological studies indicate that the locus coeruleus (LC)-norepinephrine system is activated by physiological and external stressors. This activation is mediated in part by corticotropin-releasing factor (CRF), the hypothalamic neurohormone that initiates the endocrine response to stress. We have previously shown that the central nucleus of the amygdala (CNA) provides CRF afferents to noradrenergic processes in the peri-LC area that may serve to integrate emotional and cognitive responses to stress. The bed nucleus of the stria terminalis (BNST) shares many anatomical and neurochemical characteristics with the CNA, including a high density of CRF-immunoreactive cells and fibers; however, recent studies have suggested that the CNA and the BNST may differentially regulate responses to conditioned and unconditioned fear, respectively, suggesting divergent neuroanatomical circuits underlying these processes. METHODS: In the present study, neuroanatomical substrates subserving regulation of the LC by the BNST were examined. Anterograde tract-tracing was combined with immunoelectron microscopy to test the hypotheses that BNST efferents target noradrenergic neurons of the LC and that these efferents exhibit immunolabeling for CRF. RESULTS: Ultrastructural analysis of sections that were dually labeled for the anterograde tracer biotinylated dextran amine (BDA) injected into the BNST and tyrosine hydroxylase (TH)-immunoreactivity demonstrated that BDA-labeled axon terminals formed synaptic specializations (primarily inhibitory) with TH-labeled dendrites and dendrites that lacked TH immunoreactivity. In contrast to CNA efferents that exhibited substantial immunolabeling for CRF, far fewer BDA-labeled terminals from the BNST in the rostrolateral peri-LC contained CRF. CONCLUSIONS: The present results indicate that the BNST may provide distinct neurochemical regulation of the peri-LC as compared to other limbic afferents such as the CNA. These data are interesting in light of behavioral studies showing that the CNA and BNST may be differentially involved in fear versus anxiety, respectively.     

Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

The stress-related neurohormone, corticotropin-releasing factor (CRF), also acts as a neurotransmitter to activate the brain noradrenergic nucleus, locus coeruleus (LC). Previous electrophysiological findings demonstrating that tonic CRF secretion in the LC is increased in adrenalectomized rats suggest that activity of certain CRF afferents to the LC is under inhibitory regulation by endogenous corticosteroids. The present study was designed to identify putative CRF afferents to the LC that may be regulated by glucocorticoids. Retrograde tract tracing from the rat LC and pericoerulear regions was combined with immunohistochemistry to visualize CRF and glucocorticoid receptors in the same sections of rat brain. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase coupled to gold (WGA–Au–HRP) was injected into either the nucleus LC or the rostrolateral pericoerulear region (peri-LC), where CRF-immunoreactive terminals have been demonstrated to synapse with LC dendrites. Sections were processed to visualize the tracer, as well as CRF- and glucocorticoid receptor-immunoreactivity. Following injections of WGA–Au–HRP into the nuclear LC, triple labeled neurons were observed primarily in Barrington's nucleus, where 74±4% of retrogradely labeled CRF-immunoreactive neurons colocalized glucocorticoid receptor immunoreactivity. In contrast, injections that incorporated the rostrolateral peri-LC retrogradely labeled numerous neurons that were immunoreactive for both CRF and glucocorticoid receptors in the central nucleus of the amygdala. Thus, 94±2% of the retrogradely labeled CRF-immunoreactive neurons in the central nucleus of the amygdala colocalized glucocorticoid receptor immunoreactivity. Additionally, triple labeled neurons were observed in the bed nucleus of the stria terminalis following WGA–Au–HRP injections that incorporated the rostrolateral peri-LC. The present results implicate Barrington's nucleus, the central nucleus of the amygdala and the bed nucleus of the stria terminalis as glucocorticoid-sensitive sources of CRF that can influence the LC-noradrenergic system. Alterations in glucocorticoid levels or glucocorticoid receptor function would be predicted to affect the impact of these specific CRF systems on LC activity.     

Multiple substrates for serotonergic modulation of rat locus coeruleus neurons and relationships with kainate receptors

The ultrastructural substrates of serotonin (5-hydroxytryptamine [5-HT]) immunoreactive terminals with respect to noradrenergic neurons of the locus coeruleus (LC) have only been suggested from immunocytochemical analysis in adjacent tissue sections using antisera directed against tryptophan and tyrosine hydroxylase (TH) enzymes. Here, we conducted dual immunoelectron microscopy in the same section of tissue using antisera directed against 5-HT and TH to determine cellular substrates for proposed interactions between these two transmitter systems. Axon terminals containing peroxidase labeling for 5-HT possessed small clear as well as large dense core vesicles. Of 176 5-HT-labeled axons and terminals, 19% contacted TH-labeled dendrites. When a synaptic specialization was detectable, it was more often of the asymmetric type. Electrophysiological studies have also shown that 5-HT selectively attenuates excitatory amino acid-induced activation of neurons in the LC. Thus, to further examine the cellular relationship between 5-HT-labeled axon terminals and excitatory amino acid receptors, we conducted immunogold-silver labeling of an antibody which recognized the three identified members of the kainate receptor (KAr) subunit class (GluR 5,6,7) and peroxidase localization of 5-HT. Similar proportions of 5-HT-labeled terminals (9%) were either apposed to KAr-labeled dendrites or exhibited KAr immunoreactivity. Twenty-four percent of the 5-HT axon terminals examined were apposed by glial processes that contained KAr. These data indicate that 5-HT axon terminals are in direct contact with LC neurons and also suggest pre- and postsynaptic sites for modulation of 5-HT terminals by excitatory amino acid ligands as well as indirect sites via glial processes.     

Subcellular targeting of kappa‐opioid receptors in the rat nucleus locus coeruleus

The dynorphin (DYN)‐kappa opioid receptor (κOR) system has been implicated in stress modulation, depression, and relapse to drug‐seeking behaviors. Previous anatomical and physiological data have indicated that the noradrenergic nucleus locus coeruleus (LC) is one site at which DYN may contribute to these effects. Using light microscopy, immunofluorescence, and electron microscopy, the present study investigated the cellular substrates for pre‐ and postsynaptic interactions of κOR in the LC. Dual immunocytochemical labeling for κOR and tyrosine hydroxylase (TH) or κOR and preprodynorphin (ppDYN) was examined in the same section of tissue. Light microscopic analysis revealed prominent κOR immunoreactivity in the nuclear core of the LC and in the peri‐coerulear region where noradrenergic dendrites extend. Fluorescence and electron microscopy revealed κOR immunoreactivity within TH‐immunoreactive somata and dendrites in the LC as well as localized to ppDYN‐immunoreactive processes. In sections processed for κOR and TH, ≈29% (200/688) of the κOR‐containing axon terminals identified targeted TH‐containing profiles. Approximately 49% (98/200) of the κOR‐labeled axon terminals formed asymmetric synapses with TH‐labeled dendrites. Sections processed for κOR and ppDYN showed that, of the axon terminals exhibiting κOR, 47% (223/477) also exhibited ppDYN. These findings indicate that κORs are poised to modulate LC activity by their localization to somata and dendrites. Furthermore, κORs are strategically localized to presynaptically modulate DYN afferent input to catecholamine‐containing neurons in the LC. These data add to the growing literature showing that κORs can modulate diverse afferent signaling to the LC. J. Comp. Neurol. 512:419–431, 2009. © 2008 Wiley‐Liss, Inc.     

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain

Physiological and immunohistochemical studies have suggested that corticotropin-releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold-silver localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF-labeled axon terminals consisted of both TH-labeled dendrites and dendrites lacking detectable TH-immunoreactivity. Of 113 CRF-immunoreactive axon terminals, approximately 70% were in direct contact with TH-labeled and unlabeled dendrites. Of the CRF-labeled axon terminals forming synapses with TH-labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF-labeled axon terminals. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes. © 1996 Wiley-Liss, Inc.     

Two populations of glutamatergic axons in the rat dorsal raphe nucleus defined by the vesicular glutamate transporters 1 and 2

Most glutamatergic neurons in the brain express one of two vesicular glutamate transporters, vGlut1 or vGlut2. Cortical glutamatergic neurons highly express vGlut1, whereas vGlut2 predominates in subcortical areas. In this study immunohistochemical detection of vGlut1 or vGlut2 was used in combination with tryptophan hydroxylase (TPH) to characterize glutamatergic innervation of the dorsal raphe nucleus (DRN) of the rat. Immunofluorescence labeling of both vGlut1 and vGlut2 was punctate and homogenously distributed throughout the DRN. Puncta labeled for vGlut2 appeared more numerous then those labeled for vGlut1. Ultrastructural analysis revealed axon terminals containing vGlut1 and vGlut2 formed asymmetric-type synapses 80% and 95% of the time, respectively. Postsynaptic targets of vGlut1- and vGlut2-containing axons differed in morphology. vGlut1-labeled axon terminals synapsed predominantly on small-caliber (distal) dendrites (42%, 46/110) or dendritic spines (46%, 50/110). In contrast, vGlut2-containing axons synapsed on larger caliber (proximal) dendritic shafts (> 0.5 microm diameter; 48%, 78/161). A fraction of both vGlut1- or vGlut2-labeled axons synapsed onto TPH-containing dendrites (14% and 34%, respectively). These observations reveal that different populations of glutamate-containing axons innervate selective dendritic domains of serotonergic and non-serotonergic neurons, suggesting they play different functional roles in modulating excitation within the DRN.     

大鼠Barrington核内脊髓投射神经元直接接受腰骶髓传入的电镜研究

将结合生物素的葡聚糖胺 (BDA)注射到大鼠腰骶髓后 ,在电镜下观察脑桥Barrington核内腰骶髓投射神经元与来自腰骶髓传入投射纤维间的突触联系。与先前的研究相一致 ,注射BDA到腰 6和骶 1节段后 ,光镜下可见Barrington核内出现大量顺行标记的神经末梢和一定数量的逆行标记细胞。电镜下发现标记的轴突末梢和标记的树突之间存在直接的突触连接。结果表明 ,Barrington核直接接受腰骶髓的传入投射 ,提示大鼠脑桥排尿反射的脊髓内上行投射通路中可能存在一条直接通路。     

Cellular interactions between axon terminals containing endogenous opioid peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsal pontine tegmentum

Recent evidence suggests that certain stressors release both endogenous opioids and corticotropin-releasing factor (CRF) to modulate activity of the locus coeruleus (LC)-norepinephrine (NE) system. In ultrastructural studies, axon terminals containing methionine(5)-enkephalin (ENK) or CRF have been shown to target LC dendrites. These findings suggested the hypothesis that both neuropeptides may coexist in common axon terminals that are positioned to have an impact on the LC. This possibility was examined by using immunofluorescence and immunoelectron microscopic analysis of the rat LC and neighboring dorsal pontine tegmentum. Ultrastructural analysis indicated that CRF- and ENK-containing axon terminals were abundant in similar portions of the neuropil and that approximately 16% of the axon terminals containing ENK were also immunoreactive for CRF. Dually labeled terminals were more frequently encountered in the "core" of the LC vs. its extranuclear dendritic zone, which included the medial parabrachial nucleus (mPB). Triple labeling for ENK, CRF, and tyrosine hydroxylase (TH) showed convergence of opioid and CRF axon terminals with noradrenergic dendrites as well as evidence for inputs to TH-labeled dendrites from dually labeled opioid/CRF axon terminals. One potential source of ENK and CRF in the dorsal pons is the central nucleus of the amygdala (CNA). To determine the relative contribution of ENK and CRF terminals from the CNA, the CNA was electrolytically lesioned. Light-level densitometry revealed robust decreases in CRF immunoreactivity in the LC and mPB on the side ipsilateral to the lesion but little or no change in ENK immunoreactivity, confirming previous studies of the mPB. Degenerating terminals from the CNA in lesioned rats were found to be in direct contact with TH-labeled dendrites. Together, these data indicate that ENK and CRF may be colocalized to a subset of individual axon terminals in the LC "core." The finding that the CNA provides, to dendrites in the area examined, a robust CRF innervation, but little or no opioid innervation, suggests that ENK and CRF axon terminals impacting LC neurons originate from distinct sources and that terminals that colocalize ENK and CRF are not from the CNA.     

Evidence for coexistence of enkephalin and glutamate in axon terminals and cellular sites for functional interactions of their receptors in the rat locus coeruleus

The authors previously showed that a subset of axon terminals in the locus coeruleus (LC) contains methionine5-enkephalin (ENK) and gamma-aminobutyric acid (GABA) immunoreactivities. However, numerous ENK-labeled terminals lacked GABA and exhibited synaptic specializations that were characteristic of excitatory-type transmitters. To determine whether ENK coexists with glutamate in the LC, preembedding immunoperoxidase detection of ENK or immunogold-silver was combined with postembedding identification of glutamate using a gold marker. Indeed, 28% of the ENK-labeled axon terminals examined (n = 250 axon terminals) also contained glutamate. To define further sites for functional interactions between opiate ligands and excitatory amino acid receptors, the ultrastructural localization of the mu-opioid receptor (MOR) was examined with respect to either the kainate receptor (KAR) or the R1 subunit of the N-methyl-D-aspartate (NR1)-type glutamate receptor in the LC. Gold-silver labeling for MOR and peroxidase labeling for either KAR or NR1 indicated that the MOR often was localized to the plasma membrane of dendrites that also exhibited immunolabeling for either glutamate receptor subtype. In contrast to the KAR, which was identified primarily in somata and dendrites, NR1 immunoreactivity also was found frequently in axon terminals as well as in glial processes. Glial processes containing NR1 occasionally exhibited immunolabeling for MOR and sometimes were directly apposed to MOR-containing dendrites in the LC. Furthermore, NR1-labeled receptors in axon terminals sometimes were presynaptic to MOR-labeled dendrites. The authors concluded that ENK and glutamate may be cotransmitters in LC afferents. Moreover, ligands at the KAR may modulate directly MOR-containing neurons in the LC, whereas actions at NR1 receptors may affect opioid-sensitive neurons through multiple cellular mechanisms, i.e., through presynaptic, postsynaptic, or glial actions.     

Pre- and postsynaptic sites for serotonin modulation of GABA-containing neurons in the shell region of the rat nucleus accumbens

The shell of the nucleus accumbens receives a dense serotonergic innervation and contains abundant gamma-aminobutyric acid (GABA)-immunoreactive neurons. Moreover, serotonin (5-hydroxytryptamine: 5-HT) and GABA have been implicated in a variety of common motivational and motor-related functions partially ascribed to this brain area. We used immunoelectron microscopy of antisera directed against 5-HT and GABA in the same section of tissue to examine whether there were cellular substrates that might indicate more specific sites for functional interactions involving these transmitters in the shell region of the rat nucleus accumbens. Immunogold-silver labeling for GABA was localized to perikarya, dendrites, axons and axon terminals, whereas immunoperoxidase labeling for 5-HT was restricted to axons and axon terminals. Approximately half (187/366) of the 5-HT-immunoreactive axon terminals apposed or formed synaptic junctions with postsynaptic neurons. These junctions were mainly of the symmetric-type (83/187) characteristic of inhibitory transmitters, and were equally prevalent on dendrites with and without detectable gold-silver labeling for GABA. Of the 187 5-HT-labeled axon terminals with recognized synaptic contacts, 36% also showed convergence on a common dendrite with a GABA-labeled axon terminal. In addition, 5-HT- and GABA-immunoreactive axon terminals were commonly (83/366) identified in direct apposition to one another. Within a single plane of section, 41% of the apposed GABA-immunoreactive axon terminals formed symmetric-type junctions with dendrites or somata, whereas, the apposed 5-HT-labeled axon terminals rarely showed postsynaptic contacts. These results indicate that 5-HT-containing axon terminals may postsynaptically inhibit GABAergic neurons and their targets within the shell of the rat nucleus accumbens. Additionally, our results strongly suggest that, in this brain region, appositions between 5-HT and GABA axons and axon terminals may facilitate presynaptic interactions between these transmitter systems. © 1996 Wiley-Liss, Inc.     

Ultrastructural localization of neuropeptide Y Y1 receptors in the rat medial nucleus tractus solitarius: relationships with neuropeptide Y or catecholamine neurons

Neuropeptide Y (NPY) Y1 receptor (Y1-R) agonists influence cardiovascular regulation. These actions may involve NPY- and catecholamine-containing neurons in the medial nucleus of the solitary tract (mNTS), at the level of the area postrema. The cellular sites through which Y1-R agonists may interact with NPY and catecholamines in the mNTS, however, are not known. To determine potential sites of action for Y1-R agonists, and their relationship to NPY or catecholamines in the mNTS, we used electron microscopic immunocytochemistry for the detection of sequence-specific antipeptide antisera against Y1-R alone or in combination with antisera against NPY or the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Analyses were conducted in the rat mNTS, at the level of the area postrema. Y1-R was found mainly in small unmyelinated axons and axon terminals but also in some somata and dendrites as well as a small number of glia. Within axon terminals, labeling for Y1-R was often present on dense core vesicles and small synaptic vesicles as well as extrasynaptic areas of the plasmalemma. Some Y1-R-labeled terminals also contained NPY or TH, suggesting that agonists of Y1-R may influence the release of NPY or catecholamines in the mNTS. In addition, Y1-R was found in dendrites that received asymmetric excitatory-type synapses from unlabeled axon terminals. Some of these dendrites contained NPY or TH, which indicates that Y1-R may be targeted for functional activation within NPY- or catecholamine-expressing neurons in the mNTS. These results demonstrate that Y1-R is a presynaptic receptor in NPY- or catecholamine-containing axon terminals within the mNTS as well as a postsynaptic receptor on NPY- or catecholamine-containing neurons that are contacted by axon terminals that likely contain excitatory amino acid transmitters. Agonists of Y1-R in the mNTS may thus affect cardiovascular regulation by modulating NPY, catecholamine, and excitatory amino acid transmission.     

Ultrastructural localization of serotonin 2A and N-methyl-D-aspartate receptors in somata and dendrites of single neurons within rat dorsal motor nucleus of the vagus

Both glutamate and serotonin are potent modulators of autonomic functions involving the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMNV) at the level of the area postrema. Moreover, many of the dendrites in this NTS region express both N-methyl-D-aspartate (NMDA) and serotonin (5HT) 2A receptors, and some of these dendrites may arise from the adjacent DMNV. Thus, single neurons in DMNV may also express both receptors. To test this hypothesis, we used electron microscopic immunocytochemistry for dual localization of the essential R1 subunit of the NMDA receptor (NR1) and the 5HT2A receptor in rat intermediate DMNV, a region serving mainly gastrointestinal functions. Gold particles representing NR1 and peroxidase reaction product for 5HT2A receptors were seen in the cytoplasm, as well as on distinct segments of the plasma membrane of many dendrites. Of the NR1-labeled dendrites, 31% (254/814) also contained 5HT2A immunoreactivity; among the 5HT2A-labeled dendrites, 52% (254/485) expressed NR1. The 5HT2A labeling was also present in numerous small unmyelinated axons, axon terminals, and glial processes. These profiles were largely without NR1 immunoreactivity, although NR1 was detected in some of the dendrites postsynaptic to 5HT2A-labeled terminals. Our results suggest that calcium entry through NMDA channels and 5HT2A receptor activation may dramatically affect postsynaptic excitability of single neurons in the DMNV. In addition, the findings also indicate that the 5HT2A receptor is strategically positioned for involvement in modulation of the presynaptic release of neurotransmitters affecting the postsynaptic activity of DMNV neurons responsive to NMDA activation.     

GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining.

A double immunocytochemical method combining the preembedding PAP technique and the postembedding immunogold technique was used to examine interactions between GABAergic and serotonergic neurons in the same tissue sections of the dorsal raphe nucleus of the rat. A large number of immunogold stained GABAergic axon terminals were found to be presynaptic to strongly PAP immunostained serotonergic perikarya and dendrites. The types of synapses were mostly symmetrical although a few asymmetrical ones were also found. No axo-axonic synapse between the GABAergic axon terminals and the serotonergic neuronal profiles was found. These results suggest that GABAergic neurons could modulate serotonergic neurons in the dorsal raphe nucleus through synaptic relations.     

Ultrastructural localization of the serotonin 2A receptor in dopaminergic neurons in the ventral tegmental area

Serotonin (5-hydroxytryptamine, 5-HT) 2A receptor antagonists are clinically effective antipsychotics that may differentially target mesocortical and mesolimbic dopaminergic neurons having partially segregated distribution in the parabrachial (PB) and paranigral (PN) ventral tegmental area (VTA). We examined the ultrastructural immunocytochemical localization of the 5-HT2A receptor in these subdivisions of rat VTA, to determine (1) the functional sites for receptor activation, and (2) cellular associations between the receptor and dopaminergic neurons identified by their content of tyrosine hydroxylase (TH). The mean area density of neuronal profiles containing 5-HT2A receptor labeling was not significantly different in the PB and PN VTA. In each region approximately 44% of the 5-HT2A labeled profiles were dendrites while the remainder were mainly axons. Dendritic 5-HT2A-immunoreactivity was often localized to membranous cytoplasmic organelles resembling smooth endoplasmic reticulum, and to more rarely to segments of the plasma membrane beneath contacts from unlabeled axon terminals. 5-HT2A labeling was also seen within the cytoplasm of a few axon initial segments and many small unmyelinated axons. Approximately 40% of the 5-HT2A-labeled dendritic profiles contained TH in either PB or PN VTA. Our results suggest that 5-HT2A receptors in VTA are largely cytoplasmic and play an equally important role in modulating dopaminergic neurons in PB and PN VTA. These results also implicate 5-HT2A receptors in the postsynaptic activation of non-dopaminergic neurons and possibly the presynaptic release from terminals of axons originating in, or passing through, these regions.     

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.     

Tyrosine hydroxylase in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity

We sought to determine the ultrastructural localization and the extrinsic sources of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the lateral parabrachial region (PBR) of adult male rats. In the first portion of the study, a rabbit antiserum to TH was immunocytochemically localized in coronal sections through the lateral PBR from acrolein-fixed brains using the peroxidase-antiperoxidase method. Electron-microscopic analysis revealed that perikarya and dendrites with peroxidase immunoreactivity for TH constituted only 17% of the total labeled profiles. Afferents to the TH-labeled perikarya and dendrites usually failed to exhibit immunoreactivity and were thus considered noncatecholaminergic. Somatic synapses were most commonly detected on small immunoreactive perikarya in the central lateral nucleus of the PBR. Other labeled perikarya located in the dorsal lateral or ventral lateral nuclei received few somatic synapses and were morphologically distinct in terms of their larger size, infolded nuclear membrane, and abundance of cytoplasmic organelles. Axons and axon terminals with peroxidase immunoreactivity constituted the remaining labeled profiles in the lateral PBR. These terminals primarily formed symmetric synapses with unlabeled and a few labeled dendrites. The labeled axon terminals were categorized into 2 types: Type I was small (0.3-0.6 micron), contained many small clear vesicles, and exhibited few well-defined synaptic densities. The second type was large (0.8-1.4 micron), contained both small clear and large dense core vesicles, and exhibited well-defined synaptic densities. The 2 types of terminals were morphologically similar to dopaminergic terminals. The location of catecholaminergic neurons contributing to the TH-labeled terminals was determined by combining peroxidase-antiperoxidase immunocytochemistry for TH with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The tracer was unilaterally injected into the PBR of anesthetized adult rats. Immunocytochemical labeling for TH was seen as a brown reaction product within neurons in known catecholaminergic cell groups. A black granular reaction product formed by a cobalt-intensified and diaminobenzidine-stabilized tetramethyl benzidine reaction for WGA-HRP was evident within many TH-labeled and unlabeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)