Virginia M. Pickel

Corticotrophin‐releasing factor (CRF) is expressed in the central nucleus of the amygdala (CeA), where the CRF receptor (CRFr) plays an important role in anxiety‐ and stress‐related behaviors. To determine the subcellular sites of CRFr activation in this region, we examined the electron microscopic immunolabeling of antisera recognizing CRF or CRFr. The ultrastructural analysis was principally conducted in the lateral subdivision of the rat CeA, with comparisons being made in mice so as to optimally utilize mutant mice in control experiments. The CRFr labeling was seen in many small dendrites and dendritic spines as well as in a few somata, large dendrites, axons, and axon terminals or more rarely in glial processes. Approximately 35% of the CRFr‐labeled dendrites contained CRF immunoreactivity, which was distributed diffusely throughout the cytoplasm, or specifically affiliated with either endomembranes or large dense‐core vesicles. The CRF‐immunoreactive vesicles also were present in somata and axon terminals with or without CRFr labeling. The CRF immunoreactivity was usually absent from both terminals and dendrites joined by asymmetric, excitatory‐type synapses, where a postsynaptic location of the CRFr was commonly observed. Numerous terminals containing both CRF and CRFr were seen, however, within the neuropil and sometimes apposing the excitatory synapses. These results provide ultrastructural evidence for a primary involvement of CRF receptors in modulation of the postsynaptic excitability of CeA neurons, an effect that may be limited by the availability of CRF. The findings have important implications for understanding CRF mediation of rapid responses to stress. J. Comp. Neurol. 512:323–335, 2009.

Adenosine A₁ and A_2A receptors mediate distinct cardiovascular components of defense reactions that are ascribed, in part, to opposing actions within the nucleus tractus solitarius. To assess the cellular sites of relevance to these actions, we examined the light and electron microscopic immunolabeling of adenosine A₁ and A_2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema (dmNTS-AP), a region crucial for cardiovascular regulation involving vagal baroreceptor afferents. Immunoreactivity for each receptor was independently localized to distinct segments of plasma membranes and endomembranes in somatodendritic, axonal, and glial profiles. The dendritic labeling for each receptor also was detected within and near asymmetric, excitatory-type synapses. Of all peroxidase labeled profiles exclusive of somata, ∼58% were A₁- and 39% were A_2A-labeled dendrites. Dendrites and astrocytic glia were the profiles that most often expressed both subtypes of adenosine receptors. The axonal labeling for A_2A receptors was seen mainly in unmyelinated axons, whereas the A₁ receptors were prominently localized within axon terminals. These terminals often formed single or multisynaptic excitatory-type junctions or single symmetric synapses on dendrites, a few of which expressed A₁ and A_2A receptors. These results provide the first ultrastructural evidence that A₁ and A_2A receptors have distributions conductive to their dual involvement in modulating the output of single neurons and glial function in the dmNTS-AP, where the predominate presynaptic effects of adenosine are mediated through A₁ receptors. Synapse 60:496–509, 2006.

The nucleus accumbens (Acb) shell and core are essential components of neural circuitry mediating the reward and motor effects produced by activation of dopamine D2 or cannabinoid‐1 (CB1) receptors. D2 receptors can form heterodimeric complexes with cannabinoid‐1 (CB1) receptors and are also involved in control of the availability of both dopamine and endocannabinoids. Thus, the subcellular locations of D2 and CB1 receptors with respect to each other are implicit to their physiological actions in the Acb. We used electron microscopic immunocytochemistry to determine these locations in the Acb shell and core of rat brain. In each region, many neuronal profiles showed endomembrane and plasmalemmal distributions of one or both receptors. Approximately one‐third of the labeled profiles were somata and dendrites, some of which showed overlapping subcellular distributions of D2 and CB1 immunoreactivities. The remaining labeled profiles were small axons and axon terminals containing CB1 and/or D2 receptors. Of the labeled terminals forming recognizable synapses, ∼20% of those containing CB1 receptors contacted D2‐labeled dendrites, while conversely, almost 15% of those containing D2 receptors contacted CB1‐labeled dendrites. These results provide the first ultrastructural evidence that D2 and CB1 receptors in the Acb shell and core have subcellular distributions supporting both intracellular associations and local involvement of D2 receptors in making available endocannabinoids that are active on CB1 receptors in synaptic neurons. These distributions have direct relevance to the rewarding and euphoric as well as motor effects produced by marijuana and by addictive drugs enhancing dopamine levels in the Acb. J. Comp. Neurol. 495:299–313, 2006.

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid synthesis that is present in cortical pyramidal neurons and highly implicated in control of cerebral blood flow during neural activity. We examined the electron microscopic localization of COX-2 and neuronal nitric oxide synthase (nNOS), a functionally related enzyme, in the somatosensory cortex of rat brain to determine the relevant functional sites. COX-2 immunoreactivity was detected in significantly more somatodendritic than axonal profiles, while nNOS was more often seen in axon terminals. The dendritic COX-2 was localized to endomembranes near synaptic inputs from axon terminals, some of which contained nNOS. Conversely, COX-2 terminals formed asymmetric, excitatory-type synapses with dendrites containing nNOS. The dendritic and axonal profiles containing COX-2 as well as those containing nNOS were minimally separated from penetrating arterioles and capillaries by filamentous glial processes. The perivascular COX-2 labeled terminals were among those that also formed axo-dendritic synapses, suggesting that the release of prostanoids and/or excitatory transmitters from a single terminal may simultaneously affect neuronal activity and cerebral blood flow. Thus, COX-2 has a compartmental distribution in somatosensory cortical neurons consistent with the local neuronal synthesis of prostanoids that are involved in neurovascular coupling and whose actions are modulated by nitric oxide.

The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant‐induced drug‐seeking behavior, both of which are affected by activation of the functionally opposed high‐affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory‐type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R‐immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc. Synapse 52:176–187, 2004.

Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles by the vesicular monoamine transporter‐2 (VMAT2). Somatodendritically released dopamine in this region binds to the D2 receptor (D2R) to modulate ongoing neurotransmission. Although autoregulation of mesocortical dopaminergic neurons in the parabrachial VTA (PB‐VTA) is known to be less efficacious than that of mesolimbic dopaminergic neurons in the paranigral (PN‐VTA), the cellular basis for this regional heterogeneity is not known. For this reason, we used electron microscopic immunocytochemistry to determine the subcellular localization of the dopamine storage vesicles (identified by the presence of VMAT2) in relation to the D2R in these VTA subdivisions. In both regions, D2R immunoreactivity was principally located on extrasynaptic dendritic plasma membranes near excitatory‐type synapses. Equivalent percentages (72 and 74%) of the D2R‐labeled dendrites in each region contained VMAT2‐immunoreactive tubulovesicles. Of the total VMAT2‐labeled dendrites, however, a significantly lower percentage in the PB‐VTA (26%) than in the PN‐VTA (38%) contained D2R labeling. In contrast, a significantly higher number of VMAT2 immunogold‐silver deposits was seen within individual dendrites in the PB‐VTA than in PN‐VTA. In both regions, D2R immunoreactivity was also detected in VMAT2‐negative axon terminals that formed synapses on dendrites containing VMAT2. Our results are the first to demonstrate that within VTA neurons and their afferents the D2R is strategically positioned for activation by dopamine released from dendritic storage vesicles. These findings also suggest that the potential for D2R activation may affect the expression levels of VMAT2 in VTA dendrites. Synapse 45:113–124, 2002.

Opiate‐evoked reward and motivated behaviors reflect, in part, the enhanced release of dopamine produced by activation of the μ‐opioid receptor (μOR) in the ventral tegmental area (VTA). We examined the functional sites for μOR activation and potential interactions with dopaminergic neurons within the rat VTA by using electron microscopy for the immunocytochemical localization of antipeptide antisera raised against μOR and tyrosine hydroxylase (TH), the synthesizing enzyme for catecholamines. The cellular and subcellular distribution of μOR was remarkably similar in the two major VTA subdivisions, the paranigral (VTApn) and parabrachial (VTApb) nuclei. In each region, somatodendritic profiles comprised over 50% of the labeled structures. μOR immunolabeling was often seen at extrasynaptic/perisynaptic sites on dendritic plasma membranes, and 10% of these dendrites contained TH. μOR‐immunoreactivity was also localized to plasma membranes of axon terminals and small unmyelinated axons, none of which contained TH. The μOR‐immunoreactive axon terminals formed either symmetric or asymmetric synapses that are typically associated with inhibitory and excitatory amino acid transmitters. Their targets included unlabeled (30%), μOR‐labeled (25%), and TH‐labeled (45%) dendrites. Our results suggest that μOR agonists in the VTA affect dopaminergic transmission mainly indirectly through changes in the postsynaptic responsivity and/or presynaptic release from neurons containing other neurotransmitters. They also indicate, however, that μOR agonists directly affect a small population of dopaminergic neurons expressing μOR on their dendrites in VTA and/or terminals in target regions. Synapse 41:311–328, 2001.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor‐associated NAc core. We examined the electron microscopic immunolabeling of the high‐affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR‐labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR‐immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR‐labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter‐specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR. J. Comp. Neurol. 435:142–155, 2001.