Ultrastructural evidence for prominent postsynaptic localization of alpha2C-adrenergic receptors in catecholaminergic dendrites in the rat nucleus locus coeruleus.

Alpha-2-adrenergic receptor (alpha2-AR) agonists potently inhibit the activity of noradrenergic neurons of the locus coeruleus (LC), an effect that may be mediated by the A- and/ or C-subtypes of alpha2-AR (alpha2A- and alpha2C-AR). To gain insight into the functional significance of these alpha2-AR subtypes in the LC, we have examined their ultrastructural localization by using subtype-specific antibodies. We recently demonstrated that alpha2A-ARs are localized prominently in axon terminals and catecholaminergic dendrites in the LC. In the present study, we sought to identify the subcellular substrates underlying alpha2C-AR actions in the LC by analyzing the ultrastructural distribution of alpha2C-AR immunoreactivity (alpha2C-AR-IR) in sections that were dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Alpha-2C-AR-IR was predominantly localized in dendrites, most of which also contained immunolabeling for TH. Within such dendrites, alpha2C-AR-IR was associated with the plasma membrane and occasionally Golgi cisternae and tubulovesicles. The vast majority of dendrites containing alpha2C-AR-IR received asymmetric (excitatory) contacts from unlabeled axon terminals that often contained dense core vesicles. Alpha-2C-AR-IR was observed in some unmyelinated axons and astrocytic processes that were apposed to TH-immunoreactive dendrites but was rarely associated with axon terminals. These results provide the first ultrastructural evidence that alpha2C-ARs (1) are localized postsynaptically in catecholaminergic neurons of the LC and (2) may be strategically situated to modulate the activation of LC neurons by excitatory inputs.

Hippocampal alpha2a-adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes.

Alpha-adrenergic receptor, subtype 2A (alpha2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, alpha2A-AR immunoreactivity (alpha2A-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 alpha2A-AR. By light microscopy, dense alpha2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular alpha2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, alpha2A-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 alpha2A-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): alpha2A-AR-I was found in axons and terminals (approximately 40%), glia (approximately 30%), dendritic spines (approximately 25%), and dendritic shafts (approximately 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 alpha2A-AR-I was found in axons (approximately 60%) and glia (approximately 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 alpha2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to alpha2A-AR-labeled spines and glia. These studies demonstrate that hippocampal alpha2A-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 alpha2A-ARs.

Ultrastructural localization of adenosine A2A receptors suggests multiple cellular sites for modulation of GABAergic neurons in rat striatum.

Activation of adenosine A2A receptors (A2AR) has been shown to antagonize the function of D2 dopaminergic regulation of striatal gamma-aminobutyric acid (GABA)-ergic output and, thus, locomotor activity. Adenosine A2A receptor immunoreactivity (A2A-LI) has been localized to rat striatum by light microscopy by using a previously characterized human A2AR monoclonal antibody. In this study, we evaluated the localization of A2A-LI and its colocalization with GABA immunoreactivity (GABA-LI) in dorsolateral rat striatum by immunoelectron microscopy to further characterize the potential mechanism of purinergic control of striatal output. Ultrastructural analysis demonstrated A2A-LI associated with the plasma membrane and cytoplasmic membranous structures of striatal neurons. A2A-LI was prevalent in dendrites and dendritic spines ( approximately 70% of total A2A-profiles counted) and less prevalent in axons and axon terminals (23%), soma (3%), and glia (3%). Cellular elements exhibiting both A2A-LI and GABA-LI comprised 23% of the total profiles counted; colabeling was most common in dendrites. A2A-LI was observed primarily at asymmetric synapses (n = 70) (both pre- and postsynaptically but predominantly in the postsynaptic element) and less frequently at symmetric synapses (n = 17). Of the 714 A2A-immunoreactive profiles examined, 37% were apposed to GABA-labeled profiles. The most common appositions were A2A-labeled dendrites apposed to GABA-immunoreactive dendrites (n = 132), axon terminals (n = 28), and somata (n = 22) and A2A-labeled axons apposed to GABA-labeled dendrites (n = 58), axon terminals (n = 14), and somata (n = 9). Our findings suggest that adenosine may play an important role in modulating excitatory input to striatal neurons and that A2AR may modulate GABAergic signaling at several cellular sites within the rat striatum.

Neurokinin-1 receptor-immunoreactive neurons of the ventral respiratory group in the rat.

The rostral end of the ventral respiratory group (VRG) contains neurons that are intensely neurokinin-1 receptor (NK1R) immunoreactive (ir). It has been theorized that some of these cells might be critical to respiratory rhythmogenesis (Gray et al. [1999] Science 286:1566-1568). In the present study we determined what major transmitter these NK1R-ir cells make and whether they are bulbospinal or propriomedullary. NK1R-ir neurons were found in the VRG between Bregma levels -11.7 and -13.6 mm. The highest concentration was found between Bregma -12.3 and -13.0 mm. This region overlaps with the pre-Bötzinger complex (pre-BötC) as it was found to contain many pre-inspiratory neurons, few E2-expiratory neurons, and no I-incremental neurons. VRG NK1R-ir neurons contain neither tyrosine hydroxylase (TH) nor choline acetyl-transferase (ChAT) immunoreactivity, although dual-labeled neurons were found elsewhere within the rostral medulla. GAD67 mRNA was commonly detected in the ventrolateral medulla (VLM) but rarely in the NK1R-ir neurons of the pre-BötC region (6 % of somatic profiles). GlyT2 mRNA was commonly found in the pre-BötC region but rarely within NK1R-ir neurons (1.3 %). Up to 40% of VRG NK1R-ir neurons were retrogradely labeled by Fluoro-Gold (FG) injected in the contralateral pre-BötC region. Some NK1R-ir VRG neurons located caudal to Bregma -12.6 mm were retrogradely labeled by FG injected in the spinal cord (C4-C5, T2-T4). In sum, NK1R immunoreactivity is present in many types of ventral medullary neurons. Within the VRG proper, NK1R-ir neurons are concentrated in an area that overlaps with the pre-BötC. Within this limited region of the VRG, NK1R-ir neurons are neither cholinergic nor catecholaminergic, and very few are gamma-aminobutyric acid (GABA)ergic or glycinergic. The data suggest that most NK1R-ir neurons of the pre-BötC region are excitatory. Furthermore, the more rostral NK1R-ir cells are propriomedullary, whereas some of the caudal ones project to the spinal cord.

Distribution of alpha 2A-adrenergic receptor-like immunoreactivity in the rat central nervous system.

In this study, we analyzed immunohistochemically the distribution of the A subtype of alpha 2-adrenergic receptor (alpha 2A-AR) in the rat central nervous system using light level immunohistochemistry. By using affinity-purified antisera, we found perikaryal labeling was diffuse and/or punctate; immunoreactive puncta were heterogeneous in size and number in a region-specific manner. Dense deposits of immunoreaction product were found associated with neuropil also, particularly in the lateral parabrachial nucleus, locus coeruleus, lateral septum, diagonal band, stratum lacunosum-moleculare of CA1, and various nuclei of the amygdala and extended amygdala. Prominently immunoreactive olfactory structures include the anterior olfactory nucleus and the granular layer of the olfactory bulb. The cortex was generally light to moderately labeled with greater immunoreactivity in the cingulate and insular cortices. alpha 2A-AR-like immunoreactivity was intense in the basal forebrain and continuous from the nucleus accumbens through the substantia innominata and fundus of the striatum. Most immunoreactivity in the diencephalon was restricted to the hypothalamus with light to moderate labeling in the thalamus. Generally light immunoreactivity was observed in midbrain structures. In the pons and medulla, both perikaryal and neuropil labeling were observed. Together with the accompanying paper describing the neural distribution of alpha 2C-AR-like immunoreactivity, our results provide an extensive immunohistochemical cartography of alpha 2-ARs in the adult rat central nervous system.

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system.

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Chronic imipramine administration alters the activity and phosphorylation state of tyrosine hydroxylase in dopaminergic regions of rat brain.

In the present study the influence of imipramine, a tricyclic antidepressant, on the expression and function of tyrosine hydroxylase (TH) in dopaminergic rat brain regions was examined. Chronic administration of imipramine (18 days) decreased levels of TH enzyme activity in ventral tegmental area (VTA) and substantia nigra (SN), dopaminergic cell body regions, as well as in caudate-putamen (CP), nucleus accumbens (ACB), prefrontal cortex (PFC), and olfactory tubercle (OT), dopaminergic terminal fields. These effects were dependent on chronic drug treatment, as imipramine administration for 1 or 7 days did not significantly influence levels of TH activity in either SN or VTA. In contrast to drug regulation of enzyme activity, chronic imipramine treatment did not decrease levels of TH immunoreactivity in any of the dopaminergic cell body or terminal field regions studied, although levels of TH immunoreactivity were decreased in locus coeruleus (LC) as previously reported. However, imipramine treatment increased levels of TH back phosphorylation in VTA, suggesting that the antidepressant-induced decrease in levels of TH activity is a result of decreased phosphorylation of the enzyme. These results demonstrate that imipramine treatment regulates levels of TH enzyme activity in dopaminergic brain regions, and may account for some of the previously observed effects of these drugs on dopaminergic function. Finally, imipramine regulation of TH enzyme activity in VTA and immunoreactivity in LC was observed in Sprague Dawley, but not Wistar rats, demonstrating that different rat strains exhibit different biochemical responses to antidepressant treatment.

Localization of alpha2C-adrenergic receptor immunoreactivity in catecholaminergic neurons in the rat central nervous system.

Given the importance of alpha2-adrenergic receptors in the regulation of catecholaminergic transmission, we analysed the distribution of immunoreactivity corresponding to the C-subtype of alpha2-adrenergic receptor in central catecholaminergic neurons using double-label immunohistochemistry with antibodies directed against alpha2C-adrenergic receptors and tyrosine hydroxylase. Cells exhibiting both alpha2C-adrenergic receptor and tyrosine hydroxylase immunoreactivity were found in most areas containing catecholaminergic cell groups. However, the percentage of double-labelled cells varied in a region-specific manner. In the medulla, alpha2C-adrenergic receptor immunoreactivity was characteristic of only a minority of cells exhibiting tyrosine hydroxylase immunoreactivity (40-43% in area A1/C1, 27-36% in area A2/C2, 35% in area C3) while a larger percentage of double-labelled cells was observed in the pons (65% in A5, 92% in locus coeruleus, 68% in A7). In the midbrain, alpha2C-adrenergic receptor immunoreactivity was detected in most tyrosine hydroxylase-immunoreactive cells in dopaminergic regions (63% in the retrorubral field, 77-83% in substantia nigra, 67% in ventral tegmental area). These results suggest that alpha2C-adrenergic receptors may act as autoreceptors on some central adrenergic and noradrenergic neurons. In addition, the colocalization of alpha2C-adrenergic receptor and tyrosine hydroxylase immunoreactivity in dopaminergic cell groups suggests that reported effects of alpha2-adrenergic receptor agonists in these areas may be mediated by the C-subtype.

Effects of 6-hydroxydopamine lesions of the prefrontal cortex on tyrosine hydroxylase activity in mesolimbic and nigrostriatal dopamine systems.

The effects of prefrontal cortical dopamine depletion on subcortical dopamine function in the rat were examined. 6-Hydroxydopamine lesions of the dopaminergic innervation of the prefrontal cortex did not alter concentrations of dopamine or its metabolite 3,4-dihydroxyphenylacetic acid in either the striatum or nucleus accumbens. Similarly, the activity of the catecholamine biosynthetic enzyme tyrosine hydroxylase in the striatal complex was not changed in animals with prefrontal cortical lesions. Animals sustaining neurotoxic lesions of the prefrontal cortex were challenged with haloperidol in order to activate submaximally tyrosine hydroxylase activity. The magnitude of the haloperidol-induced increase in enzyme activity in the nucleus accumbens was significantly greater in lesioned subjects than in control animals. These data suggest that lesions of the prefrontal cortical dopamine innervation do not result in significant alterations in basal dopaminergic function in the striatal complex. However, lesions of the dopaminergic innervation of the prefrontal cortex significantly increase the responsiveness of mesolimbic dopamine afferents to pharmacological challenge.

Immunohistochemical localization of alpha 2A-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat.

alpha 2-Adrenergic receptors mediate a large portion of the known inhibitory effects of catecholamines on central and peripheral neurons. Molecular cloning studies have established the identity of three alpha 2-adrenergic receptor genes from several species that encode the A, B and C subtypes of the receptor. The rat alpha 2A-adrenergic receptor, as defined by sequence similarity, is the orthologue of the human alpha 2A-adrenergic receptor. In this paper, we report the development of rabbit antisera directed against a portion of the third intracellular loop of the rat alpha 2A-adrenergic receptor and the histochemical localization of alpha 2A-adrenergic receptor-like immunoreactive material in the brainstem and spinal cord of the adult rat. Our antisera detected alpha 2A-adrenergic receptor-specific punctate staining associated with neuronal perikarya. alpha 2A-adrenergic receptor-like immunoreactivity was widely, but heterogeneously, distributed in the brainstem and spinal cord, predominantly in areas involved in the control of autonomic function. Double labelling with antisera to tyrosine hydroxylase or phenylethanolamine-N-methyl-transferase revealed that alpha 2A-adrenergic receptor-like immunoreactivity is present in most, perhaps all, noradrenergic and adrenergic cells of the brainstem. alpha 2A-Adrenergic receptor-like immunoreactivity was detected in a small percentage of the dopaminergic cells of the A9 and A10 groups. This study provides the first description of the specific immunohistochemical localization of alpha 2A-adrenergic receptors using a subtype-specific polyclonal antibody. The results support the view that alpha 2-adrenergic receptors are involved in central cardiovascular control and suggest that the catecholaminergic autoreceptors of central noradrenergic and adrenergic neurons are the A subtype of the alpha 2-adrenergic receptors.

Differential expression of a(2a), A(1)-adenosine and D(2)-dopamine receptor genes in rat peripheral arterial chemoreceptors during postnatal development.

The sensitivity of peripheral arterial chemoreceptors in the carotid body to hypoxia increases with postnatal maturation. Carotid sinus nerve activity is augmented by adenosine binding to A(2a)-adenosine receptors and attenuated by dopamine binding to D(2)-dopamine receptors. In this study, we used in situ hybridization histochemistry to determine the change in the levels of mRNA expression for A(2a) and A(1)-adenosine receptors and D(2)-dopamine receptors in the rat carotid body. We also investigated the cellular distribution and possible colocalization of these receptor mRNAs and tyrosine hydroxylase (TH) mRNAs during the first 2 weeks of postnatal development. By using immunohistocytochemistry, we detected A(2a)-adenosine receptor protein in the carotid body and petrosal ganglion. We found that A(2a)-adenosine receptor mRNA and protein are expressed in the carotid body in animals at 0, 3, 6 and 14 postnatal days. The level of A(2a)-adenosine receptor mRNA expression significantly decreased by 14 postnatal days (P<0.02 vs. day 0) while D(2)-dopamine receptor mRNA levels significantly increased by day 3 and remained greater than D(2)-dopamine receptor mRNA levels at day 0 (P<0.001 all ages vs. day 0). TH mRNA was colocalized in cells in the carotid body with A(2a) adenosine receptor and D(2)-dopamine receptor mRNAs. A(1)-adenosine receptor mRNA was not expressed in the carotid body at any of the ages examined. In the petrosal ganglion, A(1)-adenosine receptor mRNA was abundantly expressed in numerous cells, A(2a)-adenosine receptor mRNA was expressed in a moderate number of cells while D(2)-dopamine receptor mRNA was seen in a few cells in the rostral petrosal ganglion. In conclusion, using in situ hybridization histochemistry, we have shown that mRNA for both the excitatory, A(2a)-adenosine receptor, and the inhibitory, D(2)-dopamine receptor, is developmentally regulated in presumably type I cells in the carotid body which may contribute to the maturation of hypoxic chemosensitivity. Furthermore, the presence A(1)-adenosine receptor mRNAs in cell bodies of the petrosal ganglion suggests that adenosine might also have an inhibitory role in hypoxic chemotransmission.

Alpha2A-adrenergic receptors are primarily presynaptic heteroreceptors in the C1 area of the rat rostral ventrolateral medulla.

The 2A subtype of the alpha-adrenergic receptor (alpha2A-AR) is necessary for the hypotensive effects of clonidine and other sympathoinhibitory adrenergic agonists. This hypotensive response appears to be due to the inhibition of sympathoexcitatory reticulospinal neurons found in the rostral ventrolateral medulla (RVL), including neurons of the C1 adrenergic cell group. The cellular mechanisms underlying this inhibition have not been established. Thus, this study examined the ultrastructural relationships between profiles containing alpha2AAR-immunoreactivity (alpha2AAR-I) and those containing the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) to determine potential cellular substrates for alpha2A-AR inhibition of C1 neuron activity. Consistent with previous light microscopic studies, alpha2AAR-I was found in perikarya and large dendrites and the majority of these profiles also contained TH-labeling (approximately 70% of 140). However, alpha2AAR-I in these cells was primarily found within endosomes and Golgi complexes and in clusters associated with the endoplasmic reticula, probable sites for synthesis and/or trafficking of receptors. In contrast, most of the alpha2AAR-I profiles (n=646) in the RVL were axons and axon terminals (approximately 68%) which lacked TH immunoreactivity. alpha2AAR-labeled axons were small and unmyelinated and labeled terminals usually formed symmetric synapses on the shafts of catecholaminergic or unlabeled dendrites. Most of these alpha2AAR-labeled axons were found in close proximity to TH-labeled profiles and approximately one-fifth (17% of 408) of the alpha2AAR-labeled axons and axon terminals directly contacted TH-labeled profiles, mostly dendrites. These studies suggest that alpha2AARs in the C1 area of the RVL function primarily as heteroreceptors on presynaptic axons and terminals of non-catecholaminergic cells, some of which provide inhibitory synaptic input to C1 neurons. These receptors may be activated by catecholamines released either from the dendrites of C1 neurons or from the terminals of other catecholaminergic neurons via volume transmission.

alpha2A-adrenergic receptors in the rat nucleus locus coeruleus: subcellular localization in catecholaminergic dendrites, astrocytes, and presynaptic axon terminals.

To define the anatomic substrates subserving the inhibitory actions of alpha2-adrenergic receptors (alpha2-ARs) in the locus coeruleus (LC), we used dual-label immunoelectron microscopy with antibodies directed against the A-subtype of alpha2-AR (alpha2A-AR) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Of the profiles containing peroxidase labeling for alpha2A-AR (alpha2A-AR-IR) in the LC (n=735), most were dendrites ( approximately 50%), glial processes ( approximately 30%), and axon terminals ( approximately 15%). alpha2A-AR-IR was also observed in unmyelinated axons and perikarya. Within dendrites, alpha2A-AR-IR was associated with nonsynaptic regions of the plasma membrane and subsurface cisternae. Approximately 60% of dendrites with alpha2A-AR-IR were dually labeled for TH. Fifty percent of the axon terminals contacting alpha2A-AR-immunoreactive dendrites formed asymmetric (excitatory) synaptic contacts. Axon terminals with alpha2A-AR-IR were not dually labeled for TH and generally formed asymmetric synapses with TH-immunoreactive dendrites that contained or lacked alpha2A-AR-IR. Astrocytic processes exhibiting alpha2A-AR-IR were closely apposed to TH-labeled dendrites. These results extend previous ultrastructural observations of alpha2A-ARs in the LC and suggest that the inhibitory actions of norepinephrine and epinephrine in this region may be mediated by postsynaptic alpha2A-ARs on catecholaminergic dendrites and presynaptic alpha2A-ARs on excitatory inputs to catecholaminergic dendrites. In addition, the localization of alpha2A-AR-IR in astrocytic processes apposed to TH-immunoreactive dendrites suggests a role for alpha2A-ARs in functional interactions between catecholaminergic dendrites and neighboring astrocytes.

Alpha 2A-adrenergic receptors are present in lower brainstem catecholaminergic and serotonergic neurons innervating spinal cord.

A subtype-specific polyclonal antibody was used for the immunohistochemical detection of alpha 2A-adrenergic receptors (alpha 2A-ARs) in the rat lower brainstem (medulla and pons). Using dual-label fluorescence histochemistry, punctate alpha 2A-AR-like immunoreactivity (alpha 2A-AR-LIR) was identified in noradrenergic, adrenergic, and serotonergic neurons of the pontomedullary region. Confocal microscopic examination of material simultaneously labeled for TH-LIR and alpha 2A-LIR revealed that the clusters of alpha 2A-LIR were located intracellularly. Lower medullary neurons with spinal projections to segment T3 were retrogradely labeled using FITC-conjugated microbeads and the material was processed for simultaneous detection of alpha 2A-LIR and either TH-LIR or 5-HT-LIR. Using this triple-label approach, we found that virtually all medullary serotonergic cells (raphe pallidus, raphe obscurus and parapyramidal area) including those with identified spinal projections contain punctate alpha 2A-AR-LIR. In contrast, fewer than 10% of dorsal raphe serotonergic cells examined for comparison were immunoreactive. The triple labeling approach also indicated that more than 95% of the TH-immunoreactive cells of the dorsal and ventrolateral medulla, including those with demonstrable spinal projections (A5 noradrenergic and C1/C3 adrenergic) had detectable amounts of alpha 2A-AR-LIR. The presence of alpha 2A-ARs in a large fraction of bulbospinal pre-sympathetic neurons (noradrenergic A5, adrenergic C1 and C3 and serotonergic raphe cells) could explain the powerful and relatively selective effect of clonidine and other centrally acting alpha 2A-AR agonists on sympathetic efferent activity and hypertension.

Neurochemical and behavioral effects of polychlorinated biphenyls in mice.

The effects of polychlorinated biphenyls (PCBs) were assessed on some behavioral and neurochemical parameters in mice. Acute administration of a dose of 500 mg/kg (po) depressed spontaneous motor activity for a period of 15 min to 3 hr. Subchronic dosing of 30 or 100 mg/kg for 14 days was virtually without effect. PCBs given acutely had no effect on motor coordination nor on pentylenetetrazol-induced convulsions. Exposure of isolated mouse brain synaptosomes to PCBs produced a dose-dependent inhibition of neurotransmitter and precursor uptake (IC50 = 10(-5) to 10(-4) M) as well as stimulation of neurotransmitter release (EC50 = 10(-5) to 10(-4) M). Mitochondrial 45Ca2+ uptake was also enhanced (EC50 = 2.8 x 10(-4) M). Disposition of 14C-PCBs in mitochondria and synaptosomes following an oral dose of 500 mg/kg yielded levels comparable to those in isolated mitochondria and synaptosomes incubated with 14C-PCBs at concentrations that altered central neurotransmitter function in vitro.

Comparison of the effects of acute and subchronic administration of Aroclor 1254, a commercial mixture of polychlorinated biphenyls, on pentobarbital-induced sleep time and [14C]pentobarbital disposition in mice.

We have reported previously that polychlorinated biphenyls (PCBs) alter neurochemistry and suppress spontaneous locomotor activity in mice. The present study was initiated to determine whether orally administered (Aroclor 1254) would potentiate pentobarbital-induced sleep time. Sleep time was enhanced significantly by Aroclor 1254 (500 mg/kg) given 0 to 8 h prior to pentobarbital, with the peak effect occurring at 2 h. This effect was demonstrated to be dose-responsive in the range of 5 to 25 mg/kg given 2 h prior to pentobarbital, but only slightly larger increments in sleep time were observed with higher doses of PCBs (50, 100, 250, and 500 mg/kg). Administration of vehicle or Aroclor 1254 (30 or 100 mg/kg) for 14 successive days reduced sleep time when pentobarbital was given 45 min after the last dose of vehicle or Aroclor 1254, with a further reduction when pentobarbital was given 24 h after the last dose. As a correlate to the sleep-time studies, levels of pentobarbital and metabolites were measured in brain, liver, and plasma of mice that had received varying doses of Aroclor 1254 2 h prior to [14C]pentobarbital. Elevated levels of pentobarbital and decreased levels of metabolites were found after acute administration of Aroclor 1254 during a period of time when Aroclor 1254-treated mice were still asleep. These effects of Aroclor 1254 on pentobarbital disposition were found to be dose-dependent. Brain levels of pentobarbital in mice after 14 d of Aroclor 1254 treatment (30 mg/kg) were less than those in vehicle-treated animals, and these levels were consistent with the reduced sleep times. Thus, a correlation between pentobarbital brain levels and sleep time in both Aroclor 1254-treated and nontreated animals suggests that Aroclor 1254 does not alter pentobarbital narcosis by a direct action on the brain. Rather, acutely administered Aroclor 1254 may be augmenting sleep time by competing with pentobarbital for metabolic sites in the liver, while chronically administered Aroclor 1254 induces pentobarbital metabolism.

Alpha-adrenergic receptor (alpha(2 A )) is colocalized in basal forebrain cholinergic neurons: a light and electron microscopic double immunolabeling study.

A variety of data suggest that noradrenaline and acetylcholine may interact in the basal forebrain, however no morphological studies have addressed whether indeed cholinergic neurons express adrenergic receptors. We have investigated the presence of alpha-adrenergic receptor subtype alpha2A-AR in cholinergic neurons of the basal forebrain. Cholinergic neurons were identified with an antibody against choline acetyltransferase and the receptor with a polyclonal antibody raised against a 47 amino acid fragment of the third intracellular loop of the alpha2A-AR. For double labeling at the light microscopic level the Ni-DAB/DAB technique was used, and for electron microscopy an immunoperoxidase/immunogold method was applied. We detected the alpha2A-AR protein in cholinergic as well as in non-cholinergic neurons. Almost half of all cholinergic neurons contained this adrenergic receptor. Double-labeled neurons were distributed throughout the rostro-caudal extent of the basal forebrain cholinergic continuum, including the medial septum, vertical and horizontal diagonal band nuclei, pallidal regions, substantia innominata and the internal capsule. Non-cholinergic neurons that expressed the alpha2A-AR outnumbered cholinergic/alpha2A-AR neurons by several factors. Electron microscopy confirmed the presence of alpha2A-AR in cholinergic neurons in the medial septum, vertical and horizontal diagonal band nuclei. Gold particles (10 nm) indicative of alpha2A-AR were diffusely distributed in the cytoplasm and accumulated in cytoplasmic areas near the Golgi complex and cysterns of the endoplasmic reticulum and were associated with the cellular membranes at synaptic and non-synaptic locations. Since many of the alpha2A-AR+/non-cholinergic neurons we detected are likely to be GABAergic cells, our data support the hypothesis that noradrenaline may act via basal forebrain cholinergic and non-cholinergic neurons to influence cortical activity.