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Cellular Localization of Dopamine D2 Receptor Messenger RNA in the Rat Trigeminal Ganglion

Peterfreund, Robert A. MD, PhD; Kosofsky, Barry E. MD, PhD; Fink, J. Stephen MD, PhD

Regional Anesthesia and Pain Management

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

(Anesth Analg 1995;81:1181-5)

Departments of Anesthesia (Peterfreund) and Neurology, Massachusetts General Hospital, Harvard University Medical School, Boston, Massachusetts (Kosofsky, Fink).

This work was supported in part by a Research Starter Grant from the Foundation for Anesthesia Education and Research to RAP and National Institutes of Health Grants DA07496 and NS31579 to JSF. BEK was supported by a Nancy Lurie Marks Foundation Fellowship.

Presented in part at the American Society of Anesthesiologists Annual Meeting, 1991, Las Vegas, NV.

Accepted for publication June 28, 1995.

Address correspondence and reprint requests to Robert A. Peterfreund, MD, PhD, Department of Anesthesia, Clinics 3, Massachusetts General Hospital, Fruit St., Boston, MA 02114.

Dopamine is a neurotransmitter with multiple physiologic functions in the brain and periphery. The actions of dopamine on target tissues are mediated by high affinity, cell surface receptors of which five subtypes have been identified by combinations of pharmacologic and molecular methods [1]. All known members of the family of dopamine receptors belong to the superfamily of G protein-coupled receptors and exhibit the distinguishing structural feature of seven hydrophobic domains which span the cell membrane (transmembrane domains). Members of this receptor superfamily use the heterotrimeric G protein complex in their signal transduction pathway. The complementary DNA (cDNA) for the D2 subtype of dopamine receptor (D2R) was the first of the dopamine receptor family to be isolated and characterized by molecular methods [2]. The anatomic distribution of this receptor in the central nervous system of several species has been studied using the molecular methods of Northern blot analysis and in situ hybridization [3-7]. Particularly abundant expression of the receptor has been found in known projection fields of dopaminergic neurons, such as the basal ganglia, which is consistent with a postsynaptic localization of the D2R. However, detection of D2R messenger RNA (mRNA) in dopaminergic cells of the substantia nigra and ventral tegmental areas suggests the possibility of a regulatory role for dopamine autoreceptors in some cell populations [5,6].

One evaluation of the anatomic distribution of D2 dopamine receptors has detected specific D2R mRNA in the dorsal root and petrosal ganglia of the rat [8]. The abundance of D2R positive cells was greater than that of cells labeled with a probe for tyrosine hydroxylase, the rate-limiting enzyme in the pathway for dopamine biosynthesis. These findings imply that primary sensory cells express receptors for dopamine of the D2 subtype, but that these receptors do not function as autoreceptors. These data support a role for D2 receptor-mediated regulation of primary sensory neuron function.

The trigeminal ganglion contains the cell bodies of the primary sensory nerves of cranial nerve V. These pseudounipolar neurons transmit sensory information from the face, forehead, and dura to the brainstem and are thus involved in cranial pain syndromes such as headache and trigeminal neuralgia (tic douloureux). The trigeminal ganglion represents a cranial nerve analog of the dorsal root ganglion of the peripheral sensory system [9]. We therefore tested the hypothesis that the cells of the trigeminal ganglion, like those of the dorsal root (and petrosal) ganglia, express D2 dopamine receptors.

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Northern Blot Analysis

With approval from the hospital animal studies committee, adult male rats (200-250 g) were decapitated rapidly and tissues were excised and immediately frozen in liquid nitrogen. The trigeminal ganglion was identified after removing the brain to expose the base of the skull. The overlying dura and membranes were peeled away and the projecting tracts were divided approximate 2 mm from the ganglion. The ganglion was removed with care to avoid contamination from nearby pituitary tissue.

To isolate total cellular RNA, the frozen tissues were homogenized in 10-15 vol of guanidine isothiocyanate solution and centrifuged through a cesium chloride cushion to remove contaminating DNA and protein. After phenol extraction and ethanol precipitation, the RNA was resuspended in a small volume of diethylpyrocarbonate-treated water and quantified by UV absorbance. The RNA was size fractionated on 1% denaturing agarose gels in 0.02 M 3[N-morpholino] propane sulfonic acid and transferred to nylon membranes (Genescreen Registered Trademark; Dupont NEN, Boston, MA) by electroblotting. RNA was fixed to the membrane by UV illumination with a Stratolinker Registered Trademark (Stratagene, La Jolla, CA). Blots were prehybridized in a solution of 50% (vol/vol) formamide, 1% (vol/vol) sodium lauryl sulfate, 1 M NaCl with 10% (wt/vol) dextran and 10 times Denhardt's solution buffered with Tris-HCl and supplemented with 100-200 micro gram/mL boiled, sheared salmon sperm DNA at 42 degrees C for 1-6 h. This was followed by hybridization in the same solution with 0.5 times 106 cpm/mL of boiled32 P-radiolabeled cDNA probe for 12-16 h at 42 degrees C. Blots were washed twice at room temperature in 2 times SSC for 5 min (where 1 times SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) followed by two washes (30 min each) at 65 degrees C in 2 times SSC/1% sodium lauryl sulfate. The final washes were at room temperature for 30 min each in 0.1 times SSC. The membranes were exposed to Kodak XAR Trademark (Eastman Kodak, Rochester, NY) film for 1-7 days with one intensifying screen at -80 degrees C. Results are representative of data from three separately prepared Northern blots hybridized independently with D2R probes corresponding to different regions of the D2R cDNA. For reprobing, blots were stripped of probe by heating in 10 mM Tris-HCl (pH 7.5)/1 mM EDTA at >80 degrees C for 30 min followed by autoradiography to ensure complete removal of probe.

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In Situ Hybridization Analysis

The procedure was performed according to the protocol of LeMoine and Young [10]. Excised tissues were immediately placed in isopentane (-30 degrees C to -40 degrees C) and then stored at -80 degrees C. Sections (10 mu) were obtained with a cryostat at -17 degrees C and the sections were mounted on gelatin-coated slides. The sections were fixed in 4% paraformaldehyde, rinsed, acetylated, and then dehydrated and delipidated in a graded series of ethanol solutions and chloroform. Prehybridization and hybridization were performed in humidified chambers at 52-55 degrees C overnight in formamide-containing buffers with approximate 0.5 times 10 (6-1) times 106 cpm of complementary RNA (cRNA) probe per coverslipped slide. Slides were washed in 2 times SSC, "digested" in RNAase A to remove excess probe and washed again to a stringency of 0.2 times SSC at 60 degrees C for 1 h. After dehydration in graded solutions of alcohol, the sections were dipped in emulsion and exposed for 1-3 wk. Sections were then counterstained with cresyl violet or methylene blue. Results are reported from ganglia isolated from two different animals processed during three separate hybridization experiments.

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Preparation of Radiolabeled Probes

cDNA Probes. With first strand rat striatal cDNA as a template and specific rat D2R primers [2], the polymerase chain reaction (PCR) was used to amplify fragments of both the long and short forms of the D2 dopamine receptor (J. S. Fink, unpublished observations). These D2R fragments were subcloned into plasmid pGem3z (Promega, Madison, WI) and the fidelity of PCR amplification confirmed by DNA sequencing. A cDNA probe was derived by digesting one of the plasmids with Xba I (site inserted by the PCR primer) and Bal I (native site) and gel isolating the resultant products. The final fragment was 296 bp in length and includes 46 bp of 5 prime untranslated region and 250 bp of coding region which spans transmembrane domain 1 through one half of transmembrane domain 2. The probe sequence was used to search the Genbank data base [11] for homologous genes. The highest scoring matches had less than 70% nucleic acid sequence identity over the 3 prime half of the probe sequence with much lower degrees of homology in the 5 prime portion of the probe sequence.

A second cDNA probe was derived by digesting a rat cDNA (generously provided by Dr. O. Civelli) with Xma I to produce a fragment corresponding to 91 bp of the D2R predicted 5 prime untranslated region. The structure of this fragment was confirmed by sequencing the fragment after subcloning it into plasmid Bluescript II (Stratagene). This sequence had no significant homologs in the Genbank database.

cDNA probes were labeled with32 P-deoxycytosine triphosphate by the random prime method using the Klenow fragment of DNA polymerase. The probe was purified over a Sephadex column. Typical specific activity was 0.5-1 times 109 cpm/micro gram.

cRNA Probes. Fragments of the long or short forms of the rat D2 dopamine receptor cloned into pGem3z were used as templates for cRNA synthesis. For sense (control probes) cRNA synthesis, the short form of the D2R was linearized with Sac I. cRNA radiolabeled with35 S-cytosine triphosphate was synthesized with SP6 RNA polymerase to generate a predicted probe of 780 bases which spans 46 bases of predicted 5 prime untranslated region with the remainder in the coding region. For antisense cRNA probe synthesis, plasmids containing fragments of either the long or short forms of the D2 receptor were linearized at unique sites in the polylinker or the coding sequence and cRNA was synthesized with T7 RNA polymerase. The predicted probes spanned transmembrane domains 1 through 5 and were 881 bp or 736 bp in length, depending on the presence of the 87 bp (exon 5) which characterizes the long splice form [1] and the restriction site chosen for linearization. Nucleic acid sequence homology to the D4 dopamine receptor isoform was less than 50% for the region and less than 70% for the D3 dopamine receptor isoform. After synthesis, the DNA template was digested with RQ1 DNAase (Promega) and the probe was purified over a column (Stratagene).

Materials. Restriction endonucleases and polymerases were obtained from Gibco-BRL (Grand Island, NY) or Promega. Radioisotopes were obtained from Dupont-NEN. All other reagents were molecular biology grade.

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To test the hypothesis that the trigeminal ganglion expressed dopamine D2 receptor RNA, total cellular RNA from trigeminal ganglia and several brain regions were analyzed by Northern blot. A cDNA probe containing a portion of the predicted 5 prime coding sequence of the D2R hybridized to RNA from pituitary and striatum consistent with the previously reported presence of abundant D2R specific message in these tissues [4]. Hybridization signals of appropriate size, but weaker intensity, were also detected with RNA from the trigeminal ganglion and hypothalmus, but no D2R signal was detected for spleen, lung, pancreas, or liver Figure 1A.

Figure 1

Figure 1

For confirmation of these results, a second cDNA hybridization probe was prepared from the predicted 5 prime untranslated portion of the receptor. This highly specific probe also detected an abundantly hybridizing RNA species in pituitary and striatal RNA, with an appropriate sized, weaker signal in trigeminal ganglia RNA. Again, no signal was detected for several peripheral tissues Figure 1B.

To determine the distribution of cells expressing D2R specific mRNA in the trigeminal ganglion, tissues sections were analyzed by in situ hybridization. In brightfield images, the hybridization signal (arrows) was observed overlying clusters of cells in discrete groups throughout the periphery of the ganglion (Figure 2B, arrows). The cells had large, pale cytoplasms consistent with their identification as the perikarya of neurons. Darkfield images demonstrated grains of exposed emulsion which overlay the cytoplasm of labeled cells. The cells within each cluster appeared to align in a spoke-like fashion at the periphery of the ganglion (Figure 2A, arrows). In contrast to the trigeminal ganglion, the hybridization signal produced by the antisense cRNA probe in coronal brain sections through the striatum was more uniform Figure 3A. In coronal brain sections, the region of hybridization was sharply demarcated, with abundant signal over the striatum and minimal signal over surrounding corpus callosum (Figure 3A, arrows). No specific cellular hybridization signal was detected when coronal sections were hybridized to sense cRNA probes Figure 3B.

Figure 2

Figure 2

Figure 3

Figure 3

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Sensory information, including proprioception, pain and temperature, pressure, touch, and two-point discrimination is carried by neurons in one of the three periperhal branches of cranial nerve V. The dura, sinuses, cerebral vasculature, corneas, face, teeth, and mouth receive cranial nerve V sensory innervation. Thus the sensory neurons of the trigeminal ganglion mediate transmission of information relevant to cranial pain, including headache and oral and facial pain.

With the exception of afferent fibers for proprioception and stretch, the cell bodies of the sensory neurons reside in the trigeminal ganglion and have central projections to the brainstem sensory nuclei of cranial nerve V [12]. Although branches of the trigeminal nerve carry the motor fibers for mastication, the cell bodies of these neurons lie in the motor nucleus of cranial nerve V in the pons. These motor fibers traverse the trigeminal ganglion on their route to their peripheral targets. In the periphery, autonomic fibers join the motor and sensory projections. Thus the neurons whose cell bodies reside in the trigeminal ganglion appear to be exclusively sensory neurons [12].

The present findings indicate that subpopulations of trigeminal ganglion cells express the mRNA for the D2 dopamine receptor. These cells are likely to be sensory neurons. It is unlikely that the hybridization data represent an artifact of cross-hybridization between sequences of the closely related family of the G protein-coupled receptors. The coding sequence probe used for Northern blot analysis had only approximate 60% sequence identity to other receptors in its 3 prime end. The Northern blot probe derived from the 5 prime untranslated region of the receptor, where sequence is less conserved within the G protein-linked receptor family, is unlikely to cross-hybridize. Furthermore, the hybridizations and washes were performed under high stringency conditions to reduce the possibility of artifact. Although the cRNA probes used for in situ analyses span a portion of the coding sequence, and potentially could exhibit cross-hybridization to the mRNAs for other receptor species, the probe sequence is less than 70% identical to the closest related receptors. As with the Northern blot analyses, the hybridization and washes were performed under stringent conditions to reduce the possibility of cross-hybridization. We also demonstrated that the probe used for in situ hybridization to the trigeminal ganglion identifies the well described distribution of the D2R mRNA in the striatum [3-7], supporting the specificity of D2 mRNA detection in the trigeminal ganglion. We thus conclude that the anatomic data are consistent with the selective identification of D2R mRNA in the sections from the trigeminal ganglion. The present findings at the level of mRNA expression will need to be correlated with immunohistochemical or receptor binding studies to define D2R protein expression. D2R protein could be expressed on the cell bodies in the ganglion as well as on the central or peripheral projections of the trigeminal ganglion sensory neurons.

The anatomic findings support the possibility that the function of subsets of sensory neurons can be influenced by dopamine or drugs active at D2 receptors. Two classes of drugs, neuroleptics and antidepressants, potentially modulate dopamine receptor function; the neuroleptics act as receptor antagonists whereas the antidepressants potentiate catecholamine action at the synapse. Both neuroleptics [13] and tricyclic antidepressants have a therapeutic role as second-line therapy for refractory headache [14] and neuroleptic drugs such as droperidol have a role as a component of balanced anesthetic techniques in humans [15] and animals [16]. Dopamine also decreases the minimum alveolar anesthetic concentration for halothane in mice, and these effects are sensitive to selective D2R antagonism [17]. The relative role of activation and antagonism of D2Rs in analgesia and anesthesia, and the precise site of action of dopaminergic drugs for these effects, remain to be defined. D2 dopamine receptors expressed on sensory neurons may mediate some of the clinically observed responses to such drugs. Our results demonstrate an anatomic substrate for dopamine or drugs active at D2 dopamine receptors to influence sensory transmission from the face and head.

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