To determine the pattern of painless mRNA expression, whole-mount in situ hybridization on embryos was performed with anti-sense RNA probes. Beginning at stage 13 of embryonic development, painless mRNA is detected in a small number of cells in the central nervous system and in a subset of neurons of the peripheral nervous system. The cell bodies of these latter neurons are in positions suggesting that they might include sensory precursors of multidendritic (md) neurons; this was confirmed in double labeling experiments. At embryonic stage 16, prior to when dendritic process are elaborated, the painless mRNA appears to be distributed in the cytoplasm of the multidendritic neurons in a polarized manner. At stage 17 of development, when the md neurons first initiate dendritogenesis, the painless RNA becomes localized to branched projections initiating from clusters of multidendritic neurons. The structures projecting from the dorsal cluster of multidendritic neurons project dorsally. In still older embryos with a more developed dendritic arbor, the pattern of expression evolves into a more elaborate subepidermal plexus of staining. Combined, these data suggest that the painless mRNA is present in md neuron precursors at early stages and becomes localized to their dendrites at later stages. However, further experimentation will be needed to formally demonstrate the latter. In addition, strong expression is also observed in sensory neurons of the antennal maxillary complex. Weak expression of painless mRNA was also observed in chordotonal neurons. Outside of the nervous system, painless is expressed in the embryonic gonad and in the dorsal vessel, the insect heart equivalent (Tracey, 2003).
A GAL4 enhancer trap allele of painless (pain-GAL4) was generated by P element replacement. The pain-GAL4 insertion is within the painless transcription unit at an identical site to the pain1 EP insertion. Consistent with in situ hybridization results, pain-GAL4 driven expression of UAS-GFP shows fluorescence in multidendritic neurons, a subset of cells in the central nervous system, and a subset of sensory neurons in the antennal-maxillary complex. Unlike for painless mRNA, no expression of UAS-GFP driven by pain-GAL4 was detected in the dorsal vessel or in the gonad. Although chordotonal neurons express painless, these neurons are not required for nociception since atonal1 mutant larvae, which lack most chordotonal organs, show rapid, though uncoordinated, responses in both the thermal and mechanical nociception paradigms. Thus, it is hypothesized that the md-da neurons include the primary nociceptors in the larval abdominal segments (Tracey, 2003).
Immunostaining of embryos using the anti-Painless antibody shows strong staining in chordotonal organs. Consistent with the results of Western blots, which show the presence of novel bands rather than a reduction in protein levels in pain1, pain1 mutant embryos show apparently normal staining. Staining in pain2 mutants, however, is greatly reduced, again consistent with the Western analysis. In addition to the staining seen in embryonic chordotonal organs, punctate staining was seen beneath the embryonic epidermis. Given the expression pattern of pain-GAL4, it was reasoned that this staining might correspond to structures associated with the fine dendrites of the multidendritic neurons. Staining of the md neuron arbors was therefore examined in filleted preparations of third instar larvae (Tracey, 2003).
Anti-painless immunoreactivity was tightly associated with the dendritic arbors. The most intense anti-Painless staining was present in bright puncta that were juxtaposed with the dendritic arbor, but Painless was not strongly detected throughout the main branches of the arbor. The anti-Painless staining was highly localized and often clearly seen to be attached to the dendrite. The highly localized nature of Painless immunoreactivity may indicate a specialized region of the dendrite used for nociceptive signaling (Tracey, 2003).
To identify genes important for nociception, a genetic screen was perfomred for mutations that cause insensitivity to noxious heat. A collection of fly lines, carrying randomly inserted EP transposable elements, was screened. A line was considered to have impaired sensitivity to noxious heat if stimulation longer than 3 s was required to produce the rolling response (Tracey, 2003).
Among the 1500 EP lines screened, 49 were identified with reproducibly decreased sensitivity to noxious heat. EP(2)2451, among the most insensitive and carrying an insertion on the second chromosome, was chosen for further study. Larvae homozygous for EP(2)2451 had a defective response to noxious heat, some failing to roll even after 10 s. To reflect this phenotype, the mutant was named painless1 (pain1). Although painless1 larvae show a defect in the writhing response, they still showed a normal response to a light touch on the nose. Also, they did not display the highly uncoordinated movement and poor adult viability typical of the touch-insensitive mutants (Kernan, 1994) having defects in mechanosensory external sensillae (Tracey, 2003).
Nociceptors in vertebrates have been found to be divisible into several classes. Low threshold, polymodal nociceptors respond to noxious heat in the range of 42°C-48°C and to noxious mechanical stimuli, while high-threshold nociceptors respond at even higher temperatures. To test whether the painless1 mutation blocks all nociception, the response of the mutant larvae was examined over a range of temperatures. The larvae also showed a delayed response to a 48°C stimulus, but 52°C or higher elicited a rapid response, similar to that of normal larvae. Since the response to high temperature is seen even in putative null alleles of painless, this result may indicate that moderate and intense levels of noxious heat are processed via separate pathways. In addition, these data imply that the motor system needed for a rapid response is not abolished by mutations in painless; the defect is at the sensory level (Tracey, 2003).
painless mutants were also examined for the rolling response exhibited by wild-type flies given strong mechanical stimuli. To do this, larvae were stimulated with calibrated Von Frey filaments (0.2 mm diameter), which are calibrated to deliver a controlled stimulus. In the wild-type Canton S strain, rolling was not observed when larvae were stimulated with a filament delivering 10 mN of force (n = 19); instead touch responses (larva will pause or make one or more contractile waves, moving away from the stimulus) occurred. When stimulated with a 45 mN fiber, vigorous rolling was observed in 92% of the larvae (n = 36). In contrast, only 13% of painless1 mutant larvae (n = 31) rolled in response to the 45 mN fiber. Nevertheless, with an even stronger mechanical stimulus (100 mN), a high proportion (81%) of painless1 mutant larvae (n = 43) responded by rolling. These data demonstrate that the painless1 mutation results in an increased threshold for both thermal and mechanical nociception (Tracey, 2003).
The painless1 larvae that did not roll with the 45 mN fiber were not completely insensitive to the stimulus; instead, they responded by pausing their feeding movement, as in the wild-type response to light touch. Although thus defective in responding to strong mechanical stimuli, the response to light touch on the nose was unaffected in painless1. The painless1 mutation therefore genetically separates mechanical nociception from mechanosensation (Tracey, 2003).
Next, tests were performed to see whether the behavioral defect exhibited by painless1 mutants could be correlated with a specific defect in the response of abdominal primary afferents. Suction-electrode recordings were performed from sectioned abdominal nerves in third instar larvae containing axons of peripheral sensory neurons. All recordings were done blind to the genotype. At room temperature, the mean bulk spiking frequencies of nerves from wild-type and painless1 larvae were not significantly different (wild-type 8 ± 2 Hz, pain1 11 ± 4 Hz). Data were continuously recorded from the nerve as the temperature of the saline bathing the larvae was gradually increased. The bulk spiking rate of wild-type nerves increased more than 2-fold at the temperatures that elicited rolling behavior. The temperature threshold for the increase in firing rate was near 38°C; frequency at 38°C-42°C was 2.6 ± 0.8 times greater than at room temperature. By contrast, the firing rate of painless1 nerves did not increase in the noxious temperature range (Tracey, 2003).
Some of these recordings contained a variety of sufficiently distinctive spike waveforms to permit separation of spikes originating from different individual neurons. In wild-type nerves, many neurons showed a marked firing rate increase near 38°C-40°C but little spontaneous spiking activity below that temperature, as expected for thermal nociceptors. Other wild-type neurons had a lower temperature threshold. Neurons relatively insensitive to temperature were recorded simultaneously in most wild-type nerves, implying that the thermoresponses of neurons like E1 and E2 are not a nonspecific general property of insect sensory neurons. Importantly, the spiking rate of individual painless1 neurons almost never increased at elevated temperatures. Thus, Painless is required for the excitatory response of abdominal sensory neurons to noxious heat (Tracey, 2003).
To determine whether the insensitivity of painless1 was due to the presence of the EP element insertion, painless1 to a transposase line to mobilize the EP element. Of 80 excision alleles obtained, 73 were homozygous viable, and 29 of those were tested as third instar larvae for the painless phenotype. Among them, 22 alleles showed reversion of the larval response to noxious heat. Therefore, the painless phenotype can be reverted by excision of the P element (Tracey, 2003).
In addition to EP(2)2451, three other P element insertions have been identified within 16 bp of EP(2)2451 by the Berkeley Drosophila Genome Project (BDGP). Larvae homozygous for EP(2)2621 (painless2) and EP(2)2251 (painless3) were found to be strongly insensitive to noxious heat in the nociception paradigm. The fourth insertion, EP(2)2462 (painless4), showed reduced viability, but those larvae that did survive to third instar displayed the same insensitive phenotype as did the other alleles. All of the alleles failed to complement one another for the nociception defect when tested in trans, indicating that the behavioral defects of the lines were due to mutations in the same gene. pain3, like pain1, is recessive. pain2 and pain4 are semidominant and show mild nociception defects when heterozygous (Tracey, 2003).
To examine effects of the mutations on the Painless protein, rabbit antisera were raised against peptides from the Painless sequence. The affinity purified anti-serum GN6620, raised against an intracellular loop in the six-transmembrane region, stained sense organs of the peripheral nervous system, which also expressed the painless mRNA. The antibody also detects alterations on Western blots of extracts from painless1 and painless 2 (Tracey, 2003).
In wild-type extracts, the serum detects a band of 105 kd, consistent with the predicted molecular weight of Painless. That band is absent in the painless1 and painless2 strains. However, an abnormal, higher molecular weight species is detected in both mutants. The presence of this band is consistent with the finding that an upstream in-frame ATG from the painless1 mutant transcript is predicted to add 65 amino acids (18 kd) to the Painless N terminus. The data also suggest that a mutant Painless protein is produced in the painless2 strain (Tracey, 2003).
By P element transformation, transgenic flies were created containing a genomic DNA rescue fragment (P-pain-rescue). This fragment consists of the painless transcription unit, as well as 2.0 kb of upstream genomic DNA, which is hypothesized to contain critical cis-regulatory sequences. The results indicate that sequences sufficient to restore the response to noxious heat are present in the 8.5 kb of rescue DNA (Tracey, 2003).
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date revised: 25 February 2005
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