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Plasticity of histamine H3receptor expression and binding in the vestibular nuclei after labyrinthectomy in rat

Plasticity of histamine H3receptor expression and binding in the vestibular nuclei after... Background: In rat, deafferentation of one labyrinth (unilateral labyrinthectomy) results in a characteristic syndrome of ocular and motor postural disorders (e.g., barrel rotation, circling behavior, and spontaneous nystagmus). Behavioral recovery (e.g., diminished symptoms), encompassing 1 week after unilateral labyrinthectomy, has been termed vestibular compensation. Evidence suggesting that the histamine H receptor plays a key role in vestibular compensation comes from studies indicating that betahistine, a histamine-like drug that acts as both a partial histamine H receptor agonist and an H receptor antagonist, can accelerate the process of 1 3 vestibular compensation. Results: Expression levels for histamine H receptor (total) as well as three isoforms which display variable lengths of the third intracellular loop of the receptor were analyzed using in situ hybridization on brain sections containing the rat medial vestibular nucleus after unilateral labyrinthectomy. We compared these expression levels to H receptor binding densities. Total H receptor mRNA levels (detected by oligo probe H ) as well as mRNA levels of the three 3 3X receptor isoforms studied (detected by oligo probes H , H , and H ) showed a pattern of 3A 3B 3C increase, which was bilaterally significant at 24 h post-lesion for both H and H , followed by 3X 3C significant bilateral decreases in medial vestibular nuclei occurring 48 h (H and H ) and 1 week 3X 3B post-lesion (H , H , and H ). Expression levels of H was an exception to the forementioned 3A 3B 3C 3B pattern with significant decreases already detected at 24 h post-lesion. Coinciding with the decreasing trends in H receptor mRNA levels was an observed increase in H receptor binding 3 3 densities occurring in the ipsilateral medial vestibular nuclei 48 h post-lesion. Conclusion: Progressive recovery of the resting discharge of the deafferentated medial vestibular nuclei neurons results in functional restoration of the static postural and occulomotor deficits, usually occurring within a time frame of 48 hours in rats. Our data suggests that the H receptor may be an essential part of pre-synaptic mechanisms required for reestablishing resting activities 48 h after unilateral labyrinthectomy. Page 1 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 Evidence suggesting that the H receptor plays a key role Background In rat, deafferentation of one labyrinth (unilateral laby- in vestibular compensation comes from studies indicating rinthectomy) results in a characteristic syndrome of ocular that betahistine, a histamine-like drug that acts as both a motor and postural disorders. These disorders have been partial histamine H receptor agonist and an H receptor 1 3 divided into two categories : One category of symptoms, antagonist [14,35], accelerates the process of vestibular called static, includes head rotation in both the frontal compensation [32,36]. Furthermore, studies have shown and horizontal planes and ocular nystagmus [1]. The that betahistine treatment results in a reduction of [3H]N- other category, called dynamic, corresponds to a α-methylhistamine labelling in the vestibular nuclear decreased gain of the vestibulo-ocular and vestibulo-spi- complex [37]; these findings suggest that betahistine nal refelxes [1]. Behavioral recovery (e.g., diminished increases histamine turnover and release by blocking pre- symptoms), encompassing 1 week after unilateral laby- synaptic H receptors and inducing H receptor downreg- 3 3 rinthectomy, has been termed vestibular compensation ulation [37]. It is noteworthy that dynamic vestibular [2]. Moreover, the time course of recovery is very different functions can be modulated by H receptor ligands, e.g., for static and dynamic reflexes: static deficits disappear in thioperamide [38]; moreover, thioperamide can affect one week but dynamic deficits tend to take several months tonic vestibular functions as well with its demonstrated to normalize. Because unilateral labyrinthectomy results ability to attenuate barrel rotation in rats following unilat- in a permanent loss of vestibular inputs from the lesioned eral labyrinthectomy [39]. The forementioned studies side, the compensatory process is assumed to be attribut- make tenable the view that further elucidation of H able to the reorganization of the neural network in the receptor regulation is required to fully understand the central vestibular system [3,4]. Many brain regions includ- process of vestibular compensation. ing, the medial vestibular nucleus (MVe), are implicated in this process of recovery [5-7]. The detailed aim of this study was to characterize the pat- terning of mRNA expression levels for all possible mRNA The focus of our study is the histamine H receptor that splice variants of the H receptor (H ; as described in 3 3 3X was initially characterized as an autoreceptor controlling [24]) and its isoforms which display different variations histamine synthesis and release [8,9]. Subsequently, as a of the third intracellular loop (H , H , and H ; as 3A 3B 3C receptor was found to mediate pre- described in [24]) in the medial vestibular nucleus (MVe) heteroreceptor, the H synaptic inhibition of release of histamine, noradrena- during the process of vestibular compensation. line, serotonin, dopamine, glutamate, GABA and tachykinins [10-13], presumably by inhibiting calcium Moreover, in this study, we compared the aforementioned channels [14-16]. The histamine H receptor was recently trend in mRNA expression levels with H receptor binding 3 3 cloned from human [17], monkey [18], rat [19], mouse densities to reveal the possible plastic changes in the H [20], and guinea pig [21]. Moreover, the receptor was receptor which is responsible for significant constitutive found to have several isoforms [21-26] with differential activity also in vivo [40], histamine-mediated regulation of coupling to second messenger systems and a variation in neurotransmitter release, and therapeutic effects of their distribution in a region-specific manner. The iso- betahistine. forms are formed by alternative splicing of the messenger RNA (mRNA; [22,24]). This study involves analysis of Results Expression patterns of total H receptor and H receptor trends observed in mRNA expression levels for the H 3 3 3 receptor (H , the oligonucleotide probe detecting all H isoforms (H , H , and H ) 3A 3B 3C 3X 3 receptor mRNAs characterized so far) as well as three of Changes in mRNA expression levels for H receptor and the known functionally active isoforms (H , H , and the three H receptor isoforms (Figure 1A,1B,1C and 1D, 3A 3B 3 H ), described by Drutel et al. [24], during the process of respectively) occured bilaterally; that is, there were no sig- 3C post-lesional plasticity in the central nervous system nificant differences detected between the ipsilateral and (CNS). contralateral medial vestibular nuclei of animals in all groups studied (e.g., control, 4 h post-lesion, 24 h post- The primary source of histamine (e.g., ligand for H recep- lesion, 48 h post-lesion [n = 4, for each group], and 1 tors) are histaminergic perikarya located exclusively in the week post-lesion [n = 5]). tuberomammillary nuclei of the posterior hypothalamus [27,28]; these same neurons send axonal projections to We compared the total H receptor mRNA expression lev- many areas of the brain including the vestibular nuclear els (using probe H ) in ipsilateral MVe of control animals 3X complex in rat [29-32]. The fact that the rat vestibular to that of test animals from the four time points (Fig. 1A). nuclear complex is endowed with the H receptor was Fig. 2B shows scanned X-ray film depiction of H expres- 3 3X established by use of ligand binding [33,34] and in situ sion in a representative animal 24 h post-lesion. We hybridization methods [34]. found no significant rise in H mRNA levels in the 3X Page 2 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 H Figure 1 A, B, C, and D – Exp ) at 4 h, 24 h, 48 h, ression le and 1 w vels of the total histamine H eek post-lesion receptor (using probe H ) and its three isoforms (H , H , and 3C 3 3X 3A 3B A, B, C, and D – Expression levels of the total histamine H receptor (using probe H ) and its three isoforms 3 3X (H , H , and H ) at 4 h, 24 h, 48 h, and 1 week post-lesion. Data are presented as mean IOD ± SEM. Abbreviations 3A 3B 3C are as follows: ipsi, ipsilateral; contra, contralateral; cntrl, control (n = 4); 4 h, animals sacrificed 4 h post-lesion (n = 4); 48 h, animals sacrificed 48 h post-lesion (n = 4); and 1 wk, animals sacrificed 1 week post-lesion (n = 5). ***p < 0.001 and **p < 0.01 when compared to ipsilateral control. ###p < 0.001, ##p < 0.01, and #p < 0.05 when compared to contralateral control. ipsilateral MVe 4 hours after lesioning. Significant decrease of H mRNA levels does occur in the ipsilateral 3A increases in H mRNA levels were found to occur at 24 MVe 1 week after lesioning. The trend was identical when 3X and 48 hours post-lesion. After 1 week post-lesion, H comparing H mRNA levels in contralateral MVe of con- 3 3A receptor mRNA expression levels (as indicated by probe trol animals with H mRNA levels in contralateral MVe of 3A H ) returned to normal levels. The trend observed was animals from the four time points: There was no signifi- 3X similar when comparing H mRNA levels in contralateral cant increase in H mRNA levels when comparing to con- 3X 3A MVe of control animals with that of test animals from the tralateral MVe 4 hours, 24 hours, and 48 hours post- four time points. There was no significant change in total lesion; on the other hand, there is a significant decrease in H receptor mRNA levels detected in contralateral MVe 4 H mRNA levels when comparing to contralateral MVe 1 3 3A hours post-lesion, but we found a significant increase in week after lesioning. H mRNA levels detected in contralateral MVe both 24 3X hours and 48 hours post-lesion. Finally, a return to nor- No significant changes in H mRNA expression levels 3B mal levels in total H receptor mRNA level was detected were detected when we compared ipsilateral MVe in con- 3X in contralateral MVe 1 week post-lesion. trol animals to ipsilateral MVe 4 hours post-lesion (Fig. 1C). Fig. 2F shows scanned X-ray film depiction of H 3B No significant increases in H mRNA expression levels expression in a representative animal 24 h post-lesion. 3A were detected when we compared ipsilateral MVe in con- Significant decreases in mRNA levels were found in the trol animals with ipsilateral MVe 4 hours, 24 hours, and ipsilateral MVe 24 hours, 48 hours, and 1 week after 48 hours post-lesion (Fig. 1B). Fig. 2D shows scanned X- lesioning. When comparing contralateral MVe of control ray film depiction of H expression in a representative animals to contralateral MVe of animals in the other time 3A animal 24 h post-lesion. On the other hand, a significant points, there was a significant increase detected 4 hours Page 3 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 post-lesion and this was followed by significant decreases detected at 24 hours, 48 hours after lesion, and 1 week post-lesion. No significant changes in H mRNA levels were detected 3C when we compared ipsilateral MVe in control animals to ipsilateral MVe 4 hours post-lesion (Fig. 1D). Fig. 2H shows scanned X-ray film depiction of H expression in a 3C representative animal 24 h post-lesion. A significant increase was detected when comparing ipsilateral MVe of control animals to ipsilateral MVe 24 hours post-lesion; moreover, the decrease in H mRNA levels in the ipsilat- 3C eral MVe 48 hours post-lesion was not significantly differ- ent from those of the ipsilateral MVe in control animals. Finally, in comparison to the ipsilateral MVe in controls, there was a decrease in mRNA levels that was found to be significant in the ipsilateral MVe 1 week post-lesion. Results were similar when comparing the contralateral MVe in control animals to contralateral MVe from the other time points: There was no significant increase in H 3C mRNA levels when comparing to contralateral MVe 4 hours post-lesion, there was a significant increase detected when comparing to contralateral MVe 24 hours post- lesion, the decrease was not significant comparing to con- tralateral MVe 48 hours post-lesion, and a significant decrease was detected when comparing to contralateral MVe 1 week post-lesion. [ I]iodoproxyfan Binding Densities No significant changes were found in H receptor binding densities (Fig. 3) between ipsilateral MVe in control ani- mals (n = 3) and ipsilateral MVe of animals of different time points. The results were identical when H receptor binding densities in contralateral MVe in control animals were compared to that of contralateral MVe at different times post-lesion. On the other hand, when comparing the ipsilateral to contralateral MVe 48 hours post-lesion, a significant increase in binding densities occurred on the receptor ipsilateral side (Fig. 4 shows an example of H binding densities in a representative animal 48 h post- lesion). Discussion Knowing that there is no reliable method to determine the efficiency of a probe for its targetted sequence in an mRNA b Figure 2 ing co A secti h pr ly)ocking contr o bridiza Location of the MVe as visual bon and on x-ray image entro s: B) tion experimen H l F) H oC) H l G) Hblocking control D) ts with bslocking c of pa the vario ized on cresyl v rallel sections used in ontro ul H) s ol H igonucl HE) H iolet st I) H eoti block- ained in si de tu 3X 3B 3X 3B 3A 3C 3A 3C of interest, this study focuses instead on the pattern of A) Location of the MVe as visualized on cresyl violet stained section and on x-ray images of parallel sec- expression for the total H receptor (detected by using tions used in in situ hybridization experiments with probe H ) and its isoforms (H , H , and H ) occurring 3X 3A 3B 3C the various oligonucleotide probes: B) H C) H during the process of post-lesional plasticity in the CNS. 3X 3X blocking control D) H E) H blocking control F) 3A 3A Moreover, the probes used in this study were designed to H G) H blocking control H) H I) H blocking 3B 3B 3C 3C detect unique areas in the transcripts (H ), or junctional 3A control. Sections are from a representative animal sacrificed areas in deletion isoforms (probes H and H ) which 3B 3C 24 h post-lesion. Abbreviations are as follows: MVe, medial would make it highly unlikely for non-specific hybridiza- vestibular nucleus and Pr, prepositus nucleus. Scale bars are tion would occur. This study also includes a comparison 100 µm. Page 4 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 omiz Figure 3 [ I]iodoproxyfan binding ed rats densities in MVe of labyrinthect- [ I]iodoproxyfan binding densities in MVe of laby- rinthectomized rats. Data are presented as mean IOD ± SEM. Abbreviations are as stated in Figure 1 legend. Sample sizes are as follows: control, n = 3; 4 h post-lesion, n = 4; 24 h post-lesion, n = 4; 48 h post-lesion, n = 4; 1 week post- lesion, n = 5. *p = 0.0193. of the aforementioned patterns of expression with bind- ing densities for the H receptor. The prepositus nuclei are delineated and mentioned in legends for figures 2 and 4. These nuclei are delineated for iFigure 4 A) Location of B) section and C) on x-ray images of [ng contro at mag I]iodop nification used in an l is roxy repre the fan MVe as b siente nding experim d in this visual alyized a s image es on cresyl violet stained par ents; D) n ta high ma llel sec otn-specific bin ions u gnification a sed in nd d - the sole purpose of giving the reader an idea of the dors- A) Location of the MVe as visualized at high magnifi- oventral extent of the MVe at the levels depicted in figures cation and B) at magnification used in analyses on 2 and 4. Noteworthy, is that these nuclei are not included cresyl violet stained section and C) on x-ray images in the main functional projections in the vestibulo-ocular of parallel sections used in [ I]iodoproxyfan binding and vestibulo-spinal pathways from the brainstem vestib- experiments; D) non-specific binding control is rep- ular nucleus in mammals reviewed by Smith and resented in this image. Sections are from a representative animal sacrificed 48 h post-lesion. Abbreviations are as stated Curthoys [2]. in Figure 2 legend. Scale bars are 100 µm. There are three previously described types of neurons in the medial vestibular nucleus [41,42]; in vitro, all three types are depolarized by histamine [43,44]. Moreover, this depolarization has been shown to be exclusively mediated through postsynaptic H receptors [43,44] sug- ated inhibition of GABA release from inhibitory interneu- gesting a presynaptic localization of H receptors in the rons or glutamate release from terminals of second order medial vestibular nuclei [38]. Consequently, there are excitatory MVe neurons may underlie the restoration of three possible locations for H receptors in the MVe: 1) resting activity in the deafferentated MVe; more precisely, On the histaminergic or other incoming fibers innervating H receptor action would result in disinhibition of neu- the MVe [29-32]. 2) On terminals of the inhibitory rons in the deafferentated MVe interneurons in the MVe that make synaptic contacts on second order excitatory neurons-defined as neurons in the With respect to the first possible location, it has been vestibular nuclei that receive inputs from sensory afferents established that histamine fibers are endowed with H [45]. 3) On the terminals of second order excitatory MVe receptors that function as autoreceptors and inhibit hista- neurons making cross-commissural synaptic contacts on mine release [11,13]. Support for the notion that H contralateral MVe inhibitory interneurons [45]. With receptors are at the next two possible locations (i.e., either respect to MVe on the lesioned side, an H receptor medi- at the terminals of the inhibitory interneurons or at the Page 5 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 terminals of second order MVe neurons) comes from tor and H , and H isoform mRNA levels, with H 3A 3C 3B work showing that betahistine antagonizes the excitatory mRNA levels already increasing at 4 hours post-lesion and effect of histamine on vestibular neurons from in vitro decreasing at 24 h post-lesion, coinciding with a signifi- slice preparations of the dorsal brainstem of the rat [46]. cant increase in H receptor binding densities in the ipsi- This finding is significant given that H receptors mediate lateral MVe detected 48 hours post-lesion suggests the presynaptic inhibition of release of other neurotransmit- occurrence of one or a combination of events in the ipsi- ters including: noradrenaline, serotonin, dopamine, lateral MVe: 1) an increase in translation has occurred. 2) glutamate, GABA and tachykinins [10-13]. Unilateral lab- A change in receptor trafficking between intracellular yrinthectomy induced changes in expression levels of stores and cell membrane has occurred so that it can be receptors for glutamate (e.g., NR1 and NR2A-D subunits detected as an increase in H receptor receptor binding of the NMDA receptor [47] and GluR2-4 subunits of the densities. In either case, an increase in functional H AMPA receptor [48]) have been studied in the vestibular receptor protein coupled with an increase in receptor nuclei. Moreover, the existence of both GABA and GABA activity may lead to a restoration of resting activity in the A B receptors in the vestibular nuclei and their involvement in deafferentated MVe by scenarios already mentioned in vestibular compensation has been demonstrated by either this section. unilateral perfusion or microinjection of GABAergic ago- nists and antagonists (e.g., GABA, muscimol, and bicucul- Conclusions line) [49]. Our findings are significant given that normalization of resting activities in neurons located in the ipsilateral MVe Consequently, an H receptor antagonist such as betahis- has been shown to occur 48 hours after unilateral laby- tine could either facilitate GABA release from inhibitory rinthectomy [50]. Moreover, by 48 hours post-lesion, interneurons located in the MVe that make synaptic con- commissural disinhibition has been observed to occur tacts with second order neurons or facilitate glutamate [51]. Placement of H receptors at the terminals of either release from terminals of second order MVe neurons that GABAergic inhibitory MVe interneurons or on terminals synapse on inhibitory interneurons in the contralateral of glutamatergic second order MVe neurons with contral- MVe [31,45]. Either scenario would lead to an inhibition ateral projections should result in the observed commis- receptor activity would of second order neurons in the MVe and this would sural disinhibition as increased H explain the observation that betahistine antagonizes the result in an inhibition of synaptic release of excitatory effect of histamine on vestibular neurons [46]. neurotransmitters. On the other hand, betahistine administration is reported Our data would thus suggest that H receptors are to induce recovery with a time benefit of 2 weeks relative involved in presynaptic mechanisms resulting in a nor- to control animals after unilateral vestibular neurectomy malization of resting activities in ipsilateral MVe neurons [32,36]; this is thought to be due to a bilateral increase in which would balance discharge activity in MVe on both histamine release in the MVe [32,37]. The increased hista- sides. mine would be bound by H receptors on the perikarya of MVe neurons ipsilateral and contralateral to the lesion Methods resulting in a bilateral increase in activity. This should Animals and surgery This study was approved by the Local Committee for Ani- facilitate behavioral recovery as it is thought that an imbalance in discharge of MVe neurons (30–40 spikes/s mal Experiments and the Provincial State Office of West- in normal animals [4]) ipsilateral and contralateral to the ern Finland; in addition, animal experiments were in lesion underlies the static postural and occulomotor accordance with the European Convention (1986) guide- deficits triggered by unilateral labyrinthectomy [45]. Yet, lines and approved by the Animal Ethics Committee of as stated before, the actions of betahistine would also Abo Akademi Unviversity. Adult male Sprague Dawley extend to H receptors located on glutamatergic terminals rats (200–250 g) were used. Intraperitoneal (ip) injection of contralateral MVe neurons or on GABAergic terminals of pentobarbital (45 mg/kg ip; Mebunat, Orion, Finland) of inhibitory MVe interneurons. Antagonism of H recep- was used as an anesthetic; in addition, local anesthetic tors at either site may also act to speed recovery by increas- Lidocain (Orion, Finland) was infiltrated under the skin ing the amount of GABA output from terminals of and periosteum prior the procedure. Three steps initiated inhibitory interneurons and, as a consequence, equalizing the surgical procedure to left side of the animal's head: ret- neuronal discharge activity of ipsilateral and contralateral roauricular skin incision, opening of the middle ear bulla MVe. with a drill, and removal of the ossicular chain with the aid of a microscope. Unilateral labyrinthectomy was car- The trends toward bilateral increases (24 h post-lesion) ried out by opening the horizontal semicircular canal duct followed by decreases (48 h post-lesion) in total H recep- in the temporal bone, drilling through the horizontal and Page 6 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 anterior semicircular canal ampullae and aspirating the tion (10 cpm/ml) was carried out at 50°C for 16 to 20 contents of the vestibule. 100% ethanol was injected into hours in a humidified chamber. Posthybridization washes the opened labyrinth to finalize the procedure; finally, the were carried out as described previously [55]. Brain sec- wound was sutured. The sham operation entailed opening tions from control animals and animals 4 h, 24 h, 48 h, the middle ear bulla and leaving the ossicular chain intact and 1 week post-lesion were treated simultaneously with prior to suturing the wound, as described in Cameron and their respective oligonucleotide probe. Sections and car- Dutia [52]; the control animals (n = 4) that underwent the bon-14 standards were exposed to Kodak BioMax X-ray sham operation were killed 4 h later. The method is films (Kodak, USA) for 10 days. explained hereafter. All rats included in the experiments displayed symptoms characteristic of animals that have Receptor binding autoradiography undergone unilateral labyrinthectomy (e.g., barrel rota- Autoradiographic localization of [ I]iodoproxyfan bind- tion, circling behavior, and spontaneuos nystagmus). ing sites has been described before [56]. Briefly, slide These symptoms gadually disappeared during the first two mounted tissue sections were preincubated for 15 min in or three days and were completely absent within one 50 mM Na HPO -KH PO phosphate buffer, pH 6.8, con- 2 4 2 4 gas and killed by week. Animals were stunned by CO taining 0.1% bovine serum albumin and 1 µM S132 (a 1- decapitation 4 h (n = 4), 24 h (n = 4), 48 h (n = 4) and 1 substituted imidazole derivative displaying a very low week (n = 5) after labyrinthectomy. affinity at H receptors and used to decrease non-specific labelling). The sections were then incubated for 1 hour at Tissue preparation room temperature in the same buffer containing 15 pM After the mentioned decapitation, brains were removed, [ I]iodoproxyfan (Amersham Pharmacia Biotech UK frozen in isopentane (-25°C), and stored at -70°C. Tis- Limited, England). Non-specific binding was determined sues were then cut to 20 µm thick cryosections, thaw by incubating consecutive sections in the presence of 1 mounted onto poly-L-lysine coated slides (Menzel-Gläser, µM (R)α-methylhistamine (Sigma-Aldrich, Germany). At Germany), and stored at -70°C until used. the completion of incubation, the tissues were washed four times (4 min each) in the same fresh ice-cold buffer, In-situ hybridization histochemistry dipped into ice-cold water, and dried under a current of The oligonucleotide probes used for in situ hybridization air. Brain sections from control animals and animals 4 h, were designed so that they specifically recognized the dif- 24 h, 48 h, and 1 week post-lesion were treated simulta- ferent H receptor isoform mRNAs (H , H , and H ; as neously. Dried sections, along with standards, were 3 3A 3B 3C described in [24]); an additional oligonucleotide probes exposed to Kodak BioMax X-ray films (Kodak, USA) for 2 was used to detect all characterized H receptor isoforms days. (H ; as described in [24]). Sequences for H , H , H , 3X 3X 3A 3B and H probes have been previously published [24]. It is Image analysis and statistics 3C noteworthy that the isoform-specific probes detect selec- Autoradiographic films were quantified by digitizing the tively the various deletion forms of the third intracellular film images with a computer based MCID 5+ image anal- loop, but do not differentiate between the possible alter- ysis system (Imaging Research, Canada) and by measur- native C-termini of the H receptor isoforms [53]. How- ing gray scale pixel values. The relative optic density was ever, it has been shown that the differences in the third converted to integrated optic density (IOD) based on a intracellular loop are significant for coupling to intracellu- standard curve derived from standards exposed to films. lar second messengers [24]. As a control, we used a nor- Gray scale values were determined from four sections for mal hybridization mixture with a 100-fold excess of each animal, measurements from white matter of each unlabeled specific probes. As an additional control, we respective section were subtracted to obtain the final val- used a Staphylococcus aureus chloramphenicol acetyltrans- ues, and the data were analyzed using either a paired t-test ferase-specific oligonucleotide probe. The hybridization or one-way ANOVA combined with Bonferroni's Multiple procedure used has been described before and was used Comparison Test as a post-hoc test. Significance was with minor modifications [54,55]. All probes were determined when p < 0.05. labeled with [ S]deoxyadenosine 5'-α(-thio) triphos- phate (New England Nuclear, USA) at their 3' ends using Authors' contributions terminal deoxynucleotide transferase (Promega, USA). AL assisted with the surgeries and tissue collection, sec- Nonincorporated nucleotides were removed by purifica- tioned all of the brains, carried out the in situ hybridiza- tion through Sephadex G-50 columns. tion and binding studies, performed all of the image and statistical analyses, and participated in the design of the Before hybridization, cryosections were taken from the - study. AAA performed the surgeries, assisted with tissue 70°C environment and kept at room temeprature for 10 collection, assisted with image analyses, and participated min and treated with UV light for 5 min. The hybridiza- in the design of the study. KK established the method of Page 7 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 pharmacological profiles. J Pharmacol Exp Ther 2000, in situ hybridization in the lab, designed the oligonucle- 293:771-778. otide probes, assisted with surgeries and tissue collection, 20. Chen J, Liu C, Lovenberg TW: Molecular and pharmacological and participated in the design of the study. HS synthe- characterization of the mouse histamine H3 receptor. Eur J Pharmacol 2003, 467:57-65. sized and supplied rare chemicals required for the binding 21. Tardivel-Lacombe J, Rouleau A, Heron A, Morisset S, Pillot C, study. PP acquired funding, coordinated the study, and Cochois V, Schwartz JC, Arrang JM: Cloning and cerebral expres- sion of the guinea pig histamine H3 receptor: evidence for participated in its design. All authors read and approved two isoforms. Neuroreport 2000, 11:755-759. the final manuscript. 22. 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Histo- chem 1992, 98:39-49. 55. Lintunen M, Sallmen T, Karlstedt K, Fukui H, Eriksson KS, Panula P: Postnatal expression of H -receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures. Eur J Neurosci 1998, 10:2287-2301. 56. Ligneau X, Garbarg M, Vizuete ML, Diaz J, Purand K, Stark H, Schu- nack W, Schwartz JC: [ I]Iodoproxyfan, a new antagonist to label and visualize cerebral histamine H receptors. J Pharma- col Exp Ther 1994, 271:452-459. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Plasticity of histamine H3receptor expression and binding in the vestibular nuclei after labyrinthectomy in rat

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Springer Journals
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Copyright © 2004 by Lozada et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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1471-2202
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10.1186/1471-2202-5-32
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15361262
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Abstract

Background: In rat, deafferentation of one labyrinth (unilateral labyrinthectomy) results in a characteristic syndrome of ocular and motor postural disorders (e.g., barrel rotation, circling behavior, and spontaneous nystagmus). Behavioral recovery (e.g., diminished symptoms), encompassing 1 week after unilateral labyrinthectomy, has been termed vestibular compensation. Evidence suggesting that the histamine H receptor plays a key role in vestibular compensation comes from studies indicating that betahistine, a histamine-like drug that acts as both a partial histamine H receptor agonist and an H receptor antagonist, can accelerate the process of 1 3 vestibular compensation. Results: Expression levels for histamine H receptor (total) as well as three isoforms which display variable lengths of the third intracellular loop of the receptor were analyzed using in situ hybridization on brain sections containing the rat medial vestibular nucleus after unilateral labyrinthectomy. We compared these expression levels to H receptor binding densities. Total H receptor mRNA levels (detected by oligo probe H ) as well as mRNA levels of the three 3 3X receptor isoforms studied (detected by oligo probes H , H , and H ) showed a pattern of 3A 3B 3C increase, which was bilaterally significant at 24 h post-lesion for both H and H , followed by 3X 3C significant bilateral decreases in medial vestibular nuclei occurring 48 h (H and H ) and 1 week 3X 3B post-lesion (H , H , and H ). Expression levels of H was an exception to the forementioned 3A 3B 3C 3B pattern with significant decreases already detected at 24 h post-lesion. Coinciding with the decreasing trends in H receptor mRNA levels was an observed increase in H receptor binding 3 3 densities occurring in the ipsilateral medial vestibular nuclei 48 h post-lesion. Conclusion: Progressive recovery of the resting discharge of the deafferentated medial vestibular nuclei neurons results in functional restoration of the static postural and occulomotor deficits, usually occurring within a time frame of 48 hours in rats. Our data suggests that the H receptor may be an essential part of pre-synaptic mechanisms required for reestablishing resting activities 48 h after unilateral labyrinthectomy. Page 1 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 Evidence suggesting that the H receptor plays a key role Background In rat, deafferentation of one labyrinth (unilateral laby- in vestibular compensation comes from studies indicating rinthectomy) results in a characteristic syndrome of ocular that betahistine, a histamine-like drug that acts as both a motor and postural disorders. These disorders have been partial histamine H receptor agonist and an H receptor 1 3 divided into two categories : One category of symptoms, antagonist [14,35], accelerates the process of vestibular called static, includes head rotation in both the frontal compensation [32,36]. Furthermore, studies have shown and horizontal planes and ocular nystagmus [1]. The that betahistine treatment results in a reduction of [3H]N- other category, called dynamic, corresponds to a α-methylhistamine labelling in the vestibular nuclear decreased gain of the vestibulo-ocular and vestibulo-spi- complex [37]; these findings suggest that betahistine nal refelxes [1]. Behavioral recovery (e.g., diminished increases histamine turnover and release by blocking pre- symptoms), encompassing 1 week after unilateral laby- synaptic H receptors and inducing H receptor downreg- 3 3 rinthectomy, has been termed vestibular compensation ulation [37]. It is noteworthy that dynamic vestibular [2]. Moreover, the time course of recovery is very different functions can be modulated by H receptor ligands, e.g., for static and dynamic reflexes: static deficits disappear in thioperamide [38]; moreover, thioperamide can affect one week but dynamic deficits tend to take several months tonic vestibular functions as well with its demonstrated to normalize. Because unilateral labyrinthectomy results ability to attenuate barrel rotation in rats following unilat- in a permanent loss of vestibular inputs from the lesioned eral labyrinthectomy [39]. The forementioned studies side, the compensatory process is assumed to be attribut- make tenable the view that further elucidation of H able to the reorganization of the neural network in the receptor regulation is required to fully understand the central vestibular system [3,4]. Many brain regions includ- process of vestibular compensation. ing, the medial vestibular nucleus (MVe), are implicated in this process of recovery [5-7]. The detailed aim of this study was to characterize the pat- terning of mRNA expression levels for all possible mRNA The focus of our study is the histamine H receptor that splice variants of the H receptor (H ; as described in 3 3 3X was initially characterized as an autoreceptor controlling [24]) and its isoforms which display different variations histamine synthesis and release [8,9]. Subsequently, as a of the third intracellular loop (H , H , and H ; as 3A 3B 3C receptor was found to mediate pre- described in [24]) in the medial vestibular nucleus (MVe) heteroreceptor, the H synaptic inhibition of release of histamine, noradrena- during the process of vestibular compensation. line, serotonin, dopamine, glutamate, GABA and tachykinins [10-13], presumably by inhibiting calcium Moreover, in this study, we compared the aforementioned channels [14-16]. The histamine H receptor was recently trend in mRNA expression levels with H receptor binding 3 3 cloned from human [17], monkey [18], rat [19], mouse densities to reveal the possible plastic changes in the H [20], and guinea pig [21]. Moreover, the receptor was receptor which is responsible for significant constitutive found to have several isoforms [21-26] with differential activity also in vivo [40], histamine-mediated regulation of coupling to second messenger systems and a variation in neurotransmitter release, and therapeutic effects of their distribution in a region-specific manner. The iso- betahistine. forms are formed by alternative splicing of the messenger RNA (mRNA; [22,24]). This study involves analysis of Results Expression patterns of total H receptor and H receptor trends observed in mRNA expression levels for the H 3 3 3 receptor (H , the oligonucleotide probe detecting all H isoforms (H , H , and H ) 3A 3B 3C 3X 3 receptor mRNAs characterized so far) as well as three of Changes in mRNA expression levels for H receptor and the known functionally active isoforms (H , H , and the three H receptor isoforms (Figure 1A,1B,1C and 1D, 3A 3B 3 H ), described by Drutel et al. [24], during the process of respectively) occured bilaterally; that is, there were no sig- 3C post-lesional plasticity in the central nervous system nificant differences detected between the ipsilateral and (CNS). contralateral medial vestibular nuclei of animals in all groups studied (e.g., control, 4 h post-lesion, 24 h post- The primary source of histamine (e.g., ligand for H recep- lesion, 48 h post-lesion [n = 4, for each group], and 1 tors) are histaminergic perikarya located exclusively in the week post-lesion [n = 5]). tuberomammillary nuclei of the posterior hypothalamus [27,28]; these same neurons send axonal projections to We compared the total H receptor mRNA expression lev- many areas of the brain including the vestibular nuclear els (using probe H ) in ipsilateral MVe of control animals 3X complex in rat [29-32]. The fact that the rat vestibular to that of test animals from the four time points (Fig. 1A). nuclear complex is endowed with the H receptor was Fig. 2B shows scanned X-ray film depiction of H expres- 3 3X established by use of ligand binding [33,34] and in situ sion in a representative animal 24 h post-lesion. We hybridization methods [34]. found no significant rise in H mRNA levels in the 3X Page 2 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 H Figure 1 A, B, C, and D – Exp ) at 4 h, 24 h, 48 h, ression le and 1 w vels of the total histamine H eek post-lesion receptor (using probe H ) and its three isoforms (H , H , and 3C 3 3X 3A 3B A, B, C, and D – Expression levels of the total histamine H receptor (using probe H ) and its three isoforms 3 3X (H , H , and H ) at 4 h, 24 h, 48 h, and 1 week post-lesion. Data are presented as mean IOD ± SEM. Abbreviations 3A 3B 3C are as follows: ipsi, ipsilateral; contra, contralateral; cntrl, control (n = 4); 4 h, animals sacrificed 4 h post-lesion (n = 4); 48 h, animals sacrificed 48 h post-lesion (n = 4); and 1 wk, animals sacrificed 1 week post-lesion (n = 5). ***p < 0.001 and **p < 0.01 when compared to ipsilateral control. ###p < 0.001, ##p < 0.01, and #p < 0.05 when compared to contralateral control. ipsilateral MVe 4 hours after lesioning. Significant decrease of H mRNA levels does occur in the ipsilateral 3A increases in H mRNA levels were found to occur at 24 MVe 1 week after lesioning. The trend was identical when 3X and 48 hours post-lesion. After 1 week post-lesion, H comparing H mRNA levels in contralateral MVe of con- 3 3A receptor mRNA expression levels (as indicated by probe trol animals with H mRNA levels in contralateral MVe of 3A H ) returned to normal levels. The trend observed was animals from the four time points: There was no signifi- 3X similar when comparing H mRNA levels in contralateral cant increase in H mRNA levels when comparing to con- 3X 3A MVe of control animals with that of test animals from the tralateral MVe 4 hours, 24 hours, and 48 hours post- four time points. There was no significant change in total lesion; on the other hand, there is a significant decrease in H receptor mRNA levels detected in contralateral MVe 4 H mRNA levels when comparing to contralateral MVe 1 3 3A hours post-lesion, but we found a significant increase in week after lesioning. H mRNA levels detected in contralateral MVe both 24 3X hours and 48 hours post-lesion. Finally, a return to nor- No significant changes in H mRNA expression levels 3B mal levels in total H receptor mRNA level was detected were detected when we compared ipsilateral MVe in con- 3X in contralateral MVe 1 week post-lesion. trol animals to ipsilateral MVe 4 hours post-lesion (Fig. 1C). Fig. 2F shows scanned X-ray film depiction of H 3B No significant increases in H mRNA expression levels expression in a representative animal 24 h post-lesion. 3A were detected when we compared ipsilateral MVe in con- Significant decreases in mRNA levels were found in the trol animals with ipsilateral MVe 4 hours, 24 hours, and ipsilateral MVe 24 hours, 48 hours, and 1 week after 48 hours post-lesion (Fig. 1B). Fig. 2D shows scanned X- lesioning. When comparing contralateral MVe of control ray film depiction of H expression in a representative animals to contralateral MVe of animals in the other time 3A animal 24 h post-lesion. On the other hand, a significant points, there was a significant increase detected 4 hours Page 3 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 post-lesion and this was followed by significant decreases detected at 24 hours, 48 hours after lesion, and 1 week post-lesion. No significant changes in H mRNA levels were detected 3C when we compared ipsilateral MVe in control animals to ipsilateral MVe 4 hours post-lesion (Fig. 1D). Fig. 2H shows scanned X-ray film depiction of H expression in a 3C representative animal 24 h post-lesion. A significant increase was detected when comparing ipsilateral MVe of control animals to ipsilateral MVe 24 hours post-lesion; moreover, the decrease in H mRNA levels in the ipsilat- 3C eral MVe 48 hours post-lesion was not significantly differ- ent from those of the ipsilateral MVe in control animals. Finally, in comparison to the ipsilateral MVe in controls, there was a decrease in mRNA levels that was found to be significant in the ipsilateral MVe 1 week post-lesion. Results were similar when comparing the contralateral MVe in control animals to contralateral MVe from the other time points: There was no significant increase in H 3C mRNA levels when comparing to contralateral MVe 4 hours post-lesion, there was a significant increase detected when comparing to contralateral MVe 24 hours post- lesion, the decrease was not significant comparing to con- tralateral MVe 48 hours post-lesion, and a significant decrease was detected when comparing to contralateral MVe 1 week post-lesion. [ I]iodoproxyfan Binding Densities No significant changes were found in H receptor binding densities (Fig. 3) between ipsilateral MVe in control ani- mals (n = 3) and ipsilateral MVe of animals of different time points. The results were identical when H receptor binding densities in contralateral MVe in control animals were compared to that of contralateral MVe at different times post-lesion. On the other hand, when comparing the ipsilateral to contralateral MVe 48 hours post-lesion, a significant increase in binding densities occurred on the receptor ipsilateral side (Fig. 4 shows an example of H binding densities in a representative animal 48 h post- lesion). Discussion Knowing that there is no reliable method to determine the efficiency of a probe for its targetted sequence in an mRNA b Figure 2 ing co A secti h pr ly)ocking contr o bridiza Location of the MVe as visual bon and on x-ray image entro s: B) tion experimen H l F) H oC) H l G) Hblocking control D) ts with bslocking c of pa the vario ized on cresyl v rallel sections used in ontro ul H) s ol H igonucl HE) H iolet st I) H eoti block- ained in si de tu 3X 3B 3X 3B 3A 3C 3A 3C of interest, this study focuses instead on the pattern of A) Location of the MVe as visualized on cresyl violet stained section and on x-ray images of parallel sec- expression for the total H receptor (detected by using tions used in in situ hybridization experiments with probe H ) and its isoforms (H , H , and H ) occurring 3X 3A 3B 3C the various oligonucleotide probes: B) H C) H during the process of post-lesional plasticity in the CNS. 3X 3X blocking control D) H E) H blocking control F) 3A 3A Moreover, the probes used in this study were designed to H G) H blocking control H) H I) H blocking 3B 3B 3C 3C detect unique areas in the transcripts (H ), or junctional 3A control. Sections are from a representative animal sacrificed areas in deletion isoforms (probes H and H ) which 3B 3C 24 h post-lesion. Abbreviations are as follows: MVe, medial would make it highly unlikely for non-specific hybridiza- vestibular nucleus and Pr, prepositus nucleus. Scale bars are tion would occur. This study also includes a comparison 100 µm. Page 4 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 omiz Figure 3 [ I]iodoproxyfan binding ed rats densities in MVe of labyrinthect- [ I]iodoproxyfan binding densities in MVe of laby- rinthectomized rats. Data are presented as mean IOD ± SEM. Abbreviations are as stated in Figure 1 legend. Sample sizes are as follows: control, n = 3; 4 h post-lesion, n = 4; 24 h post-lesion, n = 4; 48 h post-lesion, n = 4; 1 week post- lesion, n = 5. *p = 0.0193. of the aforementioned patterns of expression with bind- ing densities for the H receptor. The prepositus nuclei are delineated and mentioned in legends for figures 2 and 4. These nuclei are delineated for iFigure 4 A) Location of B) section and C) on x-ray images of [ng contro at mag I]iodop nification used in an l is roxy repre the fan MVe as b siente nding experim d in this visual alyized a s image es on cresyl violet stained par ents; D) n ta high ma llel sec otn-specific bin ions u gnification a sed in nd d - the sole purpose of giving the reader an idea of the dors- A) Location of the MVe as visualized at high magnifi- oventral extent of the MVe at the levels depicted in figures cation and B) at magnification used in analyses on 2 and 4. Noteworthy, is that these nuclei are not included cresyl violet stained section and C) on x-ray images in the main functional projections in the vestibulo-ocular of parallel sections used in [ I]iodoproxyfan binding and vestibulo-spinal pathways from the brainstem vestib- experiments; D) non-specific binding control is rep- ular nucleus in mammals reviewed by Smith and resented in this image. Sections are from a representative animal sacrificed 48 h post-lesion. Abbreviations are as stated Curthoys [2]. in Figure 2 legend. Scale bars are 100 µm. There are three previously described types of neurons in the medial vestibular nucleus [41,42]; in vitro, all three types are depolarized by histamine [43,44]. Moreover, this depolarization has been shown to be exclusively mediated through postsynaptic H receptors [43,44] sug- ated inhibition of GABA release from inhibitory interneu- gesting a presynaptic localization of H receptors in the rons or glutamate release from terminals of second order medial vestibular nuclei [38]. Consequently, there are excitatory MVe neurons may underlie the restoration of three possible locations for H receptors in the MVe: 1) resting activity in the deafferentated MVe; more precisely, On the histaminergic or other incoming fibers innervating H receptor action would result in disinhibition of neu- the MVe [29-32]. 2) On terminals of the inhibitory rons in the deafferentated MVe interneurons in the MVe that make synaptic contacts on second order excitatory neurons-defined as neurons in the With respect to the first possible location, it has been vestibular nuclei that receive inputs from sensory afferents established that histamine fibers are endowed with H [45]. 3) On the terminals of second order excitatory MVe receptors that function as autoreceptors and inhibit hista- neurons making cross-commissural synaptic contacts on mine release [11,13]. Support for the notion that H contralateral MVe inhibitory interneurons [45]. With receptors are at the next two possible locations (i.e., either respect to MVe on the lesioned side, an H receptor medi- at the terminals of the inhibitory interneurons or at the Page 5 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 terminals of second order MVe neurons) comes from tor and H , and H isoform mRNA levels, with H 3A 3C 3B work showing that betahistine antagonizes the excitatory mRNA levels already increasing at 4 hours post-lesion and effect of histamine on vestibular neurons from in vitro decreasing at 24 h post-lesion, coinciding with a signifi- slice preparations of the dorsal brainstem of the rat [46]. cant increase in H receptor binding densities in the ipsi- This finding is significant given that H receptors mediate lateral MVe detected 48 hours post-lesion suggests the presynaptic inhibition of release of other neurotransmit- occurrence of one or a combination of events in the ipsi- ters including: noradrenaline, serotonin, dopamine, lateral MVe: 1) an increase in translation has occurred. 2) glutamate, GABA and tachykinins [10-13]. Unilateral lab- A change in receptor trafficking between intracellular yrinthectomy induced changes in expression levels of stores and cell membrane has occurred so that it can be receptors for glutamate (e.g., NR1 and NR2A-D subunits detected as an increase in H receptor receptor binding of the NMDA receptor [47] and GluR2-4 subunits of the densities. In either case, an increase in functional H AMPA receptor [48]) have been studied in the vestibular receptor protein coupled with an increase in receptor nuclei. Moreover, the existence of both GABA and GABA activity may lead to a restoration of resting activity in the A B receptors in the vestibular nuclei and their involvement in deafferentated MVe by scenarios already mentioned in vestibular compensation has been demonstrated by either this section. unilateral perfusion or microinjection of GABAergic ago- nists and antagonists (e.g., GABA, muscimol, and bicucul- Conclusions line) [49]. Our findings are significant given that normalization of resting activities in neurons located in the ipsilateral MVe Consequently, an H receptor antagonist such as betahis- has been shown to occur 48 hours after unilateral laby- tine could either facilitate GABA release from inhibitory rinthectomy [50]. Moreover, by 48 hours post-lesion, interneurons located in the MVe that make synaptic con- commissural disinhibition has been observed to occur tacts with second order neurons or facilitate glutamate [51]. Placement of H receptors at the terminals of either release from terminals of second order MVe neurons that GABAergic inhibitory MVe interneurons or on terminals synapse on inhibitory interneurons in the contralateral of glutamatergic second order MVe neurons with contral- MVe [31,45]. Either scenario would lead to an inhibition ateral projections should result in the observed commis- receptor activity would of second order neurons in the MVe and this would sural disinhibition as increased H explain the observation that betahistine antagonizes the result in an inhibition of synaptic release of excitatory effect of histamine on vestibular neurons [46]. neurotransmitters. On the other hand, betahistine administration is reported Our data would thus suggest that H receptors are to induce recovery with a time benefit of 2 weeks relative involved in presynaptic mechanisms resulting in a nor- to control animals after unilateral vestibular neurectomy malization of resting activities in ipsilateral MVe neurons [32,36]; this is thought to be due to a bilateral increase in which would balance discharge activity in MVe on both histamine release in the MVe [32,37]. The increased hista- sides. mine would be bound by H receptors on the perikarya of MVe neurons ipsilateral and contralateral to the lesion Methods resulting in a bilateral increase in activity. This should Animals and surgery This study was approved by the Local Committee for Ani- facilitate behavioral recovery as it is thought that an imbalance in discharge of MVe neurons (30–40 spikes/s mal Experiments and the Provincial State Office of West- in normal animals [4]) ipsilateral and contralateral to the ern Finland; in addition, animal experiments were in lesion underlies the static postural and occulomotor accordance with the European Convention (1986) guide- deficits triggered by unilateral labyrinthectomy [45]. Yet, lines and approved by the Animal Ethics Committee of as stated before, the actions of betahistine would also Abo Akademi Unviversity. Adult male Sprague Dawley extend to H receptors located on glutamatergic terminals rats (200–250 g) were used. Intraperitoneal (ip) injection of contralateral MVe neurons or on GABAergic terminals of pentobarbital (45 mg/kg ip; Mebunat, Orion, Finland) of inhibitory MVe interneurons. Antagonism of H recep- was used as an anesthetic; in addition, local anesthetic tors at either site may also act to speed recovery by increas- Lidocain (Orion, Finland) was infiltrated under the skin ing the amount of GABA output from terminals of and periosteum prior the procedure. Three steps initiated inhibitory interneurons and, as a consequence, equalizing the surgical procedure to left side of the animal's head: ret- neuronal discharge activity of ipsilateral and contralateral roauricular skin incision, opening of the middle ear bulla MVe. with a drill, and removal of the ossicular chain with the aid of a microscope. Unilateral labyrinthectomy was car- The trends toward bilateral increases (24 h post-lesion) ried out by opening the horizontal semicircular canal duct followed by decreases (48 h post-lesion) in total H recep- in the temporal bone, drilling through the horizontal and Page 6 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 anterior semicircular canal ampullae and aspirating the tion (10 cpm/ml) was carried out at 50°C for 16 to 20 contents of the vestibule. 100% ethanol was injected into hours in a humidified chamber. Posthybridization washes the opened labyrinth to finalize the procedure; finally, the were carried out as described previously [55]. Brain sec- wound was sutured. The sham operation entailed opening tions from control animals and animals 4 h, 24 h, 48 h, the middle ear bulla and leaving the ossicular chain intact and 1 week post-lesion were treated simultaneously with prior to suturing the wound, as described in Cameron and their respective oligonucleotide probe. Sections and car- Dutia [52]; the control animals (n = 4) that underwent the bon-14 standards were exposed to Kodak BioMax X-ray sham operation were killed 4 h later. The method is films (Kodak, USA) for 10 days. explained hereafter. All rats included in the experiments displayed symptoms characteristic of animals that have Receptor binding autoradiography undergone unilateral labyrinthectomy (e.g., barrel rota- Autoradiographic localization of [ I]iodoproxyfan bind- tion, circling behavior, and spontaneuos nystagmus). ing sites has been described before [56]. Briefly, slide These symptoms gadually disappeared during the first two mounted tissue sections were preincubated for 15 min in or three days and were completely absent within one 50 mM Na HPO -KH PO phosphate buffer, pH 6.8, con- 2 4 2 4 gas and killed by week. Animals were stunned by CO taining 0.1% bovine serum albumin and 1 µM S132 (a 1- decapitation 4 h (n = 4), 24 h (n = 4), 48 h (n = 4) and 1 substituted imidazole derivative displaying a very low week (n = 5) after labyrinthectomy. affinity at H receptors and used to decrease non-specific labelling). The sections were then incubated for 1 hour at Tissue preparation room temperature in the same buffer containing 15 pM After the mentioned decapitation, brains were removed, [ I]iodoproxyfan (Amersham Pharmacia Biotech UK frozen in isopentane (-25°C), and stored at -70°C. Tis- Limited, England). Non-specific binding was determined sues were then cut to 20 µm thick cryosections, thaw by incubating consecutive sections in the presence of 1 mounted onto poly-L-lysine coated slides (Menzel-Gläser, µM (R)α-methylhistamine (Sigma-Aldrich, Germany). At Germany), and stored at -70°C until used. the completion of incubation, the tissues were washed four times (4 min each) in the same fresh ice-cold buffer, In-situ hybridization histochemistry dipped into ice-cold water, and dried under a current of The oligonucleotide probes used for in situ hybridization air. Brain sections from control animals and animals 4 h, were designed so that they specifically recognized the dif- 24 h, 48 h, and 1 week post-lesion were treated simulta- ferent H receptor isoform mRNAs (H , H , and H ; as neously. Dried sections, along with standards, were 3 3A 3B 3C described in [24]); an additional oligonucleotide probes exposed to Kodak BioMax X-ray films (Kodak, USA) for 2 was used to detect all characterized H receptor isoforms days. (H ; as described in [24]). Sequences for H , H , H , 3X 3X 3A 3B and H probes have been previously published [24]. It is Image analysis and statistics 3C noteworthy that the isoform-specific probes detect selec- Autoradiographic films were quantified by digitizing the tively the various deletion forms of the third intracellular film images with a computer based MCID 5+ image anal- loop, but do not differentiate between the possible alter- ysis system (Imaging Research, Canada) and by measur- native C-termini of the H receptor isoforms [53]. How- ing gray scale pixel values. The relative optic density was ever, it has been shown that the differences in the third converted to integrated optic density (IOD) based on a intracellular loop are significant for coupling to intracellu- standard curve derived from standards exposed to films. lar second messengers [24]. As a control, we used a nor- Gray scale values were determined from four sections for mal hybridization mixture with a 100-fold excess of each animal, measurements from white matter of each unlabeled specific probes. As an additional control, we respective section were subtracted to obtain the final val- used a Staphylococcus aureus chloramphenicol acetyltrans- ues, and the data were analyzed using either a paired t-test ferase-specific oligonucleotide probe. The hybridization or one-way ANOVA combined with Bonferroni's Multiple procedure used has been described before and was used Comparison Test as a post-hoc test. Significance was with minor modifications [54,55]. All probes were determined when p < 0.05. labeled with [ S]deoxyadenosine 5'-α(-thio) triphos- phate (New England Nuclear, USA) at their 3' ends using Authors' contributions terminal deoxynucleotide transferase (Promega, USA). AL assisted with the surgeries and tissue collection, sec- Nonincorporated nucleotides were removed by purifica- tioned all of the brains, carried out the in situ hybridiza- tion through Sephadex G-50 columns. tion and binding studies, performed all of the image and statistical analyses, and participated in the design of the Before hybridization, cryosections were taken from the - study. AAA performed the surgeries, assisted with tissue 70°C environment and kept at room temeprature for 10 collection, assisted with image analyses, and participated min and treated with UV light for 5 min. The hybridiza- in the design of the study. KK established the method of Page 7 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:32 http://www.biomedcentral.com/1471-2202/5/32 pharmacological profiles. J Pharmacol Exp Ther 2000, in situ hybridization in the lab, designed the oligonucle- 293:771-778. otide probes, assisted with surgeries and tissue collection, 20. Chen J, Liu C, Lovenberg TW: Molecular and pharmacological and participated in the design of the study. HS synthe- characterization of the mouse histamine H3 receptor. Eur J Pharmacol 2003, 467:57-65. sized and supplied rare chemicals required for the binding 21. Tardivel-Lacombe J, Rouleau A, Heron A, Morisset S, Pillot C, study. PP acquired funding, coordinated the study, and Cochois V, Schwartz JC, Arrang JM: Cloning and cerebral expres- sion of the guinea pig histamine H3 receptor: evidence for participated in its design. All authors read and approved two isoforms. Neuroreport 2000, 11:755-759. the final manuscript. 22. 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