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Urotensin II acts as a modulator of mesopontine cholinergic neurons
Brain Research 1059 (2005) 139 – 148 www.elsevier.com/locate/brainres
Research Report
Urotensin II acts as a modulator of mesopontine cholinergic neurons Stewart D. Clark a,f, Hans-Peter Nothacker b, Charles D. Blaha c, Christopher J. Tyler d, Dee M. Duangdao b, Stephen L. Grupke d, David R. Helton e, Christopher S. Leonard d, Olivier Civelli a,b,* a
Department of Developmental and Cell Biology, University of California, Irvine, CA 92697-4625, USA b Department of Pharmacology, University of California, 369 MSRII, Irvine, CA 92697-4625, USA c Department of Psychology, University of Memphis, Memphis, TN 38152, USA d Department of Physiology, New York Medical College, Valhalla, NY 10595, USA e Cenomed Inc., 22865 Lake Forest Drive, Lake Forest, CA 92630, USA f Laboratory of Molecular Neurobiology, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8 Accepted 9 August 2005 Available online 23 September 2005
Abstract Urotensin II (UII) is a vasomodulatory peptide that was not predicted to elicit CNS activity. However, because we have recently shown that the urotensin II receptor (UII-R) is selectively expressed in rat mesopontine cholinergic (MPCh) neurons, we hypothesize that UII may have a central function. The present study demonstrates that the UII system is able to modulate MPCh neuron activity. Brain slice experiments demonstrate that UII excites MPCh neurons of the mouse laterodorsal tegmentum (LDTg) by activating a slow inward current. Furthermore, microinfusion of UII into the ventral tegmental area produces a sustained increase in dopamine efflux in the nucleus accumbens, as measured by in vivo chronoamperometry. In agreement with UII activation of MPCh neurons, intracerebroventricular injections of UII significantly modulate ambulatory movements in both rats and mice but do not significantly affect startle habituation or prepulse inhibition. The present study establishes that UII is a neuromodulator that may be exploited to target disorders involving MPCh dysfunction. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptides: anatomy and physiology Keywords: Urotensin II; Mesopontine cholinergic; Electrophysiology; Chronoamperometry; Locomotion; Prepulse inhibition
1. Introduction Urotensin II (UII) is a vasomodulatory peptide that has been recently shown to be the natural ligand of a G proteincoupled receptor formerly called GPR14 [1,25,28,30], now
Abbreviations: Icv, intracerebroventricular; LDTg, laterodorsal tegmentum; MPCh, mesopontine cholinergic; NAc, nucleus accumbens; PPTg, pedunculopontine tegmentum; PPI, prepulse inhibition; UII, urotensin II; UII-R, urotensin II receptor; VTA, ventral tegmental area * Corresponding author. Department of Pharmacology, University of California, 369 MSRII, Irvine, CA 92697-4625, USA. Fax: +1 949 824 4855. E-mail address: [email protected] (O. Civelli). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.026
known as the urotensin II receptor (UII-R). Although UII is well known for its vascular effects (for review see [9]), several reports have indicated that it may also have effects in the central nervous system [7,11,17,25,27]. In particular, we have shown that the UII-R is selectively expressed in mesopontine cholinergic (MPCh) neurons of the rat brain [7], inferring that UII can modulate the activity of MPCh neurons. In addition, the fact that UII-R activation elicits increases in cytoplasmic calcium ion concentrations in vitro [1,24,30] indicates that UII could promote cell depolarization. Studies have measured the effects of UII after injections into the brain [17,23,25,27], and our recent data indicate UII promotes REM sleep by exciting MPCh neurons of the pedunculopontine tegmental nucleus (PPTg; [18]). Here, we
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have extended this analysis by examining UII actions on an additional group of MPCh neurons in the laterodorsal tegmental (LDTg) nucleus and on other functions associated with MPCh neurons. Activation of the PPTg and LDTg is thought to contribute to a number of behavioral and physiological phenomena. For example, Garcia-Rill et al. [15] demonstrated that electrical stimulation of these regions elicited stepping behavior in the rat. In addition, lesions or electrical inhibition of the mesopontine tegmentum in non-human primates can induce ataxia [22,29]. Together, these studies have suggested that the brainstem area encompassing MPCh neurons profoundly influences motor function in mammals [38]. MPCh neurons have also been implicated in the modulation of the sensorimotor reactivity. Garcia-Rill et al. [16] predicted that activation of neurons in the mesopontine would reduce habituation of the startle reflex to a loud auditory stimulus. MPCh neurons are also intimately involved in prepulse inhibition (PPI), a phenomenon in which the amplitude of the startle reflex is reduced by the presence of a preceding muted warning stimulus (for review see [10,20]). Elucidating the role of MPCh neurons in PPI is of great interest because PPI deficits are a hallmark and diagnostic symptom in many neuropsychiatric disorders (for review see [20]). It is believed that the MPCh input from the PPTg to the caudal pontine nucleus inhibits the startle reflex [21], as destruction of the PPTg impairs PPI [21,34]. Therefore, we predict that agents such as UII, which potentially modulate MPCh activity, should influence both the startle reflex and PPI. Several diverse techniques were employed to establish UII as a modulator of MPCh function. First, the response of MPCh neurons to UII stimulation was determined in vitro. Then, to test whether UII activates neurons in vivo, chronoamperometric recordings of dopamine efflux in the nucleus accumbens (NAc) were carried out after UII microinjection into the ventral tegmental area (VTA). Electrical stimulation of MPCh neurons located in the region of the PPTg and LDTg has been shown to induce increases in basal dopamine efflux in the striatum and NAc [12,13]. Furthermore, the MPCh neurons constitute the only known cholinergic excitatory input to the VTA dopaminergic cells [31]. Given that our previous study showed that the VTA contains binding sites for radiolabeled UII [7], it was expected that UII injected into the VTA would produce changes in NAc dopamine efflux. Lastly, behavioral paradigms associated with MPCh activity were used to extend the anatomical and neurochemical findings.
2. Materials and methods 2.1. In situ hybridization Sources of the following materials were: bovine serum albumin, polyvinylpyrolidone, poly-l-lysine, RNase A (Sigma, St. Louis, MO); pBluescript SK (Stratagene, La
Jolla, CA); pCR 4-TOPO (Invitrogen, Carlsbad, CA); antidigoxygenin (dig)-AP Fab antibody, dig-dUTP, Genius system nonradioactive nucleic acid detection kit, restriction enzymes, T3, T7 polymerases, proteinase K, and yeast tRNA (Roche Molecular Biochemicals, Indianapolis, IN); formamide (Fluka, Ronkonkoma, NY); dextran sulfate (Pharmacia, Piscataway, NJ); sodium acetate (Fisher Scientific, Pittsburgh, PA); Hyperfilm hmax (Amersham, Arlington Heights, IL); nuclear track emulsion (NTB2) (Kodak, Rochester, NY); [35S]-Uridine triphosphate ([35S]-UTP) (Dupont NEN, Boston, MA). 2.1.1. Tissue preparation CD-1 mice weighing approximately 18 –30 g (Charles River, San Diego, CA), kept on a 12-h light/dark cycle with food and water ad libitum, were decapitated and brains were removed. Tissues were frozen immediately by immersion in 20 -C isopentane, then stored at 70 -C until used. All tissue removal procedures were approved by the Institutional Animal Care and Use Committee and were consistent with Federal guidelines. Twenty-micron sections were cut using a cryostat and mounted onto Vectabond (Vector Laboratories) coated slides. Fixation was performed (4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4) for 1 h at 22 -C. Slides were then rinsed in PB, air dried, and subsequently stored with desiccate at 20 -C. 2.1.2. Hybridization All sections and probes were prepared as described by Clark et al. [7]. Briefly, sections were incubated with proteinase K (1 mg/mL) for 10 min at 22 -C, then acetylated, and dehydrated through graded ethanol, followed by air-drying. Sections were exposed to a 1:1 dilution of digoxigenin-labeled anti-sense choline acetyltransferase (ChAT) riboprobe (0.2 ng/mL):[35S]-labeled UII-R sense or anti-sense probes (2 107 cpm/mL) in hybridization solution (50% formamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA, 500 mg/mL tRNA, 10 mM DTT, 0.3 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) then incubated overnight at 60 -C. Incubation with RNase A (20 mg/mL) for 30 min at 37 -C followed the hybridization. The sections were then washed 2 5 min and 2 10 min in solutions of decreasing salinity at 22 -C and a 30 min wash in 0.1 SSC at 68 -C. After the hot wash, the slides were processed for digoxigenin labeling as per the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Slides were then apposed to Bmax film for development of autoradiograms. Following film development, slides were coated with 3% parlodion in isoamylacetate and dipped in liquid NTB2 emulsion on the reference date of the [35S]-UTP. Slides for each probe were incubated according to the exposure time on film: 1 day on film, 1 week incubation with emulsion. After the appropriate exposure period, slides were developed in Kodak D19, fixed, coverslipped, and analyzed.
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2.2. Slice and patch clamp recording Brain slices were prepared and whole-cell recordings were made as described previously in detail [6]. Briefly, 300 Am slices containing the LDTg were prepared from isofluorane anesthetized C57/BL6 mice (P14 –P21; Taconic, Charles River) in ice-cold, carbogen-equilibrated, artificial cerebrospinal fluid (ACSF) which contained the following (in mM): 121 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 1.2 MgSO4, 26 NaHCO3, 20 dextrose, 4.2 lactic acid. Using a fixed-stage microscope (Olympus BX50WI), the boundaries of LDTg were first determined by low magnification inspection and then neurons to be recorded were visualized with a nuvicon camera (Dage VE-1000) using a 40 objective and IR-DIC optics. The submerged-slice recording chamber was perfused at 3 – 5 mL/min with room temperature ACSF. Human urotensin II (Bachem) was dissolved into the perfusate (300 nM) just before the experiment and was applied by bath superfusion. Giga-seal whole-cell voltage-clamp recordings of LDTg neurons were made with pipettes pulled from 1.5-mmdiameter glass capillary tubing (Corning 7052, A-M systems) using an Axopatch 200B amplifier (Axon Instruments). The pipette solution contained (in mM) 144 Kgluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 0.3 NaGTP, 4 Na2ATP. Biocytin Alexa Fluor 594 (25 AM; Molecular Probes) was included in the patch solution. Membrane voltages and currents were controlled and recorded with a computer running PCLAMP8 software (Axon Instruments). Access resistance (R acc) was estimated on-line using the PCLAMP8 membrane-test routine. The quality of recorded cells was assessed by monitoring input resistance, holding current, and capacitance and recordings were terminated if the estimated R acc was >30 MV, became unstable, or changed by more that 20% between measurements. Recordings were uncompensated for series resistance errors since the recorded currents and associated voltage errors were small. Current measurements were filtered at 2 KHz and sampled at 5 KHz. Extracellular spiking was recorded from visualized neurons in current-clamp mode with the same pipettes used for whole-cell recording. The pipettes were filled with extracellular solution and were loosely attached to the soma of the visualized neuron by weak suction for the duration of the recording. 2.2.1. Immunocytochemistry Cell identification was performed by immunocytochemistry as previously described in detail for bNOS [6]. Briefly, following successful removal of the patch pipette from the Alexa Fluor-filled neuron, the slice was fixed in 4% paraformaldehyde for 1 – 3 days at 4 -C, cryoprotected by equilibration in 30% sucrose (0.01 M PBS) and resectioned (40 Am) on a freezing microtome. Sections were incubated in rabbit polyclonal anti-bNOS and visualized with FITC-conjugated goat anti-rabbit IgGs
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(Chemicon; 1:50 in PBS) to determine if recorded cells were bNOS-immunoreactive. 2.3. Chronoamperometric recordings Male hooded Wistar rats, weighing 200 –250 g (Animal Resources Center, Adelaide, SA, Australia), were housed in pairs with constant temperature (22 T 0.5 -C), 12-h light/ dark cycle (lights on at 08.00 h), and food and water ad libitum. Rats were mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) after anesthetic plane was reached (1.5 g/kg i.p. urethane). Body temperature was maintained by use of temperature regulated heating pad (TC-831, CWE, NY, USA). For complete methods and materials, please refer to earlier publications by CD Blaha [12]. Bilateral 31 g stainless steel guide cannulae were implanted 1 mm above the VTA (interaural coordinates: AP + 2.96, ML + 0.5 mm, and DV + 1.7 mm; [32]). To facilitate the measurement of in vivo changes in dopamine oxidation current without interference from other oxidizable compounds in brain extracellular fluid [2,4,5], a single stearate-modified graphite paste electrochemical recording electrode [3] was implanted into the NAc (coordinates: AP + 1.2 mm from bregma, ML + 1.2 mm and DV 6.5 mm from dura; [32]) and an Ag/AgCl reference and stainless-steel auxiliary electrode combination was placed in contact with contralateral cortical tissue 4 mm posterior to the bregma. Repetitive chronoamperometric measurements of dopamine oxidation current using an electrometer (Echempro, GMA Tech. Inc., Vancouver, BC, Canada) were made by applying a potential pulse from 0.15 V to +0.3 V vs. Ag/AgCl to the recording electrode for 1 s at 30-s intervals, and monitoring the oxidation current at the end of each 1-s pulse [5]. At the end of at least 60 min of baseline chronoamperometric recording, UII or vehicle was applied at a rate of 0.5 AL/min with a final volume of 1 AL in concentrations appropriate to administer 1, 10, or 100 nmol of UII into the VTA. The magnitude of drug-induced changes in NAc dopamine efflux was calculated from pre-injection baseline dopamine oxidation current values normalized to zero current and was expressed as a percent change with respect to a mean baseline current value derived as in Forster and Blaha [12]. For each UII dose condition, the resulting peak oxidation current values were averaged across animals and compared with respective pre-drug baseline levels using two-tailed paired t tests (alpha level set at 0.01), using separate animals for each injection and each dose. UII was from the same source and prepared in the same manner as for the behavioral studies. Rats were euthanized with a 0.5 mL cardial infusion of urethane (350 mg/mL). Brains were removed and immersed overnight in 10% buffered formalin, and then were cryoprotected by storing the tissue in a 30% sucrose/10% formalin solution. Coronal sections (60 Am) were cut on a freezing microtome, and subsequently processed for cresyl
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violet stain. Tracts due to cannulae and electrodes were visualized on a light microscope. 2.4. Behavioral studies Animal experiments were performed on male CD-1 mice (Charles River, California) approximately 8– 12 weeks of age weighing 18– 30 g at the start of experiments. All behavioral and injection procedures were approved by the Institutional Animal Care and Use Committee and were consistent with federal guidelines. Mice were housed 4 per cage with water and food available ad libitum, in a room maintained under constant temperature (20 – 21 -C) and humidity (40 – 45%). Mice were maintained on a 12-h light/dark cycle with lights on –off at 07:00 – 19:00 h and all behavioral testing was conducted during the light phase (between the hours of 09:00 and 17:00). Sprague – Dawley rats were purchased with implanted intracerebroventricular (icv) cannula (Charles River, California) weighing approximately 240 –270 g, and were housed individually. The rats were housed in separate rooms as the mice, but under the same conditions. 2.4.1. Drugs Human urotensin II was obtained from Bachem (California), and mixed with 0.9% saline for a final concentration of 4 mM. The pH was adjusted with NaOH to a final pH of 6.9. Further dilutions as required for treatment were done using 0.9% saline. UII was delivered into the lateral ventricle of the rat through a chronically implanted cannula (10 AL over 2 min). The injection of UII into mice (5 AL) was performed by a freehand icv injection method. The resulting puncture in the skull was verified to be 2 T 0.5 mm from bregma and 2 T 0.5 mm from midline by visual inspection of euthanized subjects at the completion of the experiment. The following drugs were obtained from Sigma RBI, St. Louis, MO: apomorphine and phencyclidine (PCP), and were dissolved in 0.9% saline containing 1% Tween-80 for s.c. injections. 2.4.2. Spontaneous activity Activity levels (ambulatory and nonambulatory) were recorded using a Hamilton-Kinder Activity Monitor (with the infrared beams in a 4 8 configured frame, San Diego, California). Activity levels were quantified in transparent polycarbonate cages (32 26 20 cm) placed within the activity frames. Animals were acclimatized to the novel home cage environment for 60 min before injections, then were removed for injections and immediately replaced to the same cage for monitoring. Activity was measured for 60 min after the administration of either vehicle or test compound. 2.4.3. Sensorimotor reactivity Sensorimotor reactivity (startle response) is a crossspecies intrinsic reflex response exhibited when a sudden, high-density acoustic stimulus is presented to a subject. The amplitude of the startle response was measured in Newtons
by accelerometers within the chambers (Hamilton-Kinder, California). Habituation to the startling stimulus was calculated for each dose group by taking the average of the responses to the 120-dB stimulus trials in bins of five from a session that included 25 120-dB stimulus trials separated by 8-s intervals, preceded by a 5-min acclimatization period (65-dB background). UII was administered either 5 or 25 min prior to placement of the subject into the chamber. 2.4.4. Prepulse inhibition Prepulse inhibition refers to a reduction in startle amplitude when a startle-inducing stimulus is preceded by a weaker non-startling warning stimulus. Computer-controlled startle chambers (Hamilton-Kinder, San Diego, California) were used for this assay. Sound levels were checked with a decibel meter prior to each day’s experimentation. The noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (PCP) and the dopamine agonist apomorphine reduce PPI of the acoustic startle response in rodents. In apomorphine challenged trials, UII was administered 25 min prior to placement of the animals in the chambers, with apomorphine being injected subcutaneous (s.c.) 20 min after UII. PCP was administered s.c. 30 min prior to placing the animals into the chambers, with UII being injected icv 10 min after PCP. Following a 5-min acclimatization period with background white noise (65 dB), animals were exposed to six different trial types. Trials were presented ten times each in a quasi-random order, with quasi-randomized 5- to 25-s inter-trial intervals. Trials were: stimulus only trial (120 dB white noise, 50 ms stimulus); three different prepulse + pulse trials, in which a 20 ms 5-dB, 10-dB, or 15-dB stimulus above a 65-dB background preceded the 120-dB pulse by 120 ms (onset to onset); a 15-dB prepulse without a 120-dB pulse; and a no stimulus trial, in which only the background noise was presented. The average response for each dose group and each trial type was calculated and presented. The amount of PPI was calculated as a percentage score for each animal per prepulse trial type: % prepulse inhibition = [(stimulus only 5-dB or 10-dB stimuli) / stimulus only] 100. 2.4.5. Statistical analysis The results of the behavioral tests were analyzed by a between groups analysis of variance (ANOVA), with subsequent Tukey – Kramer HSD. The software used to perform these analyses was JMP ver. 4.0.0 (SAS Institute, Inc.).
3. Results 3.1. Expression of the UII-R in mouse MPCh neurons To determine whether UII-R is selectively expressed in mouse MPCh neurons as reported for the rat, in situ hybridization of the UII-R was performed. Coronal sections
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of mouse brains were hybridized with UII-R [35S]-cRNA to produce a brain map of UII-R mRNA expression. This resulted in specific anti-sense signal in the PPTg and LDTg (Fig. 1A). In subsequent double in situ hybridizations, with a DIG-labeled cRNA probe for ChAT, it was found that UIIR colocalizes with the cholinergic neurons of the MPCh (Figs. 1C, D). This colocalization of UII-R with ChAT was seen in the majority of cases of UII-R anti-sense labeled cell bodies (non-quantitative observations). These findings demonstrate that the mouse UII-R mRNA localization mirrors that of the rat [7] and make possible the comparison of UII-induced behaviors in mice and rats. 3.2. Slice recordings of LDTg neurons Due to the selective expression of UII-R in the LDTg and PPTg, we hypothesized that UII would excite cholinergic LDTg and PPTg neurons. Therefore, we examined the effect of bath superfusion of UII on LDTg neurons under wholecell voltage-clamp conditions ( 60 mV holding potential) in a brain slice preparation in normal ACSF. Superfusion with ACSF containing 300 nM UII evoked a slow inward shift of the holding current in 12/19 LDTg neurons ( 31.0 T 5.4 pA; mean T SEM; n = 12; Fig. 2A). Seven of these Alexa Fluor-filled neurons were recovered and all were bNOS+ (n = 7). Since this inward current would depolarize unclamped neurons, these data suggest that UII excites MPCh neurons. Indeed, UII application approximately doubled the firing of a spontaneously firing LDTg neuron recorded extracellularly (Figs. 2D – F). Of the 7 LDTg neurons that did not respond to UII, five were recovered.
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Interestingly, two were bNOS+ and the other three were bNOS . These data indicate that UII excites some but not all bNOS positive LDTg neurons and suggest that bNOSLDTg neurons do not respond to UII. 3.3. Chronoamperometry recordings To determine whether UII can induce VTA-mediated dopamine efflux in the NAc, UII was microinjected into the VTA. All injections of UII produced changes in basal dopamine oxidation currents, with increasing doses increasing both the magnitude and duration of the response. When administered at a dose of 1 nmol, UII produced a peak change in basal extracellular dopamine levels of 45% from baseline at 13 min (n = 8), while 10 nmol showed maximal change (185%) at 60 min post-injection (n = 6) (Fig. 3). After injection of 100 nmol, the change in basal dopamine concentrations steadily rose for the entire 3-h recording period to 600% of baseline levels and appeared to plateau at that time (n = 6) (Fig. 3). All three doses display the same onset dynamic, and therefore are most likely the same phenomena, but of different intensity. If the 100 nmol dose continues to act with the same dynamic as the lower doses, it would be expected that dopamine oxidation currents would return to baseline at 7.5 h, well beyond the ethical and practical endpoint of the animal preparation. Doses used for the chronoamperometric studies were kept similar to those that were used for behavioral studies. These doses were used to counter a number of factors which could serve to dampen the effectiveness of the peptide infused in vivo, as apposed to bath applied in vitro. Firstly, peptides do not diffuse easily through extracellular space, being somewhat restricted to the site of injection. Second, the degradation kinetics of UII in the CNS is unknown. It is also possible that the anesthetic (urethane) may have dampened the effect and that in an awake animal the effects of 1 nmol may have been more dramatic. In pilot studies, it was also observed that the administration of UII into the PPTg increased dopamine oxidation currents in the striatum. UII can elicit robust changes in dopamine efflux in a dose-dependent manner through established MPCh circuitry, by the stimulation of brain areas previously shown to express UII-R. 3.4. Spontaneous locomotion
Fig. 1. UII-R is expressed in mouse MPCh neurons. Autoradiogram of a single representative CD-1 mouse brain section processed by double in situ hybridization, showing specific hybridization of [35S]-UII-R anti-sense cRNA (A), with the signal being absent when the [35S]-UII-R sense cRNA probe is used (B). Emulsion dipped slides reveal that the UII-R (dark field) (C) is localized in ChAT containing neurons (D) (DIG labeled ChAT cRNA probe, anti-sense) (bright field).
Animals were acclimatized to the testing situation for 1 h to lessen exploratory activity. Mice exhibited a very profound reduction in spontaneous movement immediately after injection that lasted for up to 20 min (Fig. 4A). It was visually observed that UII-treated mice would stop movement mid-stride or when rearing with forepaws on the sides of the enclosure, and these phenomena were seen at doses as low as 10 pmol (data not shown). At high doses, an increase in spontaneous movement was seen in both rats and mice (Figs. 4A and B), with the largest effect at 25 –30 min postinjection in mice, and a doubling seen rats (Fig. 4C). There
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Fig. 2. UII excites LDTg neurons by activating a slow inward current. (A) Membrane current from a mouse LDTg neuron clamped near resting membrane potential ( 60 mV holding potential) before and after bath superfusion of 300 nM UII. The slow inward current, which would cause membrane depolarization in unclamped neurons, was observed in bNOS+ neurons. (B) Low-power fluorescent image of a coronal section. Green fluorescent neurons illustrate the distribution of bNOS+ neurons of the left and right LDTg from the recorded brain slice. The recorded neuron (C) was in the right LDTg. (C) Left panel shows the neuron from which the recording in panel A was obtained (Alexa-594; red). Center panel shows the same field with cholinergic neurons visualized by bNOS immunocytochemistry (FITC, green). Right panel shows the two images merged revealing that the recorded neuron was immunoreactive for bNOS (yellow). Bath application of UII increased the firing of an LDTg neuron recorded extracellularly. (D) DIC image of extracellularly recorded neuron in the LDTg. (E) 10-s epochs of spiking recorded from cell illustrated in panel D taken before (top trace) and after (bottom trace) UII application. (F) Histogram (10 s bins) of average firing rate shows the slow and long lasting increase in firing produced by UII. Arrows indicate epochs illustrated in panel E before (1) and after (2) application. UII approximately doubled the spiking in this neuron. Abbreviations: Aq, aqueduct; LDTg, laterodorsal tegmental nucleus; DT, dorsal tegmental nucleus.
is an early onset species-specific reduction in movement immediately after UII injection into mice. As well, there is an increase in spontaneous movement with a high dose of UII (10 nmol) in both rats and mice. We and others [17] have found that approximately 1 nmol is the highest dose that does not produce a significant effect on ambulations. Therefore, 1 nmol was used for subsequent studies, where ambulations or heightened activity could interfere with measures of startle and PPI. 3.4.1. Startle habituation and prepulse inhibition MPCh neurons are thought to be involved in the modulation of the startle reflex, and specifically PPI. Therefore, due to the selective expression of UII-R by MPCh neurons, it was hypothesized that UII administration would modulate the animals’ response to startling auditory stimuli. UII had no effect on startle habituation in mice and rats, when tested 5 (data not shown) or 25 min after injection (Figs. 5A, B). There was also no modulation of PPI when increasing doses of UII were administered 5 min (data not shown) or 25 min prior to testing (Fig. 5C). Furthermore, UII was unable to rescue the PPI deficit
induced by either apomorphine injection (0.3 mg/kg) (Fig. 5D) or PCP (3 mg/kg) (Fig. 5E).
4. Discussion To determine whether the selective expression of the UIIR in MPCh neurons is a species-independent phenomenon, UII-R expression was analyzed in the mouse brain by in situ hybridization. It was found that UII-R is expressed in mouse MPCh neurons, as has been previously reported in the rat [7]. This led us to determine whether UII can modulate synaptic transmission in the mouse. Superfusion of UII over mouse brain slices was found to cause depolarization of cholinergic LDTg neurons. These data, and similar data collected in rats [18], are the first demonstration that UII excites supraspinal neurons. To further analyze whether UII is able to regulate neurotransmitter release, we chose to study the effects of UII on dopamine release. Principal excitatory inputs to the VTA arise from MPCh neurons. These neurons are thought to be able to release either glutamate or acetylcholine [13] (along with other ligands [36,37]) to modulate the function of dopaminergic neurons.
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Fig. 3. UII microinjected into the VTA increases NAc dopamine oxidation current. Microinjections of UII (1 AL of indicated concentration) into the VTA were done using separate animals for each injection and each dose. The magnitude of the change in dopamine oxidation currents recorded in the NAc increased with increased concentration of UII injected into the VTA. A dose of 1 nmol UII (n = 8) produced a 45% increase in basal NAc dopamine efflux. 10 nmol (n = 6) and 100 nmol (n = 6) of UII increased dopamine efflux in the NAc by 185% and 600%, respectively.
The stimulation of the dopaminergic neurons of the VTA is known to increase dopamine efflux in the NAc [12]. In our previous study, it was found that [125I]-UII bound to receptors located in the VTA [7]. Therefore, it was expected that microinfusion of UII into the VTA would modulate the firing of these dopaminergic neurons. Indeed, we found that UII was able to dose-dependently increase basal levels of dopamine efflux in the NAc upon discreet injection into the VTA. Due to the fact that the VTA does not express UII-R transcripts, we believe that this response results from the activation of presynaptic UII-Rs. Further studies will be needed to confirm this hypothesis. Together with the neural activation studies, the selective expression of UII-R in MPCh neurons suggests that exogenous UII when administered in vivo should facilitate MPCh-mediated behavioral phenomena in rats and mice. Our demonstration that UII is able to induce VTA-mediated dose-dependent dopamine efflux in the NAc suggests that UII may have a role in motor function, learning, and motivation (see [39] for review). Schwienbacher et al. [33] have demonstrated that extracellular dopamine levels increase in the NAc during locomotion, and Kelley et al. [19] showed that amphetamine injected into the NAc produced a dose-dependent increase in locomotion. Microinfusion studies into the VTA of awake animals will be necessary to determine whether UII-induced increases in NAc dopamine correlate with the measured locomotor activity.
Fig. 4. UII-induced locomotor effects. Animals were acclimatized for 1 h, then UII was injected (icv) and ambulatory movements were recorded for 1 h. There was a dramatic decrease in the number of ambulatory movements when UII was injected into mice, as compared to when saline was injected (A) (n = 8) (*P < 0.05). A later increase in spontaneous movement was seen (A). In rats, UII dose-dependently increased the number of ambulations (B) (n = 10). This phenomenon was significant when the total number of ambulations for the 1 h was compared to when saline was injected (C) (*P < 0.05).
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Fig. 5. UII influence on startle habituation and prepulse inhibition. Animals were injected icv 25 min prior to placement into the startle monitors. There were no significant changes in habituation to a startling stimulus in either mice (A) (n = 10) or rats (B) (n = 9). When rats were pretreated with UII 25 min before beginning the PPI trials, there was no significant differences in PPI (C). Rats were pretreated with UII (20 min before apomorphine) then injected s.c. with 0.3 mg/kg of apomorphine 5 min prior to placement in the startle monitor. When PPI is expressed as a percent of the stimulus only group (D), there was a significant effect of apomorphine ( P < 0.05), but no significant influence of UII either on its own or when challenged with apomorphine (n = 10) ( P < 0.05). Rats were pretreated with PCP 5 min prior to UII, which was administered 25 min prior to placing the animal into the startle monitor. PCP significantly disrupted PPI ( P < 0.05). However, there was no significant improvement of PPI when PCP was challenged with UII (E) (n = 11).
In view of the important cardiovascular function of UII, it is also possible that the behavioral effects described here may be consequences of UII effects on cortical blood flow. Lin et al. [23] have found that UII icv injections in conscious rats lead to increases in heart rate but these were only observed at higher doses (10 nmol). At the dose that we used (1 nmol), regulation of heart rate is not expected to be a factor but furQ
ther studies will need to be carried out to address this issue. Taken together with the studies that have shown that movement is elicited or inhibited by stimulation or lesioning the mesopontine tegmentum, respectively [14,22], it is not surprising that UII modulates spontaneous locomotion. Though, studies where spontaneous movement is measured during microinfusion of UII into the MPCh must be performed.
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In the present study, the potent depression of movement in mice during the first 15 min after UII injection was not observed in the Sprague –Dawley rats. Previously, Gartlon et al. [17] observed mild tonic seizures, followed by hypoactivity, after icv injections of UII in Sprague – Dawley rats, at doses of UII higher than those used here. In addition, unpublished reports from other labs suggest that Wistar rats also show a significant initial decrease in spontaneous movement after UII injections (icv) [26]. Therefore, the deficit in spontaneous movement seen in CD-1 mice may not be a species-specific phenomenon, but may rather reflect differences in dose sensitivity between strains. Despite the ability of UII to activate mesopontine tegmental neurons, UII did not alter or modulate PPI. Several factors may bear this observation. First, UII may be unable to enhance the function of the PPTg over that of the normal state and hence not produce PPI deficits that are demonstrative of a pathological state. Also, the pharmacological challenges that were used to disrupt PPI may not be sufficient to reveal UII’s ability to modulate PPTg function. Alternatively, the disruption of endogenous UII signaling may produce PPI deficits, as does the lesioning of the PPTg [21,34]. However, the lack of a commercially available, selective, high potency, UII-R antagonist has so far impeded the investigation of this possibility. The MPCh neurons are thought to regulate PPI via an efferent to the pontine reticular formation [21]. It could be that the icv injection of UII activates all CNS UII-Rs closely in time, producing an overall activation of the UII system that masks a specific MPCh-pontine phenomenon. Perhaps the activation of this component alone or the use of UII-R antagonists would be more enlightening as to whether UII has a role in modulating startle habituation or PPI. The MPCh system is also known to produce and maintain cortical desynchronization, a phenomenon seen during both the wakeful and REM sleep periods in mammals. Recently, presumed cholinergic cells of the PPTg were recorded in freely moving rats and it was found that discharges of these cells correlate with wakefulness and REM state [8]. Electrical stimulation of the LDTg has also been shown to increase REM sleep in cats [35]. Due to the ability of UII to stimulate MPCh neurons, there should be observable effects on cortical desynchronization, and this may contribute to the effects that were measured in the present study. We have begun to investigate this possibility. The present study demonstrates that the UII-Rs are selectively expressed by MPCh neurons of the mouse just as it is in the rat. It shows for the first time that UII can excite cholinergic mesopontine tegmental neurons of the LDTg and stimulates forebrain dopaminergic transmission. It further illustrates that UII acts in vivo to influence locomotion in both mice and rats. The discrete localization of the UII-R in the MPCh and UII’s ability to depolarize these neurons may make UII a useful tool for investigating MPCh function.
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Acknowledgments We thank our colleagues Rainer Reinscheid, Steven Lin and Zhiwei Wang for advice during the course of this project. This work was supported by grants from NIH (MH60231 (OC), DK63001 (OC), HL64150 (CSL), NS27881 (CSL)) and from the Stanley Research Foundation (03R-415 (OC)).
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Research Report
Urotensin II acts as a modulator of mesopontine cholinergic neurons Stewart D. Clark a,f, Hans-Peter Nothacker b, Charles D. Blaha c, Christopher J. Tyler d, Dee M. Duangdao b, Stephen L. Grupke d, David R. Helton e, Christopher S. Leonard d, Olivier Civelli a,b,* a
Department of Developmental and Cell Biology, University of California, Irvine, CA 92697-4625, USA b Department of Pharmacology, University of California, 369 MSRII, Irvine, CA 92697-4625, USA c Department of Psychology, University of Memphis, Memphis, TN 38152, USA d Department of Physiology, New York Medical College, Valhalla, NY 10595, USA e Cenomed Inc., 22865 Lake Forest Drive, Lake Forest, CA 92630, USA f Laboratory of Molecular Neurobiology, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8 Accepted 9 August 2005 Available online 23 September 2005
Abstract Urotensin II (UII) is a vasomodulatory peptide that was not predicted to elicit CNS activity. However, because we have recently shown that the urotensin II receptor (UII-R) is selectively expressed in rat mesopontine cholinergic (MPCh) neurons, we hypothesize that UII may have a central function. The present study demonstrates that the UII system is able to modulate MPCh neuron activity. Brain slice experiments demonstrate that UII excites MPCh neurons of the mouse laterodorsal tegmentum (LDTg) by activating a slow inward current. Furthermore, microinfusion of UII into the ventral tegmental area produces a sustained increase in dopamine efflux in the nucleus accumbens, as measured by in vivo chronoamperometry. In agreement with UII activation of MPCh neurons, intracerebroventricular injections of UII significantly modulate ambulatory movements in both rats and mice but do not significantly affect startle habituation or prepulse inhibition. The present study establishes that UII is a neuromodulator that may be exploited to target disorders involving MPCh dysfunction. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptides: anatomy and physiology Keywords: Urotensin II; Mesopontine cholinergic; Electrophysiology; Chronoamperometry; Locomotion; Prepulse inhibition
1. Introduction Urotensin II (UII) is a vasomodulatory peptide that has been recently shown to be the natural ligand of a G proteincoupled receptor formerly called GPR14 [1,25,28,30], now
Abbreviations: Icv, intracerebroventricular; LDTg, laterodorsal tegmentum; MPCh, mesopontine cholinergic; NAc, nucleus accumbens; PPTg, pedunculopontine tegmentum; PPI, prepulse inhibition; UII, urotensin II; UII-R, urotensin II receptor; VTA, ventral tegmental area * Corresponding author. Department of Pharmacology, University of California, 369 MSRII, Irvine, CA 92697-4625, USA. Fax: +1 949 824 4855. E-mail address: [email protected] (O. Civelli). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.026
known as the urotensin II receptor (UII-R). Although UII is well known for its vascular effects (for review see [9]), several reports have indicated that it may also have effects in the central nervous system [7,11,17,25,27]. In particular, we have shown that the UII-R is selectively expressed in mesopontine cholinergic (MPCh) neurons of the rat brain [7], inferring that UII can modulate the activity of MPCh neurons. In addition, the fact that UII-R activation elicits increases in cytoplasmic calcium ion concentrations in vitro [1,24,30] indicates that UII could promote cell depolarization. Studies have measured the effects of UII after injections into the brain [17,23,25,27], and our recent data indicate UII promotes REM sleep by exciting MPCh neurons of the pedunculopontine tegmental nucleus (PPTg; [18]). Here, we
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have extended this analysis by examining UII actions on an additional group of MPCh neurons in the laterodorsal tegmental (LDTg) nucleus and on other functions associated with MPCh neurons. Activation of the PPTg and LDTg is thought to contribute to a number of behavioral and physiological phenomena. For example, Garcia-Rill et al. [15] demonstrated that electrical stimulation of these regions elicited stepping behavior in the rat. In addition, lesions or electrical inhibition of the mesopontine tegmentum in non-human primates can induce ataxia [22,29]. Together, these studies have suggested that the brainstem area encompassing MPCh neurons profoundly influences motor function in mammals [38]. MPCh neurons have also been implicated in the modulation of the sensorimotor reactivity. Garcia-Rill et al. [16] predicted that activation of neurons in the mesopontine would reduce habituation of the startle reflex to a loud auditory stimulus. MPCh neurons are also intimately involved in prepulse inhibition (PPI), a phenomenon in which the amplitude of the startle reflex is reduced by the presence of a preceding muted warning stimulus (for review see [10,20]). Elucidating the role of MPCh neurons in PPI is of great interest because PPI deficits are a hallmark and diagnostic symptom in many neuropsychiatric disorders (for review see [20]). It is believed that the MPCh input from the PPTg to the caudal pontine nucleus inhibits the startle reflex [21], as destruction of the PPTg impairs PPI [21,34]. Therefore, we predict that agents such as UII, which potentially modulate MPCh activity, should influence both the startle reflex and PPI. Several diverse techniques were employed to establish UII as a modulator of MPCh function. First, the response of MPCh neurons to UII stimulation was determined in vitro. Then, to test whether UII activates neurons in vivo, chronoamperometric recordings of dopamine efflux in the nucleus accumbens (NAc) were carried out after UII microinjection into the ventral tegmental area (VTA). Electrical stimulation of MPCh neurons located in the region of the PPTg and LDTg has been shown to induce increases in basal dopamine efflux in the striatum and NAc [12,13]. Furthermore, the MPCh neurons constitute the only known cholinergic excitatory input to the VTA dopaminergic cells [31]. Given that our previous study showed that the VTA contains binding sites for radiolabeled UII [7], it was expected that UII injected into the VTA would produce changes in NAc dopamine efflux. Lastly, behavioral paradigms associated with MPCh activity were used to extend the anatomical and neurochemical findings.
2. Materials and methods 2.1. In situ hybridization Sources of the following materials were: bovine serum albumin, polyvinylpyrolidone, poly-l-lysine, RNase A (Sigma, St. Louis, MO); pBluescript SK (Stratagene, La
Jolla, CA); pCR 4-TOPO (Invitrogen, Carlsbad, CA); antidigoxygenin (dig)-AP Fab antibody, dig-dUTP, Genius system nonradioactive nucleic acid detection kit, restriction enzymes, T3, T7 polymerases, proteinase K, and yeast tRNA (Roche Molecular Biochemicals, Indianapolis, IN); formamide (Fluka, Ronkonkoma, NY); dextran sulfate (Pharmacia, Piscataway, NJ); sodium acetate (Fisher Scientific, Pittsburgh, PA); Hyperfilm hmax (Amersham, Arlington Heights, IL); nuclear track emulsion (NTB2) (Kodak, Rochester, NY); [35S]-Uridine triphosphate ([35S]-UTP) (Dupont NEN, Boston, MA). 2.1.1. Tissue preparation CD-1 mice weighing approximately 18 –30 g (Charles River, San Diego, CA), kept on a 12-h light/dark cycle with food and water ad libitum, were decapitated and brains were removed. Tissues were frozen immediately by immersion in 20 -C isopentane, then stored at 70 -C until used. All tissue removal procedures were approved by the Institutional Animal Care and Use Committee and were consistent with Federal guidelines. Twenty-micron sections were cut using a cryostat and mounted onto Vectabond (Vector Laboratories) coated slides. Fixation was performed (4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4) for 1 h at 22 -C. Slides were then rinsed in PB, air dried, and subsequently stored with desiccate at 20 -C. 2.1.2. Hybridization All sections and probes were prepared as described by Clark et al. [7]. Briefly, sections were incubated with proteinase K (1 mg/mL) for 10 min at 22 -C, then acetylated, and dehydrated through graded ethanol, followed by air-drying. Sections were exposed to a 1:1 dilution of digoxigenin-labeled anti-sense choline acetyltransferase (ChAT) riboprobe (0.2 ng/mL):[35S]-labeled UII-R sense or anti-sense probes (2 107 cpm/mL) in hybridization solution (50% formamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA, 500 mg/mL tRNA, 10 mM DTT, 0.3 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) then incubated overnight at 60 -C. Incubation with RNase A (20 mg/mL) for 30 min at 37 -C followed the hybridization. The sections were then washed 2 5 min and 2 10 min in solutions of decreasing salinity at 22 -C and a 30 min wash in 0.1 SSC at 68 -C. After the hot wash, the slides were processed for digoxigenin labeling as per the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Slides were then apposed to Bmax film for development of autoradiograms. Following film development, slides were coated with 3% parlodion in isoamylacetate and dipped in liquid NTB2 emulsion on the reference date of the [35S]-UTP. Slides for each probe were incubated according to the exposure time on film: 1 day on film, 1 week incubation with emulsion. After the appropriate exposure period, slides were developed in Kodak D19, fixed, coverslipped, and analyzed.
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2.2. Slice and patch clamp recording Brain slices were prepared and whole-cell recordings were made as described previously in detail [6]. Briefly, 300 Am slices containing the LDTg were prepared from isofluorane anesthetized C57/BL6 mice (P14 –P21; Taconic, Charles River) in ice-cold, carbogen-equilibrated, artificial cerebrospinal fluid (ACSF) which contained the following (in mM): 121 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 1.2 MgSO4, 26 NaHCO3, 20 dextrose, 4.2 lactic acid. Using a fixed-stage microscope (Olympus BX50WI), the boundaries of LDTg were first determined by low magnification inspection and then neurons to be recorded were visualized with a nuvicon camera (Dage VE-1000) using a 40 objective and IR-DIC optics. The submerged-slice recording chamber was perfused at 3 – 5 mL/min with room temperature ACSF. Human urotensin II (Bachem) was dissolved into the perfusate (300 nM) just before the experiment and was applied by bath superfusion. Giga-seal whole-cell voltage-clamp recordings of LDTg neurons were made with pipettes pulled from 1.5-mmdiameter glass capillary tubing (Corning 7052, A-M systems) using an Axopatch 200B amplifier (Axon Instruments). The pipette solution contained (in mM) 144 Kgluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 0.3 NaGTP, 4 Na2ATP. Biocytin Alexa Fluor 594 (25 AM; Molecular Probes) was included in the patch solution. Membrane voltages and currents were controlled and recorded with a computer running PCLAMP8 software (Axon Instruments). Access resistance (R acc) was estimated on-line using the PCLAMP8 membrane-test routine. The quality of recorded cells was assessed by monitoring input resistance, holding current, and capacitance and recordings were terminated if the estimated R acc was >30 MV, became unstable, or changed by more that 20% between measurements. Recordings were uncompensated for series resistance errors since the recorded currents and associated voltage errors were small. Current measurements were filtered at 2 KHz and sampled at 5 KHz. Extracellular spiking was recorded from visualized neurons in current-clamp mode with the same pipettes used for whole-cell recording. The pipettes were filled with extracellular solution and were loosely attached to the soma of the visualized neuron by weak suction for the duration of the recording. 2.2.1. Immunocytochemistry Cell identification was performed by immunocytochemistry as previously described in detail for bNOS [6]. Briefly, following successful removal of the patch pipette from the Alexa Fluor-filled neuron, the slice was fixed in 4% paraformaldehyde for 1 – 3 days at 4 -C, cryoprotected by equilibration in 30% sucrose (0.01 M PBS) and resectioned (40 Am) on a freezing microtome. Sections were incubated in rabbit polyclonal anti-bNOS and visualized with FITC-conjugated goat anti-rabbit IgGs
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(Chemicon; 1:50 in PBS) to determine if recorded cells were bNOS-immunoreactive. 2.3. Chronoamperometric recordings Male hooded Wistar rats, weighing 200 –250 g (Animal Resources Center, Adelaide, SA, Australia), were housed in pairs with constant temperature (22 T 0.5 -C), 12-h light/ dark cycle (lights on at 08.00 h), and food and water ad libitum. Rats were mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) after anesthetic plane was reached (1.5 g/kg i.p. urethane). Body temperature was maintained by use of temperature regulated heating pad (TC-831, CWE, NY, USA). For complete methods and materials, please refer to earlier publications by CD Blaha [12]. Bilateral 31 g stainless steel guide cannulae were implanted 1 mm above the VTA (interaural coordinates: AP + 2.96, ML + 0.5 mm, and DV + 1.7 mm; [32]). To facilitate the measurement of in vivo changes in dopamine oxidation current without interference from other oxidizable compounds in brain extracellular fluid [2,4,5], a single stearate-modified graphite paste electrochemical recording electrode [3] was implanted into the NAc (coordinates: AP + 1.2 mm from bregma, ML + 1.2 mm and DV 6.5 mm from dura; [32]) and an Ag/AgCl reference and stainless-steel auxiliary electrode combination was placed in contact with contralateral cortical tissue 4 mm posterior to the bregma. Repetitive chronoamperometric measurements of dopamine oxidation current using an electrometer (Echempro, GMA Tech. Inc., Vancouver, BC, Canada) were made by applying a potential pulse from 0.15 V to +0.3 V vs. Ag/AgCl to the recording electrode for 1 s at 30-s intervals, and monitoring the oxidation current at the end of each 1-s pulse [5]. At the end of at least 60 min of baseline chronoamperometric recording, UII or vehicle was applied at a rate of 0.5 AL/min with a final volume of 1 AL in concentrations appropriate to administer 1, 10, or 100 nmol of UII into the VTA. The magnitude of drug-induced changes in NAc dopamine efflux was calculated from pre-injection baseline dopamine oxidation current values normalized to zero current and was expressed as a percent change with respect to a mean baseline current value derived as in Forster and Blaha [12]. For each UII dose condition, the resulting peak oxidation current values were averaged across animals and compared with respective pre-drug baseline levels using two-tailed paired t tests (alpha level set at 0.01), using separate animals for each injection and each dose. UII was from the same source and prepared in the same manner as for the behavioral studies. Rats were euthanized with a 0.5 mL cardial infusion of urethane (350 mg/mL). Brains were removed and immersed overnight in 10% buffered formalin, and then were cryoprotected by storing the tissue in a 30% sucrose/10% formalin solution. Coronal sections (60 Am) were cut on a freezing microtome, and subsequently processed for cresyl
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violet stain. Tracts due to cannulae and electrodes were visualized on a light microscope. 2.4. Behavioral studies Animal experiments were performed on male CD-1 mice (Charles River, California) approximately 8– 12 weeks of age weighing 18– 30 g at the start of experiments. All behavioral and injection procedures were approved by the Institutional Animal Care and Use Committee and were consistent with federal guidelines. Mice were housed 4 per cage with water and food available ad libitum, in a room maintained under constant temperature (20 – 21 -C) and humidity (40 – 45%). Mice were maintained on a 12-h light/dark cycle with lights on –off at 07:00 – 19:00 h and all behavioral testing was conducted during the light phase (between the hours of 09:00 and 17:00). Sprague – Dawley rats were purchased with implanted intracerebroventricular (icv) cannula (Charles River, California) weighing approximately 240 –270 g, and were housed individually. The rats were housed in separate rooms as the mice, but under the same conditions. 2.4.1. Drugs Human urotensin II was obtained from Bachem (California), and mixed with 0.9% saline for a final concentration of 4 mM. The pH was adjusted with NaOH to a final pH of 6.9. Further dilutions as required for treatment were done using 0.9% saline. UII was delivered into the lateral ventricle of the rat through a chronically implanted cannula (10 AL over 2 min). The injection of UII into mice (5 AL) was performed by a freehand icv injection method. The resulting puncture in the skull was verified to be 2 T 0.5 mm from bregma and 2 T 0.5 mm from midline by visual inspection of euthanized subjects at the completion of the experiment. The following drugs were obtained from Sigma RBI, St. Louis, MO: apomorphine and phencyclidine (PCP), and were dissolved in 0.9% saline containing 1% Tween-80 for s.c. injections. 2.4.2. Spontaneous activity Activity levels (ambulatory and nonambulatory) were recorded using a Hamilton-Kinder Activity Monitor (with the infrared beams in a 4 8 configured frame, San Diego, California). Activity levels were quantified in transparent polycarbonate cages (32 26 20 cm) placed within the activity frames. Animals were acclimatized to the novel home cage environment for 60 min before injections, then were removed for injections and immediately replaced to the same cage for monitoring. Activity was measured for 60 min after the administration of either vehicle or test compound. 2.4.3. Sensorimotor reactivity Sensorimotor reactivity (startle response) is a crossspecies intrinsic reflex response exhibited when a sudden, high-density acoustic stimulus is presented to a subject. The amplitude of the startle response was measured in Newtons
by accelerometers within the chambers (Hamilton-Kinder, California). Habituation to the startling stimulus was calculated for each dose group by taking the average of the responses to the 120-dB stimulus trials in bins of five from a session that included 25 120-dB stimulus trials separated by 8-s intervals, preceded by a 5-min acclimatization period (65-dB background). UII was administered either 5 or 25 min prior to placement of the subject into the chamber. 2.4.4. Prepulse inhibition Prepulse inhibition refers to a reduction in startle amplitude when a startle-inducing stimulus is preceded by a weaker non-startling warning stimulus. Computer-controlled startle chambers (Hamilton-Kinder, San Diego, California) were used for this assay. Sound levels were checked with a decibel meter prior to each day’s experimentation. The noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (PCP) and the dopamine agonist apomorphine reduce PPI of the acoustic startle response in rodents. In apomorphine challenged trials, UII was administered 25 min prior to placement of the animals in the chambers, with apomorphine being injected subcutaneous (s.c.) 20 min after UII. PCP was administered s.c. 30 min prior to placing the animals into the chambers, with UII being injected icv 10 min after PCP. Following a 5-min acclimatization period with background white noise (65 dB), animals were exposed to six different trial types. Trials were presented ten times each in a quasi-random order, with quasi-randomized 5- to 25-s inter-trial intervals. Trials were: stimulus only trial (120 dB white noise, 50 ms stimulus); three different prepulse + pulse trials, in which a 20 ms 5-dB, 10-dB, or 15-dB stimulus above a 65-dB background preceded the 120-dB pulse by 120 ms (onset to onset); a 15-dB prepulse without a 120-dB pulse; and a no stimulus trial, in which only the background noise was presented. The average response for each dose group and each trial type was calculated and presented. The amount of PPI was calculated as a percentage score for each animal per prepulse trial type: % prepulse inhibition = [(stimulus only 5-dB or 10-dB stimuli) / stimulus only] 100. 2.4.5. Statistical analysis The results of the behavioral tests were analyzed by a between groups analysis of variance (ANOVA), with subsequent Tukey – Kramer HSD. The software used to perform these analyses was JMP ver. 4.0.0 (SAS Institute, Inc.).
3. Results 3.1. Expression of the UII-R in mouse MPCh neurons To determine whether UII-R is selectively expressed in mouse MPCh neurons as reported for the rat, in situ hybridization of the UII-R was performed. Coronal sections
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of mouse brains were hybridized with UII-R [35S]-cRNA to produce a brain map of UII-R mRNA expression. This resulted in specific anti-sense signal in the PPTg and LDTg (Fig. 1A). In subsequent double in situ hybridizations, with a DIG-labeled cRNA probe for ChAT, it was found that UIIR colocalizes with the cholinergic neurons of the MPCh (Figs. 1C, D). This colocalization of UII-R with ChAT was seen in the majority of cases of UII-R anti-sense labeled cell bodies (non-quantitative observations). These findings demonstrate that the mouse UII-R mRNA localization mirrors that of the rat [7] and make possible the comparison of UII-induced behaviors in mice and rats. 3.2. Slice recordings of LDTg neurons Due to the selective expression of UII-R in the LDTg and PPTg, we hypothesized that UII would excite cholinergic LDTg and PPTg neurons. Therefore, we examined the effect of bath superfusion of UII on LDTg neurons under wholecell voltage-clamp conditions ( 60 mV holding potential) in a brain slice preparation in normal ACSF. Superfusion with ACSF containing 300 nM UII evoked a slow inward shift of the holding current in 12/19 LDTg neurons ( 31.0 T 5.4 pA; mean T SEM; n = 12; Fig. 2A). Seven of these Alexa Fluor-filled neurons were recovered and all were bNOS+ (n = 7). Since this inward current would depolarize unclamped neurons, these data suggest that UII excites MPCh neurons. Indeed, UII application approximately doubled the firing of a spontaneously firing LDTg neuron recorded extracellularly (Figs. 2D – F). Of the 7 LDTg neurons that did not respond to UII, five were recovered.
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Interestingly, two were bNOS+ and the other three were bNOS . These data indicate that UII excites some but not all bNOS positive LDTg neurons and suggest that bNOSLDTg neurons do not respond to UII. 3.3. Chronoamperometry recordings To determine whether UII can induce VTA-mediated dopamine efflux in the NAc, UII was microinjected into the VTA. All injections of UII produced changes in basal dopamine oxidation currents, with increasing doses increasing both the magnitude and duration of the response. When administered at a dose of 1 nmol, UII produced a peak change in basal extracellular dopamine levels of 45% from baseline at 13 min (n = 8), while 10 nmol showed maximal change (185%) at 60 min post-injection (n = 6) (Fig. 3). After injection of 100 nmol, the change in basal dopamine concentrations steadily rose for the entire 3-h recording period to 600% of baseline levels and appeared to plateau at that time (n = 6) (Fig. 3). All three doses display the same onset dynamic, and therefore are most likely the same phenomena, but of different intensity. If the 100 nmol dose continues to act with the same dynamic as the lower doses, it would be expected that dopamine oxidation currents would return to baseline at 7.5 h, well beyond the ethical and practical endpoint of the animal preparation. Doses used for the chronoamperometric studies were kept similar to those that were used for behavioral studies. These doses were used to counter a number of factors which could serve to dampen the effectiveness of the peptide infused in vivo, as apposed to bath applied in vitro. Firstly, peptides do not diffuse easily through extracellular space, being somewhat restricted to the site of injection. Second, the degradation kinetics of UII in the CNS is unknown. It is also possible that the anesthetic (urethane) may have dampened the effect and that in an awake animal the effects of 1 nmol may have been more dramatic. In pilot studies, it was also observed that the administration of UII into the PPTg increased dopamine oxidation currents in the striatum. UII can elicit robust changes in dopamine efflux in a dose-dependent manner through established MPCh circuitry, by the stimulation of brain areas previously shown to express UII-R. 3.4. Spontaneous locomotion
Fig. 1. UII-R is expressed in mouse MPCh neurons. Autoradiogram of a single representative CD-1 mouse brain section processed by double in situ hybridization, showing specific hybridization of [35S]-UII-R anti-sense cRNA (A), with the signal being absent when the [35S]-UII-R sense cRNA probe is used (B). Emulsion dipped slides reveal that the UII-R (dark field) (C) is localized in ChAT containing neurons (D) (DIG labeled ChAT cRNA probe, anti-sense) (bright field).
Animals were acclimatized to the testing situation for 1 h to lessen exploratory activity. Mice exhibited a very profound reduction in spontaneous movement immediately after injection that lasted for up to 20 min (Fig. 4A). It was visually observed that UII-treated mice would stop movement mid-stride or when rearing with forepaws on the sides of the enclosure, and these phenomena were seen at doses as low as 10 pmol (data not shown). At high doses, an increase in spontaneous movement was seen in both rats and mice (Figs. 4A and B), with the largest effect at 25 –30 min postinjection in mice, and a doubling seen rats (Fig. 4C). There
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Fig. 2. UII excites LDTg neurons by activating a slow inward current. (A) Membrane current from a mouse LDTg neuron clamped near resting membrane potential ( 60 mV holding potential) before and after bath superfusion of 300 nM UII. The slow inward current, which would cause membrane depolarization in unclamped neurons, was observed in bNOS+ neurons. (B) Low-power fluorescent image of a coronal section. Green fluorescent neurons illustrate the distribution of bNOS+ neurons of the left and right LDTg from the recorded brain slice. The recorded neuron (C) was in the right LDTg. (C) Left panel shows the neuron from which the recording in panel A was obtained (Alexa-594; red). Center panel shows the same field with cholinergic neurons visualized by bNOS immunocytochemistry (FITC, green). Right panel shows the two images merged revealing that the recorded neuron was immunoreactive for bNOS (yellow). Bath application of UII increased the firing of an LDTg neuron recorded extracellularly. (D) DIC image of extracellularly recorded neuron in the LDTg. (E) 10-s epochs of spiking recorded from cell illustrated in panel D taken before (top trace) and after (bottom trace) UII application. (F) Histogram (10 s bins) of average firing rate shows the slow and long lasting increase in firing produced by UII. Arrows indicate epochs illustrated in panel E before (1) and after (2) application. UII approximately doubled the spiking in this neuron. Abbreviations: Aq, aqueduct; LDTg, laterodorsal tegmental nucleus; DT, dorsal tegmental nucleus.
is an early onset species-specific reduction in movement immediately after UII injection into mice. As well, there is an increase in spontaneous movement with a high dose of UII (10 nmol) in both rats and mice. We and others [17] have found that approximately 1 nmol is the highest dose that does not produce a significant effect on ambulations. Therefore, 1 nmol was used for subsequent studies, where ambulations or heightened activity could interfere with measures of startle and PPI. 3.4.1. Startle habituation and prepulse inhibition MPCh neurons are thought to be involved in the modulation of the startle reflex, and specifically PPI. Therefore, due to the selective expression of UII-R by MPCh neurons, it was hypothesized that UII administration would modulate the animals’ response to startling auditory stimuli. UII had no effect on startle habituation in mice and rats, when tested 5 (data not shown) or 25 min after injection (Figs. 5A, B). There was also no modulation of PPI when increasing doses of UII were administered 5 min (data not shown) or 25 min prior to testing (Fig. 5C). Furthermore, UII was unable to rescue the PPI deficit
induced by either apomorphine injection (0.3 mg/kg) (Fig. 5D) or PCP (3 mg/kg) (Fig. 5E).
4. Discussion To determine whether the selective expression of the UIIR in MPCh neurons is a species-independent phenomenon, UII-R expression was analyzed in the mouse brain by in situ hybridization. It was found that UII-R is expressed in mouse MPCh neurons, as has been previously reported in the rat [7]. This led us to determine whether UII can modulate synaptic transmission in the mouse. Superfusion of UII over mouse brain slices was found to cause depolarization of cholinergic LDTg neurons. These data, and similar data collected in rats [18], are the first demonstration that UII excites supraspinal neurons. To further analyze whether UII is able to regulate neurotransmitter release, we chose to study the effects of UII on dopamine release. Principal excitatory inputs to the VTA arise from MPCh neurons. These neurons are thought to be able to release either glutamate or acetylcholine [13] (along with other ligands [36,37]) to modulate the function of dopaminergic neurons.
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Fig. 3. UII microinjected into the VTA increases NAc dopamine oxidation current. Microinjections of UII (1 AL of indicated concentration) into the VTA were done using separate animals for each injection and each dose. The magnitude of the change in dopamine oxidation currents recorded in the NAc increased with increased concentration of UII injected into the VTA. A dose of 1 nmol UII (n = 8) produced a 45% increase in basal NAc dopamine efflux. 10 nmol (n = 6) and 100 nmol (n = 6) of UII increased dopamine efflux in the NAc by 185% and 600%, respectively.
The stimulation of the dopaminergic neurons of the VTA is known to increase dopamine efflux in the NAc [12]. In our previous study, it was found that [125I]-UII bound to receptors located in the VTA [7]. Therefore, it was expected that microinfusion of UII into the VTA would modulate the firing of these dopaminergic neurons. Indeed, we found that UII was able to dose-dependently increase basal levels of dopamine efflux in the NAc upon discreet injection into the VTA. Due to the fact that the VTA does not express UII-R transcripts, we believe that this response results from the activation of presynaptic UII-Rs. Further studies will be needed to confirm this hypothesis. Together with the neural activation studies, the selective expression of UII-R in MPCh neurons suggests that exogenous UII when administered in vivo should facilitate MPCh-mediated behavioral phenomena in rats and mice. Our demonstration that UII is able to induce VTA-mediated dose-dependent dopamine efflux in the NAc suggests that UII may have a role in motor function, learning, and motivation (see [39] for review). Schwienbacher et al. [33] have demonstrated that extracellular dopamine levels increase in the NAc during locomotion, and Kelley et al. [19] showed that amphetamine injected into the NAc produced a dose-dependent increase in locomotion. Microinfusion studies into the VTA of awake animals will be necessary to determine whether UII-induced increases in NAc dopamine correlate with the measured locomotor activity.
Fig. 4. UII-induced locomotor effects. Animals were acclimatized for 1 h, then UII was injected (icv) and ambulatory movements were recorded for 1 h. There was a dramatic decrease in the number of ambulatory movements when UII was injected into mice, as compared to when saline was injected (A) (n = 8) (*P < 0.05). A later increase in spontaneous movement was seen (A). In rats, UII dose-dependently increased the number of ambulations (B) (n = 10). This phenomenon was significant when the total number of ambulations for the 1 h was compared to when saline was injected (C) (*P < 0.05).
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Fig. 5. UII influence on startle habituation and prepulse inhibition. Animals were injected icv 25 min prior to placement into the startle monitors. There were no significant changes in habituation to a startling stimulus in either mice (A) (n = 10) or rats (B) (n = 9). When rats were pretreated with UII 25 min before beginning the PPI trials, there was no significant differences in PPI (C). Rats were pretreated with UII (20 min before apomorphine) then injected s.c. with 0.3 mg/kg of apomorphine 5 min prior to placement in the startle monitor. When PPI is expressed as a percent of the stimulus only group (D), there was a significant effect of apomorphine ( P < 0.05), but no significant influence of UII either on its own or when challenged with apomorphine (n = 10) ( P < 0.05). Rats were pretreated with PCP 5 min prior to UII, which was administered 25 min prior to placing the animal into the startle monitor. PCP significantly disrupted PPI ( P < 0.05). However, there was no significant improvement of PPI when PCP was challenged with UII (E) (n = 11).
In view of the important cardiovascular function of UII, it is also possible that the behavioral effects described here may be consequences of UII effects on cortical blood flow. Lin et al. [23] have found that UII icv injections in conscious rats lead to increases in heart rate but these were only observed at higher doses (10 nmol). At the dose that we used (1 nmol), regulation of heart rate is not expected to be a factor but furQ
ther studies will need to be carried out to address this issue. Taken together with the studies that have shown that movement is elicited or inhibited by stimulation or lesioning the mesopontine tegmentum, respectively [14,22], it is not surprising that UII modulates spontaneous locomotion. Though, studies where spontaneous movement is measured during microinfusion of UII into the MPCh must be performed.
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In the present study, the potent depression of movement in mice during the first 15 min after UII injection was not observed in the Sprague –Dawley rats. Previously, Gartlon et al. [17] observed mild tonic seizures, followed by hypoactivity, after icv injections of UII in Sprague – Dawley rats, at doses of UII higher than those used here. In addition, unpublished reports from other labs suggest that Wistar rats also show a significant initial decrease in spontaneous movement after UII injections (icv) [26]. Therefore, the deficit in spontaneous movement seen in CD-1 mice may not be a species-specific phenomenon, but may rather reflect differences in dose sensitivity between strains. Despite the ability of UII to activate mesopontine tegmental neurons, UII did not alter or modulate PPI. Several factors may bear this observation. First, UII may be unable to enhance the function of the PPTg over that of the normal state and hence not produce PPI deficits that are demonstrative of a pathological state. Also, the pharmacological challenges that were used to disrupt PPI may not be sufficient to reveal UII’s ability to modulate PPTg function. Alternatively, the disruption of endogenous UII signaling may produce PPI deficits, as does the lesioning of the PPTg [21,34]. However, the lack of a commercially available, selective, high potency, UII-R antagonist has so far impeded the investigation of this possibility. The MPCh neurons are thought to regulate PPI via an efferent to the pontine reticular formation [21]. It could be that the icv injection of UII activates all CNS UII-Rs closely in time, producing an overall activation of the UII system that masks a specific MPCh-pontine phenomenon. Perhaps the activation of this component alone or the use of UII-R antagonists would be more enlightening as to whether UII has a role in modulating startle habituation or PPI. The MPCh system is also known to produce and maintain cortical desynchronization, a phenomenon seen during both the wakeful and REM sleep periods in mammals. Recently, presumed cholinergic cells of the PPTg were recorded in freely moving rats and it was found that discharges of these cells correlate with wakefulness and REM state [8]. Electrical stimulation of the LDTg has also been shown to increase REM sleep in cats [35]. Due to the ability of UII to stimulate MPCh neurons, there should be observable effects on cortical desynchronization, and this may contribute to the effects that were measured in the present study. We have begun to investigate this possibility. The present study demonstrates that the UII-Rs are selectively expressed by MPCh neurons of the mouse just as it is in the rat. It shows for the first time that UII can excite cholinergic mesopontine tegmental neurons of the LDTg and stimulates forebrain dopaminergic transmission. It further illustrates that UII acts in vivo to influence locomotion in both mice and rats. The discrete localization of the UII-R in the MPCh and UII’s ability to depolarize these neurons may make UII a useful tool for investigating MPCh function.
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Acknowledgments We thank our colleagues Rainer Reinscheid, Steven Lin and Zhiwei Wang for advice during the course of this project. This work was supported by grants from NIH (MH60231 (OC), DK63001 (OC), HL64150 (CSL), NS27881 (CSL)) and from the Stanley Research Foundation (03R-415 (OC)).
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