Research ReportUrotensin II acts as a modulator of mesopontine cholinergic neurons
Introduction
Urotensin II (UII) is a vasomodulatory peptide that has been recently shown to be the natural ligand of a G protein-coupled receptor formerly called GPR14 [1], [25], [28], [30], now 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 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.
Section snippets
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); anti-digoxygenin (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
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 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 UII-R colocalizes with the cholinergic neurons of
Discussion
To determine whether the selective expression of the UII-R 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,
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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