Diederik Moechars

Vesicular Glutamate Transporter VGLUT2 Expression Levels Control Quantal Size and Neuropathic Pain

Uptake of l-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1–VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

Vesicular Glutamate Transporter VGLUT1 Has a Role in Hippocampal Long-Term Potentiation and Spatial Reversal Learning

Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) show largely complementary distribution in the mature rodent brain and tend to segregate to synapses with different physiological properties. In the hippocampus, VGLUT1 is the dominate subtype in adult animals, whereas VGLUT2 is transiently expressed during early postnatal development. We generated and characterized VGLUT1 knockout mice in order to examine the functional contribution of this transporter to hippocampal synaptic plasticity and hippocampus-dependent spatial learning. Because complete deletion of VGLUT1 resulted in postnatal lethality, we used heterozygous animals for analysis. Here, we report that deletion of VGLUT1 resulted in impaired hippocampal long-term potentiation (LTP) in the CA1 region in vitro. In contrast, heterozygous VGLUT2 mice that were investigated for comparison did not show any changes in LTP. The reduced ability of VGLUT1-deficient mice to express LTP was accompanied by a specific deficit in spatial reversal learning in the water maze. Our data suggest a functional role of VGLUT1 in forms of hippocampal synaptic plasticity that are required to adapt and modify acquired spatial maps to external stimuli and changes.

Dissecting the role of sodium currents in visceral sensory neurons in a model of chronic hyperexcitability using Nav1.8 and Nav1.9 null mice.

Tetrodotoxin‐resistant (TTX‐R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX‐R currents are available, this study employs Nav1.8 and Nav1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Nav1.9 and Nav1.8 null, wild‐type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20–24 post‐infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Nav1.9 and Nav1.8 currents were recorded. DRG neuronal excitability was significantly increased in post‐infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 ± 0.3, infected = 3.6 ± 0.7 APs at 2× rheobase, P= 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX‐R currents. Hyperexcitability was maintained in Nav1.9−/− mice, but hyperexcitability was absent and APs were blunted in Nav1.8−/− mice. This study identifies a critical role for Nav1.8 in chronic post‐infectious visceral hyperexcitability, with no contribution from Nav1.9. Nb infection‐induced hyperexcitability is not observed in Nav1.8−/− mice, but is still present in Nav1.9−/− mice. It is not clear whether hyperexcitability is due to a change in the function of Nav1.8 channels or a change in the number of Nav1.8 channels.

BACE1 Physiological Functions May Limit Its Use as Therapeutic Target for Alzheimer's Disease

The protease β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is required for the production of the amyloid-β (Aβ) peptide, which is central to the pathogenesis of Alzheimers disease (AD). Chronic inhibition of this protease may temper amyloid production and cure or prevent AD. However, while BACE1 inhibitors are being pushed forward as drug candidates, a remarkable gap in knowledge on the physiological functions of BACE1 and its close homolog BACE2 becomes apparent. Here we discuss the major discoveries of the past 3 years concerning BACE1 biology and to what extent these could limit the use of BACE1 inhibitors in the clinic.

Time course of tinnitus development following noise exposure in mice.

Gap‐induced prepulse inhibition of acoustic startle (GPIAS) has been used in rats and mice to study the problem of tinnitus. The current study demonstrates that similar methods can be used to study the temporal development of tinnitus over time in middle‐aged mice. Six‐month‐old mice on a mixed C57Bl6 × 129 background were anesthetized with isoflurane and exposed to unilateral noise (n = 15), or sham exposure for controls (n = 8), for 1 hr (16‐kHz octave band signal, 116‐dB SPL). Tinnitus was tested in eight different sound frequency bands before and at postexposure time points of 1, 3–4, 7, 14, 21, and 30 days and monthly thereafter until 7 months postexposure. Noise‐exposed mice displayed a number of changes in GPIAS consistent with the presence of hyperacusis and tinnitus. Noise exposure was associated with acute tinnitus measured 1 day later at several frequencies at and above the exposure frequency center. Consistent, chronic tinnitus then emerged in the 24‐kHz range. Several time points following noise exposure suggested evidence of hyperacusis, often followed temporally by the development of deficits in GPIAS (reflecting tinnitus). Temporal development of these changes following noise exposure are discussed in the context of the interactions among aging, noise exposure, and the associated neurochemical changes that occur at early stages of auditory processing.

Effect of peripheral obestatin on food intake and gastric emptying in ghrelin-knockout mice.

The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelins stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice.

Endogenous and exogenous ghrelin enhance the colonic and gastric manifestations of dextran sodium sulphate-induced colitis in mice.

Abstractu2002 Ghrelin is an important orexigenic peptide that not only exerts gastroprokinetic but also immunoregulatory effects. This study aimed to assess the role of endogenous and exogenous ghrelin in the pathogenesis of colitis and in the disturbances of gastric emptying and colonic contractility during this process. Dextran sodium sulphate colitis was induced for 5u2003days in (i) ghrelin+/+ and ghrelin−/− mice and clinical and histological parameters were monitored at days 5, 10 and 26 and (ii) in Naval Medical Research Institute non‐inbred Swiss (NMRI) mice treated with ghrelin (100u2003nmolu2003kg−1) twice daily for 5 or 10u2003days. Neural contractility changes were measured in colonic smooth muscle strips, whereas gastric emptying was measured with the 14C octanoic acid breath test. Inflammation increased ghrelin plasma levels. Body weight loss, histological damage, myeloperoxidase activity and IL‐1β levels were attenuated in ghrelin−/− mice. Whereas absence of ghrelin did not affect changes in colonic contractility, gastric emptying in the acute phase was accelerated in ghrelin+/+ but not in ghrelin−/− mice. In agreement with the studies in ghrelin knockout mice, 10u2003days treatment of NMRI mice with exogenous ghrelin enhanced the clinical disease activity and promoted infiltration of neutrophils and colonic IL‐1β levels. Unexpectedly, ghrelin treatment decreased excitatory and inhibitory neural responses in the colon of healthy but not of inflamed NMRI mice. Endogenous ghrelin enhances the course of the inflammatory process and is involved in the disturbances of gastric emptying associated with colitis. Treatment with exogenous ghrelin aggravates colitis, thereby limiting the potential therapeutic properties of ghrelin during intestinal inflammation.

Knockout mice in understanding the mechanism of action of lithium

Lithium inhibits IMPase (inositol monophosphatase) activity, as well as inositol transporter function. To determine whether one or more of these mechanisms might underlie lithiums behavioural effects, we studied Impa1 (encoding IMPase) and Smit1 (sodium-myo-inositol transporter 1)-knockout mice. In brains of adult homozygous Impa1-knockout mice, IMPase activity was found to be decreased; however, inositol levels were not found to be altered. Behavioural analysis indicated decreased immobility in the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures. These are behaviours robustly induced by lithium. In homozygous Smit1-knockout mice, free inositol levels were decreased in the frontal cortex and hippocampus. These animals behave like lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced-swim test model of depression. In contrast with OBrien et al. [OBrien, Harper, Jove, Woodgett, Maretto, Piccolo and Klein (2004) J. Neurosci. 24, 6791-6798], we could not confirm that heterozygous Gsk3b (glycogen synthase kinase 3beta)-knockout mice exhibit decreased immobility in the Porsolt forced-swim test or decreased amphetamine-induced hyperactivity in a manner mimicking lithiums behavioural effects. These data support the role of inositol-related processes rather than GSK3beta in the mechanism of the therapeutic action of lithium.

Activation of the cannabinoid 2 (CB2) receptor inhibits murine mesenteric afferent nerve activity

Abstractu2002 Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild‐type and CB2−/− mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (nu2003=u200332). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10u2003mgu2003kg−1), however, AM1241 (10u2003mgu2003kg−1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2−/− mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.

Impairment of VGLUT2 but not VGLUT1 signaling reduces neuropathy-induced hypersensitivity

Glutamate is the major excitatory neurotransmitter in the central nervous system with an important role in nociceptive processing. Storage of glutamate into vesicles is controlled by vesicular glutamate transporters (VGLUT). Null mutants for VGLUT1 and VGLUT2 were poorly viable, thus, pain‐related behavior was presently compared between heterozygote VGLUT1 and VGLUT2 mice and their respective wild‐type littermates using a test battery that included a variety of assays for thermal and mechanical acute nociception, and inflammatory and neuropathic pain syndromes. Behavioral analysis of VGLUT1 mutant mice did not show important behavioral changes in the pain conditions tested. Reduction of VGLUT2 also resulted in unaltered acute nociceptive and inflammatory‐induced pain behavior. Interestingly, VGLUT2 heterozygote mice showed an attenuation or absence of some typical neuropathic pain features (e.g., absence of mechanical and cold allodynia after spared nerve injury). Chronic constriction injury in VGLUT2 heterozygote mice showed also reduced levels of cold allodynia, but had no impact on mechanical thresholds. Together, these data suggest that VGLUT2, but not VGLUT1, plays a role in neuropathy‐induced allodynia and hypersensitivity, and might be a therapeutic target to prevent and/or treat neuropathic pain.