Higher cognitive functions such as attention have been difficult to model in genetically tractable organisms. In humans, attentiondistracting stimuli interfere with trace but not delay conditioning, two forms of associative learning. Attention has also been correlated with activation of anterior cingulate cortex (ACC), but its functional significance is unclear. Here we show that a visual distractor interferes selectively with trace but not delay auditory fear conditioning in mice. Trace conditioning is associated with increased neuronal activity in ACC, as assayed by relative levels of c-fos expression, and is selectively impaired by lesions of this structure. The effects of the ACC lesions are unlikely to be caused by indirect impairment of the hippocampus, which is required for mnemonic aspects of trace conditioning. These data suggest that trace conditioning may be useful for studying neural substrates of attention in mice, and implicate the ACC as one such substrate. Selective attention is thought to contribute to conscious awareness, but its neural basis is poorly understood. The search for the neural substrates of attention has been concentrated in the primate visual system (1, 2). Although extremely useful for identifying neural correlates of attention, primates offer limited accessibility for functional perturbation experiments, prompting a search for alternative animal models more amenable to tests of causation. Although some attentional models have been established in rats (3), they depend on operant conditioning paradigms that have proven difficult to extend to mice, and require lengthy training periods.Studies in humans have suggested that attention is required for certain forms of associative learning (4). Associative learning paradigms, such as fear conditioning, have been successfully extended from rats to mice (5). Two commonly used variants of this procedure are delay and trace conditioning. In delay fear conditioning, a conditioned stimulus (CS), such as a tone, is immediately followed by an unconditioned stimulus (US), such as a foot shock. In trace conditioning, a time gap is introduced between the end of the CS and the start of the US. In human eye blink conditioning, another associative learning paradigm, distracting stimuli interfere with trace but not delay conditioning, suggesting that attention is necessary for the former type of learning (4, 6-10). More recent studies have suggested a similar requirement for attention in trace but not delay fear conditioning in humans (11).Potential neural substrates of attention have been identified by functional imaging in humans. For example, attention has been correlated with increased activity in the anterior cingulate cortex (ACC) (12-17). Furthermore, the ACC is preferentially activated during presentation of the conditional stimulus, compared with that of a meaningless stimulus, during aversive trace conditioning (18). The ACC has also been implicated in tasks requiring visual attention in rats (3,(19)(20)(21)(22)(23). Lesion studies have show...

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Three different subtypes of Hϩ -dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT 1A -mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3-and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

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Small focal injections of manganese ion (MnThe combined features of transport along neurons and paramagnetism have been used to trace neuronal connections in mice utilizing manganese enhanced MRI (MEMRI) (4). Briefly, Mn 2ϩ in the form of MnCl 2 when washed into the naris or injected intravitreally into the eye produced positive contrast enhancement along the olfactory and visual neuronal pathways in the mouse. This has been confirmed by Frahm's group (5), who injected Mn 2ϩ into the rat eye and observed MEMRI tracings in the rat visual system. In the current model for Mn 2ϩ trans-synaptic neuronal transport: Mn 2ϩ enters neurons through Ca 2ϩ channels (6 -10); is taken up by the endoplasmic reticulum and packaged for transport, then transported along the axon by microtubules, released into the synaptic cleft, and taken up by the next neuron in the circuit (7,9,10).We demonstrate here that with nanoliter injections of MnCl 2 into the mouse brain it is possible to obtain precise placement of Mn 2ϩ that subsequently highlights both anterograde and retrograde active neuronal circuits leading from the site of focal injection. This tract tracing methodology will allow rapid mapping of neuronal connections in live animals from focal points of interest within the brain. MATERIALS AND METHODS Stereotaxic InjectionsMale C57/Bl6 mice, 6 -8 weeks old (n ϭ 5 per study), were anesthetized with ketamine (7.5 mg/ml) / xylazine (0.5 mg/ml) at a dose of 0.17 ml / 10 g body weight. Stereotaxic injections of 10 nl of 5 mM MnCl 2 into either the dorsal striatum or the basolateral amygdala were performed on anesthetized mice using a picospritzer (Parker Hannifin, Cleveland, OH). A calibrated gauge was utilized in order to ensure that 10 nl was injected into each animal. Stereotaxic coordinates for the structures were determined from a mouse brain stereotaxic atlas (11): for the dorsal striatum coordinates were: 1.2 mm rostrocaudal from bregma, -1.6 mm mesolateral from the midline, -3.8 mm ventral; the coordinates for the basolateral amygdala were: -2.2 mm rostrocaudal from bregma, -3.3 mm mesolateral from the midline, -4.53 mm ventral. After recovery from the surgery, mice were allowed to roam freely within their cages. Either 24 hr (striatum) or 48 hr or 10 days (amygdala) postinjection, in vivo MR imaging was performed on the mice. MRIT 1 -weighted, spin-echo 3D datasets were acquired from the mouse brain using a vertical bore 11.7 T Bruker Avance

Environment and experience influence defensive behaviors, but the neural circuits mediating such effects are not well understood. We describe a new experimental model in which either flight or freezing reactions can be elicited from mice by innately aversive ultrasound. Flight and freezing are negatively correlated, suggesting a competition between fear motor systems. An unfamiliar environment or a previous aversive event, moreover, can alter the balance between these behaviors. To identify potential circuits controlling this competition, global activity patterns in the whole brain were surveyed in an unbiased manner by c-fos in situ hybridization, using novel experimental and analytical methods. Mice predominantly displaying freezing behavior had preferential neural activity in the lateral septum ventral and several medial and periventricular hypothalamic nuclei, whereas mice predominantly displaying flight had more activity in cortical, amygdalar, and striatal motor areas, the dorsolateral posterior zone of the hypothalamus, and the vertical limb of the diagonal band. These complementary patterns of c-fos induction, taken together with known connections between these structures, suggest ways in which the brain may mediate the balance between these opponent defensive behaviors.

Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

The vesicular monoamine transporter type 2 gene (VMAT2) has a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of VMAT2 under control of the serotonin transporter (slc6a4) promoter. VMAT2 sertÀcre mice showed a major (À95%) depletion of 5-HT levels in the brain with no major alterations in other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2 sertÀcre mice but developed normal innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT 1A autoreceptor coupling to G protein, as assessed with agonist-stimulated [ 35 S]GTP-g-S binding, was observed in the raphe area, indicating an adaptive change to reduced 5-HT transmission. Behavioral evaluation in adult VMAT2 sertÀcre mice showed an increase in escape-like reactions in response to tail suspension and anxiolytic-like response in the novelty-suppressed feeding test. In an aversive ultrasoundinduced defense paradigm, VMAT2 sertÀcre mice displayed a major increase in escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, although in reduced amounts, in synaptosomes from VMAT2 sertÀcre mouse brain. These findings are coherent with the notion that 5-HT has an important role in anxiety, and provide new insights into the role of endogenous 5-HT in defense behaviors.