Rats learn to prefer the late-consumed flavor over the early-consumed flavor in a multi-flavored meal paired with oral glucose and corn oil.

Conditioned flavor preference (CFP) is established by association: where a neutral flavor (conditioned stimulus, CS) is paired with orosensory and post-ingestive components of nutrients, including sugar and fat (unconditioned stimulus, US). A previous study reported that rats can learn to prefer flavors that they consumed earlier and later in a multi-flavored solution paired with an intragastric infusion of glucose, but they expressed only a preference for a late-consumed flavor when they were tested after feeding (Myers and Whitney, 2011). This paradigm can be a suitable rodent model to explain how humans acquire a selective preference for routinely late-served "dessert" foods and why these foods remain attractive even in the absence of hunger. Here, we examined whether oral glucose (Experiment 1) or fat (Experiment 2) acts as a US for flavor preference learning processes in this paradigm. In Experiment 1, adult female rats under food restriction were trained in 16 daily sessions with two distinct flavor CSs in succession per session; eight CS(+) sessions in which two distinct flavor CSs (early(+), late(+)) were sequentially presented for 8 min each with oral glucose (12%) as a US, and eight CS(-) sessions in which different CSs (early(-), late(-)) were unpaired with the US. In the 30-minute two-bottle choice test, rats preferred late(+) over late(-) only when tested 90 min after consumption of normal chow (fed test) but not after overnight deprivation (hungry test). Early(+) was not preferred over early(-) in both tests. Moreover, a significant preference for late(+) over early(+) was observed only in the fed test, which is a unique feature of oral glucose-CFP. These results indicate that taste sensations of oral glucose promote a rewarding effect of late-onset glucose nutrients. In Experiment 2, separate rats were trained with the same conditioning paradigm, but used a caloric matched fat solution (5.3% corn oil) for a US. The results showed that they expressed stronger preferences for early(+) and late(+) relative to their respective CS(-) flavors in both tests. Similar to Experiment 1, it was observed in the fed test that there was a preference for late(+) over early(+) in oral fat-CFP. Taken together, the present results suggest that routine timing arrangements can cause qualitative differences in conditioned preferences between multiple flavors within a sugar or fat-containing meal in rats, and that rats prefer the late-consumed flavor over the early-consumed flavor in the absence of hunger.

Inactivation of the ventral hippocampus facilitates the attenuation of odor neophobia in rats.

Food neophobia is a behavior observed in rodents involving reduced consumption of a novel food or drink. In the absence of negative post-ingestive consequences, consumption increases with exposure (attenuation of neophobia), which is seen as an associative safe memory. Olfaction and gustation are sensory modalities essential for the development of a food preference. However, little is known about the neural mechanisms underlying neophobia to a food-related odor stimulus. In the present study, we examined the effect of pharmacological inactivation of the ventral hippocampus (vHPC) on neophobia to orally consumed solutions in rats using muscimol, a gamma aminobutyric acid type A receptor agonist. Two different types of solutions, almond odor (benzaldehyde) and sweet taste (saccharin), were prepared. In the results, microinjections of muscimol into the bilateral vHPC before the first odor and taste exposures did not alter the neophobic reactions of the rats to each stimulus. However, in the second odor, but not taste, exposure, the muscimol-injected rats showed higher consumption in comparison to that observed in the control rats, suggesting that the vHPC inactivation facilitates the attenuation of odor neophobia. On the other hand, intra-vHPC muscimol microinjections after the first odor and taste exposures did not facilitate consumption at the second exposures. These results indicate that neural activations within vHPC during orally consuming a novel odor, but not taste, solution play an inhibitory role in the subsequent attenuation of neophobia.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and ß-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

The Basolateral Nucleus of the Amygdala Executes the Parallel Processes of Avoidance and Palatability in the Retrieval of Conditioned Taste Aversion in Male Rats.

Conditioned taste aversion (CTA) is an essential behavior for animal survival. Conditioned animals show avoidance and decreased palatability to a conditioned stimulus (CS) on CTA retrieval. In this study, we aimed to determine whether the basolateral nucleus of the amygdala (BLA) is involved in CTA retrieval and whether avoidance and palatability in CTA retrieval are processed in the BLA. We developed an experimental chamber for time-course analysis of the behavior to approach a spout and lick a CS. In this experimental chamber, we analyzed the behavior of male rats following microinjections of GABAA receptor agonist muscimol or saline into the BLA. The rats showed two types of approach behavior: they either (1) approached and licked the spout or (2) approached but did not lick the spout. Muscimol injection into the BLA decreased the frequency of the latter type of approach behavior, indicating that BLA inactivation reduced avoidance to the CS. The muscimol injection into the BLA also significantly increased the consumption of the CS. Lick microstructure analysis demonstrated that intra-BLA muscimol significantly increased licking burst number and size, indicating that BLA inactivation attenuated aversion to the CS as large burst licking is an indicator of high palatability. These results suggest that the increase in CS consumption with intra-BLA muscimol injection was due to alterations in approach and aversive responses to the CS. Therefore, we conclude that the BLA plays an essential role in CTA retrieval by parallel processing of avoidance and palatability.

Inactivation of the basolateral amygdala suppresses the expression of taste neophobia but not the retrieval process in attenuation of neophobia.

Taste neophobia is a behavior seen in rodents that involves reduction in the consumption of a novel food or drink. In the absence of negative post-ingestive consequences, consumption increases with exposure. In this study, we examined the effect of pharmacological inactivation of the basolateral nucleus of the amygdala (BLA) on taste neophobia in rats using muscimol, a GABAA receptor agonist. Rats treated with muscimol infused into the bilateral BLA showed a suppressed but not totally abolished neophobic reaction to a novel saccharin solution compared to that observed in control rats. This result indicates that BLA function is important, but not essential, for expression of a neophobic reaction to a novel sweet taste solution. However, infusion of muscimol into the BLA did not disrupt attenuation of neophobia, which implies the presence of a retrieval process in safe taste memory.

Intraperitoneal injection of d-serine inhibits high-fat diet intake and preference in male mice.

d-serine is a co-agonist of the N-methyl d-aspartate (NMDA) receptor, an important modulator of glutamatergic excitatory synaptic transmission. We previously reported that oral d-serine ingestion inhibited the intake of highly preferred food and promoted the intake of less preferred food in mice. Here, we analyzed the effects of intraperitoneal (IP) d-serine injections on feeding behavior in mice. We assessed the effects of d-serine during both the acquisition and maintenance of a preference for high-fat diets (HFDs). Aversiveness of IP d-serine was analyzed in the conditioned taste aversion paradigm. The effects on food intake were assessed by providing liquid meals with different fat contents. Finally, we measured brain d-serine and l-serine levels after d-serine administration. We found that IP-injected d-serine effectively inhibited the acquisition of a HFD preference, but failed to prevent expression of a previously learned HFD preference. IP-injected d-serine was not sufficient to condition taste aversion. The effect on HFD preference acquisition was associated with increases in d-serine levels in the cerebral cortex, hypothalamus, and cerebellum. IP-injected d-serine most effectively inhibited the intake of liquid meals with high fat content. This effect was dose-dependent, but the responses varied significantly among male C57BL/6J mice. The differential responses to d-serine were consistent among multiple trials in each mouse. In summary, IP-injected d-serine inhibited HFD intake and the acquisition of an HFD preference. Individual mice with the same genetic background showed different sensitivities to d-serine; thus, d-serine sensitivity may be associated with unidentified traits.

Systemic administration of anorexic gut peptide hormones impairs hedonic-driven sucrose consumption in mice.

A number of reports suggest that gut hormones such as cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and peptide YY(3-36) (PYY3-36), which are released postprandially, suppress homeostatic food intake and result in satiety and the termination of feeding. However, it remains unclear whether these peptide hormones also suppress non-homeostatic consumption of palatable foods or fluids. To examine whether gut hormones reduce hedonically motivated sugar consumption, we assessed the effects of intraperitoneal administration of these gut hormones on the consumption of a highly palatable sucrose solution, using a mouse model we previously established for binge-like sucrose overconsumption (Yasoshima and Shimura, 2015). To reduce homeostatic hunger, chow was available at nighttime prior to testing. After a limited-access training procedure for 10days, during which access to both sucrose and chow were controlled, on the test day, control mice injected with saline consumed significantly more sucrose than during the pre-training period. In contrast, sucrose consumption on the test day in the mice injected with CCK-8 (2 and 4µg/kg), GLP-1 (500 and 1000nmol/kg), or PYY3-36 (12.5 and 25nmol/kg) was significantly less than that in saline-injected mice. In a separate cohort of mice, the higher doses of CCK-8 and GLP-1 and a greater dose of PYY3-36 (50nmol/kg) did not produce conditioned taste aversion to saccharin, suggesting that the doses of exogenous hormones in the present study do not cause aversive visceral distress. The present findings suggest that the systemic administration of these three gut hormones suppresses hedonic-driven sugar consumption due to the anorexic, but not aversive-visceral, effects of these hormones.

Midazolam impairs the retrieval of conditioned taste aversion via opioidergic transmission in mice.

Midazolam is a benzodiazepine agonist that affects the acquisition, retention, and retrieval of malaise-induced conditioned taste aversion (CTA) in rats. Our previous study suggested that the palatability-enhancing rather than amnesic effects of midazolam were responsible for impaired retrieval of conditioned aversion to palatable conditioned stimuli (CSs). However, it remains unclear whether this effect is opioid-dependent. In the present study, we examined the involvement of opioid signaling with the ability of peripheral midazolam administration to transiently impair CTA retrieval in mice. CTA was established by pairing 5mM saccharin ingestion (conditioned stimulus, CS) with an intraperitoneal (i.p.) injection of 0.15M lithium chloride (LiCl, 2% body weight) (unconditioned stimulus) for two consecutive days. Conditioned mice that received midazolam (1.5mg/kg, i.p.) before the first retention test consumed significantly more saccharin (CS) than conditioned mice that received vehicle (phosphate-buffered physiological saline, PBS; i.p.). On the next day, both conditioned groups showed strong aversions to the CS. Next, naloxone, an opioid receptor antagonist, was peripherally administered prior to the midazolam injection before the retention test. Pre-administration of naloxone but not PBS attenuated midazolam-induced increases in CS intake. Finally, we examined aversive orofacial taste reactions (TRs) to an oral infusion of the CS with pre-administration of naloxone or PBS prior to midazolam using a taste reactivity test. Conditioned mice that received midazolam showed significantly longer latencies to express aversive orofacial TRs than those that received PBS. Pre-administration of naloxone eliminated the effect of midazolam on latency to express aversive TRs. Taken together, these data suggest that midazolam activates opioidergic transmission and opioid-dependent palatability enhancement of the CS to eliminate conditioned aversion to a sweet taste.

A mouse model for binge-like sucrose overconsumption: Contribution of enhanced motivation for sweetener consumption.

Behavioral and neural features of binge-like sugar overconsumption have been studied using rat models. However, few mouse models are available to examine the interaction between neural and genetic underpinnings of bingeing. In the present study, we first aim to establish a simple mouse model of binge-like sucrose overconsumption using daytime limited access training in food-restricted male mice. Trained mice received 4-h limited access to both 0.5M sucrose solution and chow for 10 days. Three control groups received (1) 4-h sucrose and 20-h chow access, (2) 20-h sucrose and 4-h, or (3) 20-h chow access, respectively. Only the trained group showed progressively increased sucrose consumption during brief periods of time and developed binge-like excessive behavior. Next, we examined whether the present mouse model mimicked a human feature of binge eating known as "eating when not physically hungry." Trained mice consumed significantly more sucrose or non-caloric sweetener (saccharin) during post-training days even after they nocturnally consumed substantial chow prior to daytime sweetener access. In other trained groups, both a systemic administration of glucose and substantial chow consumption prior to the daytime limited sucrose access failed to reduce binge-like sucrose overconsumption. Our results suggest that even when caloric consumption is not necessarily required, limited access training shapes and triggers binge-like overconsumption of sweetened solution in trained mice. The binge-like behavior in trained mice may be mainly due to enhanced hedonic motivation for the sweetener's taste. The present study suggests that our mouse model for binge-like sugar overconsumption may mimic some human features of binge eating and can be used to investigate the roles of neural and genetic mechanisms in binge-like overconsumption of sweetened substances in the absence of physical hunger.

Efficient gene transfer via retrograde transport in rodent and primate brains using a human immunodeficiency virus type 1-based vector pseudotyped with rabies virus glycoprotein.

The primate lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) has been used for a wide range of gene therapy trials in animal models. Axonal transport in the retrograde direction, which is observed with some viral vectors, confers a considerable advantage to gene transfer into neuronal cell bodies that are localized in regions remote from the injection site of the vectors. However, retrograde axonal transport of the HIV-1-based lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein is reported to be inefficient. In the present study, we developed an efficient gene transfer system through retrograde transport in the brain with the HIV-1-based vector pseudotyped with rabies virus glycoprotein (RV-G). Injection of the RV-G-pseudotyped HIV-1 vector into the dorsal striatum of mice yielded an increase in gene transfer into neuronal populations in the cerebral cortex, thalamus, and ventral midbrain, each of which innervates the striatum. In addition, injection of the RV-G-pseudotyped vector into the monkey striatum (putamen) resulted in highly efficient transfer into neurons in the ventral midbrain (nigrostriatal dopamine neurons). Our results indicate that pseudotyping of the HIV-1 vector with RV-G enhances the efficiency of gene transfer through retrograde axonal transport in both mouse and monkey brains. This primate lentiviral vector system will provide a powerful approach to gene therapy for neurological and neurodegenerative diseases by means of enhanced retrograde transport.

Decline of striatal dopamine release in parkin-deficient mice shown by ex vivo autoradiography.

Parkin is the causal gene of autosomal recessive juvenile parkinsonism (AR-JP). Dopamine (DA) metabolism has been linked to Parkinson's disease (PD). To understand the pathogenesis of AR-JP, we generated parkin-deficient mice to assess the status of DA signaling pathway and examine DA release and DA receptor by ex vivo autoradiography. Ex vivo autoradiography using [11C]raclopride showed a clear decrease in endogenous DA release after methamphetamine challenge in parkin-deficient mice. Furthermore, parkin deficiency was associated with considerable upregulation of DA (D1 and D2) receptor binding in vivo in the striatum and increased DA levels in the midbrain. Our results suggest that dopaminergic neurons could behave abnormally before neuronal death.

Acute suppression, but not chronic genetic deficiency, of c-fos gene expression impairs long-term memory in aversive taste learning.

Several lines of evidence have indicated that the establishment of long-term memory requires protein synthesis, including the synthesis of immediate-early gene products. Although the anatomical expression patterns of the c-fos gene, a transcription factor-encoding immediate-early gene, in conditioned taste aversion (CTA) are well documented, the functional roles of c-fos gene expression and Fos-mediated transcription remain to be clarified. Using the antisense oligodeoxynucleotide (AS-ODN) method in rats and gene-targeting knockout techniques in mice (c-fos(-/-) mice), we examined the roles of c-fos gene expression in the acquisition, retrieval, and retention of CTA. Preconditioning microinfusion of AS-ODN directed against c-fos mRNA (c-fos AS-ODN) into the parabrachial nucleus (PBN) impaired the acquisition, whereas infusion of control ODNs consisting of a randomized or inverted base order had no effect. Microinfusion of c-fos AS-ODN into either the amygdala or insular cortex did not impair the acquisition, whereas it attenuated the retention. Retrieval and subsequent retention of an acquired CTA were not disrupted by c-fos AS-ODN infusion into the PBN or amygdala. Microinfusion of another AS-ODN directed against zif268 (egr-1, krox-24, NGFI-A) mRNA into the PBN or amygdala did not affect the acquisition and retention. The genetic deficiency in c-fos(-/-) mice caused normal acquisition and retention. The present results suggest that the Fos-mediated gene transcription in the PBN, amygdala, or insular cortex plays critical roles in the acquisition and/or consolidation, but not the retrieval, of long-term taste memory; nevertheless, some other factors could compensate CTA mechanism when Fos-mediated transcription is not available.

Involvement of the supramammillary nucleus in aversive conditioning.

Mechanisms for the retention and retrieval of conditioned taste aversions (CTAs) have yet to be fully defined. The authors explored relevant subcortical forebrain regions by tracking the expression of immediate early genes, c-fos and zif268. The supramammillary nucleus (SuM) was activated following both viscerally based CTA and somatically based inhibitory avoidance (IA). Excitotoxic lesions of the SuM before conditioning caused no disruption of acquisition but accelerated the extinction of both the CTA and IA. In contrast, lesions after CTA conditioning did not impair retention or retrieval. The present study indicates that the SuM is activated by memory-elicited discomfort during retrieval, suggesting that it plays a role in resisting the extinction of a long-term aversive memory.

Subthalamic neurons coordinate basal ganglia function through differential neural pathways.

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Effects of midazolam on the expression of conditioned taste aversion in rats.

In conditioned taste aversion (CTA), the animals learn to avoid a taste substance (conditioned stimulus, CS) which was previously associated with visceral distress (unconditioned stimulus, US). The present study examined the effects of administration of midazolam (MDZ), a benzodiazepine agonist, after the acquisition of CTA on the expression of CTA. After ingestion of 0.5 M sucrose (CS) was paired with an intraperitoneal (i.p.) injection of 0.15 M LiCl (US), control rats showed strong CTA to the CS. However, a systemic injection of MDZ (1.5 mg/kg, i.p.) before the retention test prevented conditioned animals from rejecting the CS, but in the subsequent retention tests where the drug was not administrated, those animals again showed strong aversions to the CS. Aversive taste reactivity patterns to the intraorally infused sucrose and 0.3 M dl-alanine in the conditioned animals were also diminished by the similar injection of MDZ, but not by a serotonergic anxiolytic agent, buspirone (2.5 or 5.0 mg/kg, i.p.). General taste sensory deficit might not be induced by MDZ because the drug injection did not impair conditioned aversions to 0.2 M NaCl and 0.01 M HCl. Infusion of MDZ into the basolateral nucleus of the amygdala (BLA) also attenuated conditioned aversions to sucrose. These results suggest that systemic or intra-BLA administrations of MDZ impair the expression of CTA selectively to sweet-tasting substances, implying that a transient MDZ-induced CTA expression deficit is due to the enhancement of palatability of CSs with preferable tastes rather than general anxiolytic or amnesic effects of MDZ.

Conditional ablation of striatal neuronal types containing dopamine D2 receptor disturbs coordination of basal ganglia function.

Dopamine (DA) exerts synaptic organization of basal ganglia circuitry through a variety of neuronal populations in the striatum. We performed conditional ablation of striatal neuronal types containing DA D2 receptor (D2R) by using immunotoxin-mediated cell targeting. Mutant mice were generated that express the human interleukin-2 receptor alpha-subunit under the control of the D2R gene. Intrastriatal immunotoxin treatment of the mutants eliminated the majority of the striatopallidal medium spiny neurons and cholinergic interneurons. The elimination of these neurons caused hyperactivity of spontaneous movement and reduced motor activation in response to DA stimulation. The elimination also induced upregulation of GAD gene expression in the globus pallidus (GP) and downregulation of cytochrome oxidase activity in the subthalamic nucleus (STN), whereas it attenuated DA-induced expression of the immediate-early genes (IEGs) in the striatonigral neurons. In addition, chemical lesion of cholinergic interneurons did not alter spontaneous movement but caused a moderate enhancement in DA-induced motor activation. This enhancement of the behavior was accompanied by an increase in the IEG expression in the striatonigral neurons. These data suggest that ablation of the striatopallidal neurons causes spontaneous hyperactivity through modulation of the GP and STN activity and that the ablation leads to the reduction in DA-induced behavior at least partly through attenuation of the striatonigral activity as opposed to the influence of cholinergic cell lesion. We propose a possible model in which the striatopallidal neurons dually regulate motor behavior dependent on the state of DA transmission through coordination of the basal ganglia circuitry.

Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development.

Dopamine (DA)-producing neurons in the ventral midbrain are generated from a specified neuronal lineage and form selective axonal pathways that mediate multiple CNS functions. Expression of the gene encoding tyrosine hydroxylase (TH), which is a key enzyme of catecholamine biosynthesis, is regulated during the development of midbrain DA neurons. In the present study, we report the developmental regulation and cell type specificity of TH gene promoter in the ventral midbrain by using a green fluorescent protein (GFP) reporter system. Transgenic mice were generated that express GFP in the majority of midbrain DA neurons under the control of the 9-kb upstream region of the rat TH gene. At an early embryonic stage, GFP expression was induced in the developing DA neurons, and the expression was then markedly down-regulated at later embryonic stages. However, the expression was reactivated and approached the adult levels during early post-natal development. These developmental changes in GFP expression patterns suggest the presence of multistep regulatory mechanisms for TH gene expression during DA neuron development. The TH promoter appears to possess transcriptional elements at least necessary for the induction of TH expression at the early embryonic stage and its reactivation during the post-natal development.