A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, Oncorhynchus keta, Using Mouse Adeno-Associated Viral Vectors.

To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Alcohol withdrawal drives depressive behaviors by activating neurons in the rostromedial tegmental nucleus.

Rostromedial tegmental nucleus (RMTg) GABA neurons exert a primary inhibitory drive onto midbrain dopamine neurons and are excited by a variety of aversive stimuli. There is, however, little evidence that the RMTg-ventral tegmental area (VTA)-nucleus accumbens shell (Acb) circuit plays a role in the aversive consequences of alcohol withdrawal. This study was performed in adult male Long-Evans rats at 48-h withdrawal from chronic alcohol drinking in the intermittent schedule. These rats displayed clear anhedonia and depression-like behaviors, as measured with the sucrose preference, and forced swimming tests. These aberrant behaviors were accompanied by a substantial increase in cFos expression in the VTA-projecting RMTg neurons, identified by a combination of immunohistochemistry and retrograde-tracing techniques. Pharmacological or chemogenetic inhibition of RMTg neurons mitigated the anhedonia and depression-like behaviors. Ex vivo electrophysiological data showed that chemogenetic inactivation of RMTg neurons reduced GABA release and accelerated spontaneous firings of VTA dopamine neurons. Finally, using a functional hemispheric disconnection procedure, we demonstrated that inhibition of unilateral RMTg, when combined with activation of D1 and D2 dopamine receptors in the contralateral (but not ipsilateral) Acb, mitigated the anhedonia and depression-like behaviors in alcohol-withdrawal rats. These data show that the integrity in the RMTg-VTA-Acb pathway in a single hemisphere is sufficient to elicit depression-like behavior during ethanol-withdrawal. Overall, the present results reveal that the RMTg-VTA-Acb pathway plays a crucial role in the depression-like behavior in animals undergoing alcohol withdrawal, further advocating the RMTg as a potential therapeutic target for alcoholism.

Nicotine aversion is mediated by GABAergic interpeduncular nucleus inputs to laterodorsal tegmentum.

Nicotine use can lead to dependence through complex processes that are regulated by both its rewarding and aversive effects. Recent studies show that aversive nicotine doses activate excitatory inputs to the interpeduncular nucleus (IPN) from the medial habenula (MHb), but the downstream targets of the IPN that mediate aversion are unknown. Here we show that IPN projections to the laterodorsal tegmentum (LDTg) are GABAergic using optogenetics in tissue slices from mouse brain. Selective stimulation of these IPN axon terminals in LDTg in vivo elicits avoidance behavior, suggesting that these projections contribute to aversion. Nicotine modulates these synapses in a concentration-dependent manner, with strong enhancement only seen at higher concentrations that elicit aversive responses in behavioral tests. Optogenetic inhibition of the IPN-LDTg connection blocks nicotine conditioned place aversion, suggesting that the IPN-LDTg connection is a critical part of the circuitry that mediates the aversive effects of nicotine.

Prenatal nicotine exposure alters postsynaptic AMPA receptors and glutamate neurotransmission within the laterodorsal tegmentum (LDT) of juvenile mice.

Despite dissemination of information regarding the harm on fetal development of smoking while pregnant, the number of pregnancies associated with nicotine exposure appears to have stagnated. Presence of nicotine during neural formulation is associated with a higher susceptibility of drug dependence, suggesting an altered development of neurons in circuits involved in saliency and motivation. The laterodorsal tegmental nucleus (LDT) plays a role in coding stimuli valence via afferents to mesolimbic nuclei. Accordingly, alterations in development of neural mechanisms in the LDT could be involved in vulnerability to drug dependency. Therefore, we examined the effect of prenatal nicotine exposure (PNE) on glutamatergic functioning of LDT neurons in mouse brain slices using whole-cell, patch clamp concurrent with fluorescence-based calcium imaging. PNE was associated with larger amplitudes of AMPA-induced currents, and greater AMPA-mediated rises in intracellular calcium. AMPA/NMDA ratios and the AMPA-current rectification index were lower and higher, respectively, consistent with changes in the functionality of AMPA receptors in the PNE, which was substantiated by a greater inhibition of evoked and spontaneous glutamatergic synaptic events by a selective inhibitor of GluA2-lacking AMPA receptors. Paired pulse ratios showed a decreased probability of glutamate release from presynaptic inputs, and fluorescent imaging indicated a decreased action potential-dependent calcium increase associated with PNE. When taken together, our data suggest that PNE alters LDT glutamatergic functioning, which could alter output to mesolimbic targets. Such an alteration could play a role in altered coding of relevancy of drug stimuli that could enhance risk for development of drug dependency.

Glucagon-Like Peptide-1 Receptor Signaling in the Lateral Dorsal Tegmental Nucleus Regulates Energy Balance.

The neurobiological substrates that mediate the anorectic effects of both endogenous glucagon-like peptide-1 (GLP-1) and exogenous GLP-1 receptor (GLP-1R) agonists are an active area of investigation. As the lateral dorsal tegmental nucleus (LDTg) expresses the GLP-1R and represents a potential neuroanatomical hub connecting the nucleus tractus solitarius (NTS), the major central source of GLP-1, with the other nuclei in the midbrain and forebrain, we tested the hypothesis that GLP-1R signaling in the LDTg controls food intake. Direct activation of LDTg GLP-1R suppresses food intake through a reduction in average meal size and independent of nausea/malaise. Immunohistochemical data show that GLP-1-producing neurons in the NTS project to the LDTg, providing anatomical evidence of endogenous central GLP-1 in the LDTg. Pharmacological blockade of LDTg GLP-1Rs with exendin-(9-39) dose-dependently increases food intake and attenuates the hypophagic effects of gastric distension. As GLP-1 mimetics are administered systemically in humans, we evaluated whether peripherally administered GLP-1R agonists access the LDTg to affect feeding. Immunohistochemical data show that a systemically administered fluorescent GLP-1R agonist accesses the LDTg and is juxtaposed with neurons. Additionally, blockade of LDTg GLP-1Rs attenuates the hypophagic effects of a systemic GLP-1R agonist. Together, these data indicate that LDTg GLP-1R signaling controls energy balance and underscores the role of the LDTg in integrating energy balance-relevant signals to modulate feeding.

Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression.

Prenatal overnutrition affects development into adulthood and influences risk of obesity. We assessed the transgenerational effect of maternal Western diet (WD) consumption on offspring physical activity. Voluntary wheel running was increased in juvenile (4-7 wk of age), but decreased in adult (16-19 wk of age), F1 female WD offspring In contrast, no wheel-running differences in F1 male offspring were observed. Increased wheel running in juvenile female WD offspring was associated with up-regulated dopamine receptor (DRD)-1 and -2 in the nucleus accumbens (NAc) and with down-regulated Lepr in the ventral tegmental area (VTA). Conversely, decreased wheel running by adult female WD offspring was associated with down-regulated DRD1 in the NAc and with up-regulated Lepr in the VTA. Body fat, leptin, and insulin were increased in male, but not in female, F1 WD offspring. Recombinant virus (rAAV) leptin antagonism in the VTA decreased wheel running in standard diet but not in WD F1 female offspring. Analysis of F2 offspring found no differences in wheel running or adiposity in male or female offspring, suggesting that changes in the F1 generation were related to in utero somatic reprogramming. Our findings indicate prenatal WD exposure leads to age-specific changes in voluntary physical activity in female offspring that are differentially influenced by VTA leptin antagonism.-Ruegsegger, G. N., Grigsby, K. B., Kelty, T. J., Zidon, T. M., Childs, T. E., Vieira-Potter, V. J., Klinkebiel, D. L., Matheny, M., Scarpace, P. J., Booth, F. W. Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression.

Muscarinic control of rostromedial tegmental nucleus GABA neurons and morphine-induced locomotion.

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express µ-opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid-induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co-localized with µ-opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno-associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D-expressing mice, clozapine-N-oxide increased morphine-induced, but not vehicle-induced, locomotion. In M3D-expressing mice, clozapine-N-oxide blocked morphine-induced, but not vehicle-induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine-dependent and dopamine-independent opioid-induced rewards and locomotion.

Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the cellular changes in the LDT induced by exposures to nicotine prenatally, when combined with alterations in other reward-related regions, could contribute to the increased susceptibility to smoking observed in the offspring.

Expression of estrogen receptors α and ß in the trigeminal mesencephalic nucleus of adult women and men.

Temporomandibular disorders are more prevalent in women than in men and phases of pain relate to the estrous cycle. Several studies described the location of estrogen receptors (ER) in the temporomandibular joint (TMJ), the masseteric muscles and cartilage, but it was unknown whether they are also expressed within the pseudounipolar neurons of the trigeminal mesencephalic nucleus, which receives direct sensory inputs from these structures. Therefore, we studied expression of ERα and ERß protein in the trigeminal mesencephalic nucleus of ten human brains (five female/five male). Both receptors were uniformly expressed on neurons, but not other cell types within the target structure. Thus, sensory inputs from the TMJ and adjacent structures are likely to be modulated by estrogen at the level of the first sensory neuron which may underlie the well-known correlation of pain incidence and phases of the estrous cycle.

Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat.

The lateral habenula (LHb) is an epithalamic structure differentiated in a medial (LHbM) and a lateral division (LHbL). Together with the rostromedial tegmental nucleus (RMTg), the LHb has been implicated in the processing of aversive stimuli and inhibitory control of monoamine nuclei. The inhibitory LHb influence on midbrain dopamine neurons has been shown to be mainly mediated by the RMTg, a mostly GABAergic nucleus that receives a dominant input from the LHbL. Interestingly, the RMTg also projects to the dorsal raphe nucleus (DR), which also receives direct LHb projections. To compare the organization and transmitter phenotype of LHb projections to the DR, direct and indirect via the RMTg, we first placed injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the LHb or the RMTg. We then confirmed our findings by retrograde tracing and investigated a possible GABAergic phenotype of DR-projecting RMTg neurons by combining retrograde tracing with in situ hybridization for GAD67. We found only moderate direct LHb projections to the DR, which mainly emerged from the LHbM and were predominantly directed to the serotonin-rich caudal DR. In contrast, RMTg projections to the DR were more robust, emerged from RMTg neurons enriched in GAD67 mRNA, and were focally directed to a distinctive DR subdivision immunohistochemically characterized as poor in serotonin and enriched in presumptive glutamatergic neurons. Thus, besides its well-acknowledged role as a GABAergic control center for the ventral tegmental area (VTA)-nigra complex, our findings indicate that the RMTg is also a major GABAergic relay between the LHb and the DR.

Paralemniscal TIP39 is induced in rat dams and may participate in maternal functions.

The paralemniscal area, situated between the pontine reticular formation and the lateral lemniscus in the pontomesencephalic tegmentum contains some tuberoinfundibular peptide of 39 residues (TIP39)-expressing neurons. In the present study, we measured a 4 times increase in the level of TIP39 mRNA in the paralemniscal area of lactating mothers as opposed to nulliparous females and mothers deprived of pups using real-time RT-PCR. In situ hybridization histochemistry and immunolabeling demonstrated that the induction of TIP39 in mothers takes place within the medial paralemniscal nucleus, a cytoarchitectonically distinct part of the paralemniscal area, and that the increase in TIP39 mRNA levels translates into elevated peptide levels in dams. The paralemniscal area has been implicated in maternal control as well as in pain perception. To establish the function of induced TIP39, we investigated the activation of TIP39 neurons in response to pup exposure as maternal, and formalin injection as noxious stimulus. Both stimuli elicited c-fos expression in the paralemniscal area. Subsequent double labeling demonstrated that 95% of neurons expressing Fos in response to pup exposure also contained TIP39 immunoreactivity and 91% of TIP39 neurons showed c-fos activation by pup exposure. In contrast, formalin-induced Fos does not co-localize with TIP39. Instead, most formalin-activated neurons are situated medial to the TIP39 cell group. Our data indicate that paralemniscal neurons may be involved in the processing of maternal and nociceptive information. However, two different groups of paralemniscal neurons participate in the two functions. In particular, TIP39 neurons may participate in the control of maternal functions.

Interleukin-1 inhibits putative cholinergic neurons in vitro and REM sleep when microinjected into the rat laterodorsal tegmental nucleus.

STUDY OBJECTIVES: REM sleep is suppressed during infection, an effect mimicked by the administration of cytokines such as interleukin-1 (IL-1). In spite of this observation, brain sites and neurochemical systems mediating IL-1-induced suppression of REM sleep have not been identified. Cholinergic neurons in the brainstem laterodorsal tegmental nucleus (LDT) are part of the neuronal circuitry responsible for REM sleep generation. Since IL-1 inhibits acetylcholine synthesis and release, the aim of this study was to test the two different, but related hypotheses. We hypothesized that IL-1 inhibits LDT cholinergic neurons, and that, as a result of this inhibition, IL-1 suppresses REM sleep. DESIGN, MEASUREMENT, AND RESULTS: To test these hypotheses, the electrophysiological activity of putative cholinergic LDT neurons was recorded in a rat brainstem slice preparation. Interleukin-1 significantly inhibited the firing rate of 76% of recorded putative cholinergic LDT neurons and reduced the amplitude of glutamatergic evoked potentials in 60% of recorded neurons. When IL-1 (1 ng) was microinjected into the LDT of freely behaving rats, REM sleep was reduced by about 50% (from 12.7% +/- 1.5% of recording time [after vehicle] to 6.1% +/- 1.4% following IL-1 administration) during post-injection hours 3-4. CONCLUSIONS: Results of this study support the hypothesis that IL-1 can suppress REM sleep by acting at the level of the LDT nucleus. Furthermore this effect may result from the inhibition of evoked glutamatergic responses and of spontaneous firing of putative cholinergic LDT neurons.

Sex differences in urocortin 1 dynamics in the non-preganglionic Edinger-Westphal nucleus of the rat.

Women have higher vulnerability to stress and stress-induced diseases than men. Estrogen may be involved in the control of sex-dependent stress adaptation via estrogen receptors alpha and beta (ERalpha/beta). Urocortin 1 (Ucn1) in the npEW plays an important role in stress adaptation. We hypothesize that the activity of npEW-Ucn1 neurons differs between sexes and is related to estrogen signalling. We here indicate by immunocytochemistry the absence of ERalpha and the presence of ERbeta in the npEW-Ucn1 neurons. Q-RT-PCR of the npEW confirmed this notion, demonstrating that in male rats ERbeta mRNA was almost 5 times higher than in females in di-estrus. Furthermore, Ucn1 mRNA in males was nearly 10 times and 1.6 times higher than in females in di- and pro-estrus, respectively, indicating a sex-dependent difference in Ucn1 biosynthetic activity. Since, at the same time, immunocytochemistry revealed that the amount of Ucn1 peptide stored in the cell bodies of the npEW-Ucn1 neurons did not differ between males and females, as judged on the basis of the number and immunosignal density of these neurons, we propose that the rate of axonal Ucn1 transport and, possibly, the strength of Ucn1 secretion, are dependent on sex to the same degree as is Ucn1 biosynthesis.

Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice.

In humans, tobacco withdrawal produces symptoms that contribute to the difficulty associated with smoking cessation. Nicotine withdrawal symptoms can also be observed in rodents. A major standing question is which nicotinic receptor subtypes and which areas of the brain are necessary for nicotine withdrawal to occur. Using knock-out mice, we previously showed that the beta4, but not the beta2 subunit of nicotinic acetylcholine receptors, is necessary for the somatic manifestations of nicotine withdrawal. Since the beta4 subunit is highly expressed in the medial habenula, we focused our studies on the medial habenula and its primary target, the interpeduncular nucleus. In particular, we studied nicotine withdrawal in mice lacking the alpha2 or the alpha5 nicotinic receptor subunits, which are highly expressed in the interpeduncular nucleus. We precipitated withdrawal by systemically injecting the nicotinic antagonist mecamylamine in mice chronically treated with nicotine. Both the alpha2 and the alpha5 null mutations abolished the somatic manifestations of nicotine withdrawal. In addition, in wild-type mice chronically treated with nicotine, mecamylamine precipitated withdrawal when microinjected into the habenula or the interpeduncular nucleus, but not into the cortex, ventral tegmental area or hippocampus. Our results demonstrate a major role for the habenulo-interpeduncular system and the nicotinic receptor subunits expressed therein, in nicotine withdrawal symptoms. Our data suggest that the efforts to develop new smoking cessation therapies should concentrate on these areas and receptor types.

The mesopontine rostromedial tegmental nucleus: A structure targeted by the lateral habenula that projects to the ventral tegmental area of Tsai and substantia nigra compacta.

Prior studies revealed that aversive stimuli and psychostimulant drugs elicit Fos expression in neurons clustered above and behind the interpeduncular nucleus that project strongly to the ventral tegmental area (VTA) and substantia nigra (SN) compacta (C). Other reports suggest that these neurons modulate responses to aversive stimuli. We now designate the region containing them as the "mesopontine rostromedial tegmental nucleus" (RMTg) and report herein on its neuroanatomy. Dense micro-opioid receptor and somatostatin immunoreactivity characterize the RMTg, as do neurons projecting to the VTA/SNC that are enriched in GAD67 mRNA. Strong inputs to the RMTg arise in the lateral habenula (LHb) and, to a lesser extent, the SN. Other inputs come from the frontal cortex, ventral striatopallidum, extended amygdala, septum, preoptic region, lateral, paraventricular and posterior hypothalamus, zona incerta, periaqueductal gray, intermediate layers of the contralateral superior colliculus, dorsal raphe, mesencephalic, pontine and medullary reticular formation, and the following nuclei: parafascicular, supramammillary, mammillary, ventral lateral geniculate, deep mesencephalic, red, pedunculopontine and laterodorsal tegmental, cuneiform, parabrachial, and deep cerebellar. The RMTg has meager outputs to the forebrain, mainly to the ventral pallidum, preoptic-lateral hypothalamic continuum, and midline-intralaminar thalamus, but much heavier outputs to the brainstem, including, most prominently, the VTA/SNC, as noted above, and to medial tegmentum, pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe, and locus ceruleus and subceruleus. The RMTg may integrate multiple forebrain and brainstem inputs in relation to a dominant LHb input. Its outputs to neuromodulatory projection systems likely converge with direct LHb projections to those structures.

Nicotine-induced monoamine neurotransmitter changes in the brain of young rats.

A number of studies in various species including man indicated a greater risk of drug preference and addictive behavior in young as compared to adults. Such age dependent preference was also found with nicotine. To examine possible mechanisms for this difference in our continuing study of reward mechanisms, we compared nicotine-induced neurotransmitter changes in the brain regions of adult and young Sprague-Dawley rats, assaying the transmitters via microdialysis in conscious freely moving animals. In general, nicotine-induced changes were significantly less in the regions measured in the young. Nicotine-induced effects on dopamine in the dorsal and ventral hippocampus (VH), prefrontal and medial temporal cortex, and superior cerebral peduncle were lower in the young than in adult, the same in the ventral tegmental area (VTA) and lateral septal nucleus (LS), and somewhat higher in the nucleus accumbens shell (NAccS). Norepinephrine levels in the young were lower in all areas except in the VH where they were the same, and serotonin levels were lower except in the VTA and LS where they remained the same, and higher in the NAccS. Age-dependent differences in the metabolites measured were more mixed. We conclude that the greater nicotine preference in young is not paralleled by a greater effect of nicotine on the release of monoamines at least in most of the brain areas assayed. Thus, increases of nicotine reward are not likely due to increases of monoamines in reward and cognitive areas. The small increase of dopamine (DA) and more significant increase of serotonin (5-HT) only in the NAccS are of significance, and would indicate a more significant role of 5-HT than of DA at least in the age difference in nicotine preference. Developmental changes in receptor composition and distribution involving several transmitter systems and other components such as neuropeptides are also likely to play a role.

Injections of urocortin 1 into the basolateral amygdala induce anxiety-like behavior and c-Fos expression in brainstem serotonergic neurons.

The amygdala plays a key role in emotional processing and anxiety-related physiological and behavioral responses. Previous studies have shown that injections of the anxiety-related neuropeptide corticotropin-releasing factor or the related neuropeptide urocortin 1 into the region of the basolateral amygdaloid nucleus induce anxiety-like behavior in several behavioral paradigms. Brainstem serotonergic systems in the dorsal raphe nucleus and median raphe nucleus may be part of a distributed neural system that, together with the basolateral amygdala, regulates acute and chronic anxiety states. We therefore investigated the effect of an acute bilateral injection of urocortin 1 into the basolateral amygdala on behavior in the social interaction test and on c-Fos expression within serotonergic neurons in the dorsal raphe nucleus and median raphe nucleus. Male rats were implanted with bilateral cannulae directed at the region of the basolateral amygdala; 72 h after surgery, rats were injected with urocortin 1 (50 fmol/100 nl) or vehicle (100 nl of 1% bovine serum albumin in distilled water). Thirty minutes after injection, a subgroup of rats from each experimental group was exposed to the social interaction test; remaining animals were left in the home cage. Two hours after injection rats were perfused with paraformaldehyde and brains were removed and processed for immunohistochemistry. Acute injection of urocortin 1 had anxiogenic effects in the social interaction test, reducing total interaction time without affecting locomotor activity or exploratory behavior. These behavioral effects were associated with increases in c-Fos expression within brainstem serotonergic neurons. In home cage rats and rats exposed to the social interaction test, urocortin 1 treatment increased the number of c-Fos-immunoreactive serotonergic neurons within subdivisions of both the dorsal raphe nucleus and median raphe nucleus. These results are consistent with the hypothesis that the basolateral amygdala and serotonergic neurons within the midbrain raphe complex are part of an integrated neural system modulating anxiety state.

Cocaine- and amphetamine-regulated transcript peptides play a role in drug abuse and are potential therapeutic targets.

Cocaine- and amphetamine-regulated transcript (CART) peptides (55 to 102 and 62 to 102) are neurotransmitters with important roles in a number of physiologic processes. They have a role in drug abuse by virtue of the fact that they are modulators of mesolimbic function. Key findings supporting a role in drug abuse are as follows. First, high densities of CART-containing nerve terminals are localized in mesolimbic areas. Second, CART 55 to 102 blunts some of the behavioral effects of cocaine and dopamine (DA). This functional antagonism suggests that CART peptides be considered as targets for medications development. Third, CREB in the nucleus accumbens has been shown to have an opposing effect on cocaine self-administration. CREB may activate CART expression in that region, and, if so, CART may mediate at least some of the effects of CREB. Fourth, in addition to the effects of CART on DA, DA can influence CART in the accumbens. Thus a complex interacting circuitry likely exists. Fifth, in humans, CART is altered in the ventral tegmental area of cocaine overdose victims, and a mutation in the CART gene associates with alcoholism. Overall, it is clear that there are functional interactions among CART, DA, and cocaine and that plausible cellular mechanisms exist to explain some of these actions. Future studies will clarify and extend these findings.

Chronic ether stress-induced response of urocortin 1 neurons in the Edinger-Westphal nucleus in the mouse.

Urocortin 1 (Ucn1) neurons, most abundantly expressed in the Edinger-Westphal nucleus (E-WN), respond to various acute challenges. In a recent study, we found that acute ether stress resulted in the strongest activation of E-WN Ucn1 cells, as revealed by immunohistochemistry for Fos (often used as a marker for neuronal activation). Although the acute stress responsiveness of E-WN Ucn1 neurons has been widely studied, the activation pattern of Fos in these neurons in response to repeated challenges has not yet been investigated. Therefore, we quantitatively studied Fos activation in E-WN neurons and measured Ucn1 mRNA levels in E-WN neurons after acute and chronic ether stress in mice. Acute stress resulted in a robust Fos response and an increase in Ucn1 mRNA as compared to non-stressed mice. In the chronic stress paradigm, Fos expression was unchanged, whereas after 2 and 3 weeks of daily ether exposure Ucn1 mRNA expression had strongly declined in the E-WN. Fos and Ucn1 mRNA were co-expressed in E-WN neurons in both acutely and chronically stressed animals. This paper is the first to demonstrate that Ucn1 mRNA-expressing neurons in the E-WN show a non-habituating Fos response to a chronic homotypic ether challenge that also resulted in a reliable down-regulation of E-WN Ucn1 mRNA levels vs. acutely stressed animals. Based on these results, we propose that the E-WN-Ucn1 system represents a novel stress adaptation pathway, which may play an important role in coping with chronic challenges.