How did smokers respond to standardised cigarette packaging with new, larger health warnings in the United Kingdom during the transition period? A cross-sectional online survey.

INTRODUCTION: In the United Kingdom, standardised packaging for cigarettes was phased in between May 2016 and May 2017. We assessed whether there was an association between using standardised packs and warning salience, thoughts about the risks of smoking, thoughts about quitting, and awareness and use of stop-smoking websites. METHODS: We conducted a cross-sectional online survey with current smokers aged 16 and over (N = 1865) recruited in two regions of England between February-April 2017, when both standardised and fully-branded packs were on the market. Participants were asked about use of standardised packs, warning salience (noticing, reading closely), and whether the packs they were using increased thoughts of the risks of smoking and quitting. They were also asked about awareness of stop-smoking websites, source of awareness (including warnings on packs), and whether they had visited a stop-smoking website. RESULTS: Most participants reported currently using standardised packs (76.4%), 9.3% were not currently using them but had previously used them, and 14.3% had never used them. Compared with never users, current users were more likely to have noticed the warnings on packs often/very often (AOR (95%CI) = 2.76 (2.10, 3.63)), read them closely often/very often (AOR(95%CI) = 2.16 (1.51, 3.10)), thought somewhat/a lot about the health risks of smoking (AOR(95%CI) = 1.92 (1.38, 2.68)), and thought somewhat/a lot about quitting (AOR(95%CI) = 1.90 (1.30, 2.77)). They were also more likely to have noticed a stop-smoking website on packs. CONCLUSIONS: Consistent with the broad objectives of standardised packaging, we found that it was associated with increased warning salience and thoughts about risks and quitting.

Awareness of Changes in E-cigarette Regulations and Behavior Before and After Implementation: A Longitudinal Survey of Smokers, Ex-smokers, and Vapers in the United Kingdom.

INTRODUCTION: In line with the European Union's Tobacco Products Directive (TPD), new regulations for electronic cigarettes implemented in the United Kingdom between May 2016 and May 2017 included limiting refills to 10 mL, tank and cartridge sizes to 2 mL, and nicotine concentrations to 20 mg/mL. AIMS: To investigate the (1) awareness of new regulations, (2) product use before and after implementation, and (3) association between use of compliant products and subsequent smoking. METHODS: A UK online longitudinal survey of smokers, ex-smokers, and vapers was conducted between May and June 2016 (wave 4) and September 2017 (wave 5).The following methods were used: (1) to assess awareness of changes, proportions were calculated by smoking and vaping status (n = 1606). (2) Comparison of refill volume, tank and cartridge volumes, nicotine concentration at waves 4 and 5 (n = 199-388) was conducted. (3) Association was studied between number of TPD-compliant products used at wave 4 and smoking at wave 5, adjusted for wave 4 vaping status, age, gender, income, urges to smoke, and device type (n = 480). RESULTS: Awareness of regulations was highest for refill volume (10.1%; 37.4% among exclusive vapers) and nicotine concentration (9.5%; 27.3%). Higher proportions used TPD-compliant refill volumes (60.0%-73.7%, χ2(1) = 10.9, p = .001) and nicotine concentrations (89.2%-93.9%, χ2(1) = 7.41, p = .007) in wave 5 than wave 4, with little change for tank or cartridge volumes (77.1-75.5%, χ2(1) = 0.38, p = .540). The likelihood of smoking was similar for those using no or one TPD-compliant products as it was for those using two (OR = 1.10, 95% CI = 0.47-2.59) or three (OR = 1.56, 95% CI = 0.69-3.55). CONCLUSION: Several months after full implementation, awareness of new regulations was low and most vapers used TPD-compliant products. Use of compliant products was not associated with subsequent smoking. IMPLICATIONS: Using a longitudinal survey at the beginning and a few months after the end of the transition period for implementation of new regulation on electronic cigarettes, this is the first study to assess awareness of regulation and use of compliant products. After full implementation, awareness of changes was low overall (smokers, ex-smokers, and vapers combined) although higher among those who vaped. Nevertheless, most vapers (74%-94%) used products that were compliant with the new regulations and the use of products compliant with incoming regulations did not predict whether they were smoking cigarettes after implementation.

Neuropeptide Y (NPY) or cocaine- and amphetamine-regulated transcript (CART) fiber innervation on central and medial amygdaloid neurons that project to the locus coeruleus and dorsal raphe in the rat.

The amygdaloid nuclear complex has been linked to the regulation of emotional behavior and energy regulation in that emotional stress might cause either reduction or enhancement of eating. We examined hypothalamic neuronal origin of feeding/arousal-related peptidergic fibers containing cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) located in the rat amygdala along with its efferent projections to the brainstem monoaminergic nuclei. First, central (CeA) as well as medial (MeA) amygdala, among several amygdaloid subdivisions, exhibited the most prominent NPY or CART immunostaining which consisted of a substantial number of somata as well as labeled fibers. When we examined hypothalamic neuronal origin of NPY or CART fibers projecting to the CeA and MeA, medial and lateral arcuate nuclei were neuronal origins of NPY and CART fibers, respectively. However, the majority (>70%) of amygdala-projecting CART neurons which co-contained melanin-concentrating hormone (MCH) originated from the lateral hypothalamus (LH), zona incerta (ZI), and dorsal hypothalamic area (DA). This observation implied that the CeA as well as the MeA might receive potent second-order (and downstream) feeding-related CART input from the lateral hypothalamic regions in addition to first-order CART or NPY input from the Arc. Second, a large number of CeA neurons projected to the locus coeruleus (LC), whereas only a small number of MeA cells projected to the dorsal raphe (DR); none of the CeA or MeA cells provided dual projections to the LC and DR. Finally, a portion of MCH cells in the LH, ZI, and DA sent divergent axon collaterals to the CeA and LC. Considering that the CeA sends substantial GABAergic input to the LC, the present observation might serve as an anatomical substrate to support the potent hypnogenic role of MCH neurons in the LH regions during cataplexy and REM sleep.

Hypothalamic neuronal origin of neuropeptide Y (NPY) or cocaine- and amphetamine-regulated transcript (CART) fibers projecting to the tuberomammillary nucleus of the rat.

Based on the importance of tuberomammillary nucleus (TMN) as a target for feeding/arousal-related functions, we aimed in the present study to investigate hypothalamic neuronal origin of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) fibers projecting to the histaminergic nucleus. In the first series of experiments, we examined NPY (or CART) fiber distribution within the boundary of adenosine deaminase (ADA)-immunoreactive (ir) TMN regions; extensive NPY (or CART)-ir axon terminals were observed in E4 (TMMd), E3 (TMMv), and E2 (TMVr) subdivisions. NPY varicosities co-contained vesicular GABA transporters (vGAT). CART boutons, however, contained either vGAT or vesicular glutamate transporters (vGLU), which suggested dual (or multiple) origins of CART fibers. Based on the previous observation on melanin-concentrating hormone (MCH)-ir neuronal elements in the TMN, their coexistence with CART peptide was examined in detail. In E4 subdivision, approximately 40.8% of MCH-ir somata co-contained CART, but the proportion was reduced to 24.1% in E3 region. In E2 and E1 (TMVc) regions, only MCH-ir axon terminals existed without any MCH-ir somata. In the second series of experiments, we investigated hypothalamic neuronal origin of NPY (or CART) fibers projecting to the TMN. The arcuate nucleus (Arc) was the sole source of hypothalamic NPY fibers projecting to the nucleus. In contrast, CART fibers in the TMN originated from the Arc as well as the other hypothalamic nuclei including the retrochiasmatic nucleus, paraventricular nucleus, lateral hypothalamus (LH), zona incerta (ZI), and dorsal hypothalamic area. Quantitative analysis showed that arcuate CART projection to the TMN occupied approximately 23.5% of the total hypothalamic CART input to the nucleus, while the rest originated mainly from the LH and ZI. The present observations suggested that the TMN might play a key role in energy balance and arousal, by receiving periphery-derived, first-order NPY (or CART) inputs from the Arc as well as second-order (and downstream) CART inputs from the other hypothalamic nuclei.

Dual projections of single orexin- or CART-immunoreactive, lateral hypothalamic neurons to the paraventricular thalamic nucleus and nucleus accumbens shell in the rat: Light microscopic study.

The paraventricular thalamic nucleus (PVT) is a major relay station to the limbic forebrain areas such as the nucleus accumbens shell (AcbSh). Both PVT and AcbSh are known to receive feeding/arousal-related peptidergic fibers including orexin (ORX) and cocaine- and amphetamine-regulated transcript (CART) peptide. In the first series of experiments, we examined the peptidergic fiber distribution in the AcbSh; the density of ORX (or CART) fibers in the AcbSh was substantially lower than that in the PVT. At the light microscopic level, ORX (or CART) terminals formed close appositions to choline acetyltransferase (ChAT)-, glutamate decarboxylase (GAD)-, or enkephalin (Enk)-immunoreactive neuronal elements in the AcbSh. In the second series of experiments, we addressed the question of whether single ORX (or CART) cells in the hypothalamus provided divergent axon collaterals to the PVT and AcbSh. ORX neurons with dual projections were found in the medial, central, and lateral subdivisions of the lateral hypothalamus (LH), which amounted to an average of 1.6% of total ORX cells. CART neurons with divergent axon collaterals were observed in the LH, zona incerta, dorsal hypothalamic area, and retrochiasmatic nucleus, which represented a mean of 2.5% of total CART cells. None of arcuate CART cells sent dual projections. These data suggested that a portion of ORX (or CART) neurons in the hypothalamus, via divergent axon collaterals, might concurrently modulate the activity of PVT and AcbSh cells to affect feeding and drug-seeking behaviors.

Hypothalamic, feeding/arousal-related peptidergic projections to the paraventricular thalamic nucleus in the rat.

Based on the importance of paraventricular thalamic nucleus (PVT) as a relay station of energy balance, arousal, and food reward, we aimed in the present study to determine projection patterns of neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and orexin (ORX)-ergic fibers to the PVT. First, the distribution of peptidergic axon terminals within the PVT was examined. NPY and CART terminals were confined within the boundary of the thalamic nucleus, exhibiting almost identical distribution. MCH terminals were rarely observed. In contrast, ORX terminals were as extensive as NPY/CART terminals, but spread into the peri-PVT region. Second, neuronal origin of feeding/arousal-related peptides projecting to the PVT was investigated. NPY neurons were observed in the medial subdivision of the arcuate nucleus (Arc), whereas CART cells were in the lateral Arc as well as other hypothalamic regions including the paraventricular hypothalamic nucleus, lateral hypothalamus (LH), dorsal hypothalamic area, and zona incerta. Both NPY- and CART-fiber projections to the PVT were bilateral; ipsilateral proportion was 54.0% ± 3.6% (n = 6) for NPY and 57.1% ± 2.5% (n = 6) for CART. The total number of CART neurons projecting to the PVT exceeded that of NPY cells; the ratio of labeled CART neurons to NPY cells was 2.4 ± 0.2 (n = 6). In contrast, ORX-ergic fiber projection to the PVT exhibited a slight ipsilateral dominance (62.7% ± 1.6%, n = 6), with majority of labeled cells located in the LH medial to the fornix (72.2% ± 2.3%, n = 6). Third, based on heavy projection from the PVT to the nucleus accumbens shell (NAcSh), the convergence of NPY and CART terminals on a single PVT neuron was identified; the proportion of labeled PVT neurons that received converging NPY/CART terminals compared with the total PVT neurons projecting to the NAcSh was 2.7% ± 0.6% (n = 3). Finally, PVT cells receiving NPY, CART, or ORX terminals provided divergent axon collaterals to NAcSh and medial prefrontal cortex. The present observations provided the anatomical evidence that the PVT might play an essential role in the integration of antagonistically-acting, feeding/arousal-related peptidergic inputs on their way to the cortical reward circuit.

Vestibular suppressants after canalith repositioning in benign paroxysmal positional vertigo.

OBJECTIVES/HYPOTHESIS: To investigate the characteristics of residual symptoms and to evaluate the effects of adjuvant vestibular suppressants on residual symptoms after successful canalith repositioning procedures (CRPs). STUDY DESIGN: Individual randomized controlled trial. METHODS: One hundred fifty patients with idiopathic benign paroxysmal positional vertigo who achieved successful CRPs on initial visit participated in this study. Dizziness Handicap Inventory (DHI) questionnaires were completed before CRPs. All study populations were divided into three groups after successful CRPs on the initial visit day: the medication (V) group (treated with a vestibular suppressant [dimenhydrinate 50 mg per day]), the placebo (P) group, and the no medication (N) group. One week after successful CRPs, residual symptoms were checked and repeated DHI questionnaires were completed to compare residual symptoms. RESULTS: Among the 138 patients who did not show positional nystagmus at follow-up, 67 (48.5%) complained of residual symptoms. The presence of residual symptoms was more prevalent in the P and N group compared with the V group (P = .035, P = .017, respectively). The most frequent residual symptom was lightheadedness (n = 42). Moreover, in the V group, lightheadedness was significantly reduced compared with the P group (P = .029). However, in the analysis of DHI, total and subscale scores did not differ across the three groups before or after successful CRP. CONCLUSIONS: Vestibular suppressants significantly reduced residual symptoms compared to both placebo and no medication after CRP. However, there was no significant reduction in DHI score compared with the control group, suggesting that the residual symptoms could not be evaluated by DHI score alone.

Reciprocal connections between CART-immunoreactive, hypothalamic paraventricular neurons and serotonergic dorsal raphe cells in the rat: Light microscopic study.

Based on the overlapping physiological roles of cocaine- and amphetamine-regulated transcript (CART) peptides and serotonin, the present study examined the anatomical connection between the hypothalamic paraventricular nucleus (PVN) and the dorsal raphe (DR). The first series of experiments were performed to investigate descending projections from the CART-immunoreactive (CART-ir) PVN to serotonergic DR cells. CART-ir varicosities made contact with serotonergic DR neurons. An anterograde tracing study revealed that varicosities originating from the PVN formed close appositions to serotonergic neuronal profiles along the entire rostro-caudal extent of the DR. A retrograde study demonstrated that CART neurons projecting to the DR were mainly localized in the caudal parvicellular PVN, comprising approximately 3.0%±0.4% (n=8) of total CART cells. A second series of experiments was performed to investigate ascending projections from the DR to CART-ir PVN cells. Serotonin transporter-ir boutons made contact with CART-ir PVN neurons. Anterograde tracing revealed that varicosities originating from the DR formed close appositions to CART-ir PVN cells. Retrograde examination demonstrated that serotonergic neurons projecting to the parvicellular PVN were located along the entire rostro-caudal extent of the DR. The present observation provided an anatomical basis for accumulating evidence in the literature that suggests a functional interaction between the CART and serotonin systems during the regulation of energy balance, emotional behavior, and arousal.

Retrograde study of CART- or NPY-neuronal projection from the hypothalamic arcuate nucleus to the dorsal raphe and/or the locus coeruleus in the rat.

The present study was designed to reveal cocaine- and amphetamine-regulated transcript (CART)- or neuropeptide Y (NPY)-immunoreactive neuronal projections from the hypothalamic arcuate nucleus (Arc) to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. Our results demonstrated that CART or NPY axon terminals formed close appositions to the neuronal profiles in the DR and the LC. Thus, arcuate sections were immunostained for the CART or NPY after the injections of green RetroBeads(™) into the DR and red tracer into the LC (or vice versa). First, retrogradely-labeled CART cells were mainly observed in the lateral Arc without colchicine. Of the total population of arcuate CART neurons, DR- and LC-projecting cells were 5.7% ± 0.9% and 6.6% ± 0.7%, respectively. In addition, a subset (3.3% ± 0.7%) of CART neurons provided divergent axon collaterals to the DR and the LC. Second, retrogradely-labeled NPY cells were observed in lateral or ventral borders of the medial Arc only after colchicine injection. Of the entire NPY cell population, DR- and LC-projecting neurons were 1.5% ± 0.3% and 1.3% ± 0.3%, respectively. Only a scanty proportion (0.1% ± 0.0%) sent axon collaterals to the DR and the LC. These observations suggested that arcuate CART or NPY system might have a potential influence on the brainstem monoaminergic nuclei, modulating their roles in feeding, nociception, emotional behaviors, arousal, and stress responses. Furthermore, a portion of arcuate CART neurons (along with only a few NPY cells) sending divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the monoaminergic nuclei.

Projections from melanin-concentrating hormone (MCH) neurons to the dorsal raphe or the nuclear core of the locus coeruleus in the rat.

Brainstem aminergic and cholinergic nuclei are essential components of reticular activating system which are under the control of hypothalamic sleep/arousal centers. In contrast to well-known role of hypocretin (Hcrt) as a potent wake-promoting substance, only recent reports stated that melanin-concentrating hormone (MCH) plays a role in the maintenance of rapid eye movement (REM) sleep. As the sequel to our report concerning the MCH/Hcrt projection to the brainstem cholinergic nuclei (Hong et al., 2011), in the present study we examined the differential projection from MCH/Hcrt neurons in medial and lateral subdivisions of the lateral hypothalamus (LH) to the dorsal raphe (DR) or the nuclear core of the locus coeruleus (LC) of the rat. Following the injection of Red Retrobeads into the LC core (n=6), the proportions of retrogradely labeled (retro-) MCH neurons over the total retro-cells were 4.4% ± 0.5% (medial subdivision) and 7.4% ±0 .6% (lateral one), whereas those of retro-Hcrt cells over the total retro-cells were 69.4% ± 3.6% (medial) and 64.4% ± 5.2% (lateral). Following midline-DR injections (n=6), the proportions of retro-MCH neurons over the total retro-cells were 14.3% ± 2.9% (medial) and 12.3% ± 1.6% (lateral), while those of retro-Hcrt cells over the total retro-cells were 46.5% ± 6.2% (medial) and 51.3% ± 9.5% (lateral). Following lateral wing-DR injections (n=3), the proportions of retro-MCH neurons over the total retro-cells were 15.5% ± 1.2% (medial) and 11.9% ± 3.1% (lateral), while those of retro-Hcrt cells over the total retro-cells were 48.5% ± 2.7% (medial) and 52.8% ± 2.3% (lateral). The statistical analysis showed that MCH neurons projecting to the LC core or DR were outnumbered by Hcrt cells (P<0.01) and that retro-MCH cells exhibited lateral predominance in LC injection cases (P<0.05). Based on our present as well as previous (Hong et al., 2011) observations, we suggested that MCH and Hcrt neurons in the LH provide preferential projections to the brainstem cholinergic and aminergic nuclei, respectively and that MCH projections to the nuclear core of the LC exhibit differential distribution within LH subdivisions.

Projection patterns of lateral hypothalamic, cocaine- and amphetamine-regulated transcript (CART) neurons to the dorsal raphe and/or the locus coeruleus in the rat.

The present study was designed to reveal the projection patterns of lateral hypothalamic (LH), cocaine- and amphetamine-regulated transcript (CART) neurons to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. After the injection of Red or Green Retrobeads into the DR or LC, LH sections were immunostained for CART and/or melanin-concentrating hormone (MCH). First, CART-immunoreactive axon terminals formed close appositions to the DR (or LC) neuronal profiles. Second, a subpopulation of CART neurons containing MCH projected to the monoaminergic nuclei; the majority of labeled neurons were observed in the dorsal hypothalamic area, the dorsal part of the posterior hypothalamic area, and the zona incerta. Cells were also observed in the perifornical part of the LH, the dorsomedial hypothalamic nucleus, the peduncular and the magnocellular parts of the LH. Of the total population of DR (or LC)-projecting cells, CART/MCH co-containing neurons were 9.5% ± 1.6% (or 10.8% ± 1.3% for LC). Finally, a subset of CART (or MCH) neurons provided divergent axon collaterals to the DR and the LC. Of the entire CART (or MCH) cell population, 3.9% ± 0.8% (or 5.6% ± 1.0% for MCH) sent axon collaterals to the DR/LC. CART/MCH co-containing neurons projecting to the DR or LC might be involved in the feeding-related regulation of arousal, stress-related responses, and emotional behaviors. Thus, CART (or MCH) cells that send divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the aminergic nuclei.

Differential distribution of melanin-concentrating hormone (MCH)- and hypocretin (Hcrt)-immunoreactive neurons projecting to the mesopontine cholinergic complex in the rat.

Hypocretin (Hcrt or orexin) and melanin-concentrating hormone (MCH) containing neurons are located in the hypothalamus and are implicated in the regulation of feeding behavior, energy homeostasis, and sleep-wake cycle. MCH and Hcrt are not co-localized within the same neuron, but these neurons project widely throughout the brain, especially to brain regions regulating arousal. Recent data indicate that HCRT and MCH neurons located medially with respect to the fornix have a differential projection pattern compared to those located lateral to the fornix. To further elucidate the projection of these neurons in the present study we use retrograde tracing methods combined with double immunofluorescence to determine the differential distribution of Hcrt- and MCH-immunoreactive neurons projecting to the pedunculopontine tegmental (PPTg) or laterodorsal tegmental (LDTg) nuclei. In rats where the retrograde tracer was confined to the PPTg/LDTg we found that there were more MCH neurons projecting to these targets compared to HCRT neurons (P<0.01). When the retrograde tracer was confined to the PPTg, there were more retrogradely labeled MCH neurons lateral to the fornix compared to MCH neurons in the medial LH subdivision (P<0.05). On the average, only about 4.5% of MCH neurons versus 6.1% of HCRT neurons project to PPTg/LDTg. Thus, very few of the MCH or HCRT neurons project to these arousal populations. Although there were significantly more MCH neurons projecting to the mesopontine cholinergic arousal zone compared to the HCRT neurons, the HCRT neurons also exert an indirect influence via the tuberomammillary nucleus. Based on the present and previous (Hong and Lee, 2011) observations, we suggest that both MCH and HCRT neurons exert a potent influence on the PPTg/LDTg, which might play an important role in arousal.

Retrograde study of projections from the tuberomammillary nucleus to the mesopontine cholinergic complex in the rat.

The mesopontine cholinergic complex comprising the pedunculopontine (PPTg) and laterodorsal (LDTg) tegmental nuclei is a key component of ascending reticular activating system. The brainstem cholinergic nuclei are yet under the control of descending projections from hypothalamic arousal centers such as the hypocretinergic, lateral hypothalamus (LH), and the histaminergic, tuberomammillary nucleus (TMN). The present study was designed to determine the differential projection pattern as well as the laterality of descending TMN projections to the PPTg and LDTg. Our results showed that each TMN subdivision provided differential projections to the mesopontine complex. The majority of PPTg-projecting neurons were located in ventrolateral TMN mainly at its subpial border, whereas LDTg-projecting cells were in dorsomedial TMN with a few in the medial border of the ventrolateral subdivision. For both PPTg and LDTg cases, retrogradely labeled neurons were more pronounced in each TMN subdivision ipsilateral to the injection site. The light microscopic observation also indicated that hypocretinergic, terminal-like boutons formed close appositions to somata as well as proximal dendrites of PPTg- or LDTg-projecting TMN neurons. Taken together, the present observations suggested that each TMN subdivision provide differential projections to the mesopontine cholinergic complex and that the LH, via the TMN, provides indirect, descending projections to the complex as well.

Dual projections of tuberomammillary neurons to whisker-related, sensory and motor regions of the rat.

The primary goal of this study was to examine in the rat whether neurons in the tuberomammillary nucleus (TMN) provide axon collaterals to whisker-related, sensorimotor regions at cortical and brainstem levels, using two different retrograde tracers. When injections were made at primary sensory (S1) barrel field/primary whisker motor (M1) cortices, dual-projecting TMN neurons were observed mainly in the ventrolateral subdivision; the projection was almost exclusively ipsilateral. On the other hand, following injections of tracers into whisker-related, principal sensory trigeminal (Pr5)/lateral facial motor (Mo7) nuclei, dual-projecting cells were observed mainly in the dorsomedial subdivision; the projection was bilateral with a slight contralateral dominance. Taken together, the present observation demonstrated that each subdivision of the TMN possessed a differential functional organization with respect to its collateral projection to whisker-related sensorimotor targets, suggesting that the histaminergic projection might play a modulatory role in vibrissal sensorimotor integration, which allows the guidance of behavioral action essential for the survival of the animal.

Collateral projection from the forebrain and mesopontine cholinergic neurons to whisker-related, sensory and motor regions of the rat.

The primary goal of this anatomical study was to examine in the rat whether cholinergic neurons provide axon collaterals to whisker-related, sensorimotor regions at cortical, thalamic, and brainstem levels, using a combined method of retrograde tracing and choline acetyltransferase (ChAT) immunostaining. First, when injections were made at primary sensory (S1) barrel field/primary whisker motor (M1) cortices, cholinergic neurons with dual projections were observed in the basal nucleus of Meynert (BM), mainly at middle level; the projection was almost exclusively ipsilateral (99%+/-0.7%, n=6). Second, following unilateral injections of tracers into ventroposteromedial (VPM) barreloids/ventrolateral (VL) thalamic nucleus, dual-projecting cells were observed in the mesopontine tegmental complex including the pedunculopontine tegmental (PTg) and laterodorsal tegmental (LDTg) nuclei, mainly at rostral to middle levels; the projection exhibited ipsilateral dominance, i.e., 67%+/-1.3% (n=6) for the PTg and 64%+/-1.2% (n=6) for the LDTg. Finally, when injections were made at whisker-related, principal sensory trigeminal (Pr5)/facial motor (Mo7) nuclei, a relatively small number of labeled neurons were observed in the PTg and the LDTg at middle to caudal levels; within LDTg, labeled cells occupied the ventral portion of the dorsal LDTg as well as the ventral LDTg (LDTgV). This projection exhibited contralateral preponderance, i.e., 67%+/-2.0% (n=6) for the PTg and 69%+/-3.2% (n=6) for the LDTg. Taken together, the present observations demonstrated that each division of the BM, PTg, and LDTg possessed a differential functional organization with respect to its collateral projection to whisker-related sensorimotor targets, suggesting that the cholinergic projection might play a modulatory role in vibrissal sensorimotor integration, which allows the guidance of behavioral action essential for the survival of the animal.

Collateral projection from the locus coeruleus to whisker-related sensory and motor brain regions of the rat.

The primary goal of this study was to examine whether the locus coeruleus (LC) provides collateral projections to whisker-related, sensorimotor brain regions. After injections of retrograde tracers into the primary sensory (S1) barrel field/primary whisker motor (M1) cortices, ventroposteromedial (VPM)/ventrolateral (VL) thalamic nuclei, or principal sensory trigeminal (Pr5)/facial motor (Mo7) nuclei, the distribution of double-labeled neurons within the LC was examined. Our observations indicated that a large number of individual LC cells provided axon collaterals to S1-M1 or VPM-VL regions, whereas only a few projected to Pr5-Mo7 nuclei. The laterality and the distribution of dual-projecting LC neurons were as follows. 1) The neurons projecting to the S1-M1 cortices were predominantly ipsilateral (96% +/- 0.7%). Labeled neurons were located ventrally at the rostral pole but were evenly distributed along the dorsoventral aspect of the principal LC. 2) The cells projecting to the VPM-VL nuclei were bilateral, with ipsilateral (68% +/- 2.3%) dominance. Neurons were observed at the rostrocaudal extent of the LC, where the labeling was most pronounced at the ventral, principal LC. 3) The neurons projecting to the Pr5-Mo7 regions exhibited slightly contralateral (56% +/- 2.9%) dominance, where labeled cells were confined within the ventral margin of the principal subdivision. Taken together, the present observations demonstrate that each subdivision of the LC possesses a differential functional organization with respect to its collateral projection to whisker-related sensorimotor targets, suggesting that the nucleus might play a modulatory role in vibrissal sensorimotor integration that allows the guidance of behavioral action essential for the survival of the animal.

The collateral projection from the dorsal raphe nucleus to whisker-related, trigeminal sensory and facial motor systems in the rat.

The primary goal of this study was to identify the collateral projection from the dorsal raphe (DR) nucleus to whisker-related, trigeminal sensory and facial motor systems in the rat. Following the injections of two retrograde tracers, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold) and Fluorogold (FG) within vibrissae-related, sensory and motor areas at the cerebral cortical, thalamic, and medullary levels, the distribution of double-labeled neurons was examined within each subdivision of the DR. The major findings were: 1) the 5-HT-immunoreactive, DR neurons projecting to vibrissae-related, primary sensory and motor cortices were mainly observed in the ventromedial subdivision, with a few cells in the dorsomedial subdivision; 2) the DR neurons projecting to ventroposteromedial and ventrolateral thalamic nuclei were observed in the lateral wing subdivision ipsilateral to the injection sites; and 3) the DR neurons projecting to vibrissae-related, principal trigeminal and facial motor nuclei were also located mainly in the lateral wing subdivision ipsilateral to the injection sites. Taken together, these observations provide evidence that midline vs. lateral wing DR subdivisions have a differential functional organization with respect to their efferent projection systems and that individual DR neurons in each subdivision might preferentially send axon collaterals to sensory and motor whisker system targets, thus providing an anatomical substrate for coordination of whisker movement and tactile sensory coding.

Organization of histamine-immunoreactive, tuberomammillary neurons projecting to the dorsal tier of the substantia nigra compacta in the rat.

Following the injection of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), into the dorsal tier of substantia nigra compacta (SNCD), histamine immunostaining was performed for the tuberomammillary nucleus (TMN) in order to investigate the projection from the TMN to the SNCD. At the rostral pole of the TMN, the retrograde labeling in the dorsomedial subdivision following medial SNCD injections was predominantly ipsilateral (78%), whereas only a few cells were located bilaterally following lateral SNCD injections. Following tracer injections along the medio-lateral location along the SNCD, the labeling at the ventrolateral TMN was bilateral with slightly ipsilateral (58-61%) dominance. At rostral and caudal TMN levels, clusters of labeled neurons were localized within two discrete columns of the ventrolateral TMN. At rostral TMN level, a lateral column of cells was located at the lateral tip of the ventrolateral TMN just medial to the internal capsule, while the medial column was close to the protruded region along the ventral, pial border. At the caudal TMN level, two columns were located on either side of the lateral mammillary nucleus. Taken together, the present study suggests that ventrolateral as well as dorsomedial TMN might provide arousal-related information to medial, intermediate, and lateral regions of the SNCD, which in turn influence extrapyramidal, behavioral functions performed by the substantia nigra compacta.

Projection patterns from the amygdaloid nuclear complex to subdivisions of the dorsal raphe nucleus in the rat.

The goal of the present study was to identify the projection from the subdivisions of the amygdaloid nuclear complex to specified subregions of the dorsal raphe (DR) nucleus and to attempt to compare the density of amygdaloid input to the DR with that of inputs from other limbic structures. Use of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), demonstrated that amygdaloid input to midline DR subdivision originates mainly from the medial portion of the medial amygdaloid nucleus, whereas that to lateral wing subdivision derives from the region extending from the lateral portion of the medial amygdaloid nucleus to the commissural stria terminalis. Use of the retrograde tracer Fluorogold (FG) produced relatively large but circumscribed injection sites comprising midline DR as well as portions of lateral wing subdivision and confirmed that the medial amygdaloid nucleus provides the major input to the DR. We also demonstrated that although amygdaloid input was not as extensive as inputs from other limbic structures such as the medial prefrontal cortex or the lateral habenular nucleus, it was comparable to input from the lateral septal nucleus. Based on these observations, we suggest that the medial amygdaloid nucleus provides substantial input to the DR and may contribute an emotional influence on sleep-wakefulness cycle or pain-stress modulation. Furthermore, it seems that the medial amygdaloid-DR projection might be anatomically and functionally distinct from the well-characterized central amygdaloid-periaqeductal gray (PAG) circuit which is essential for conditioned fear.