Number Of Double-labeled Neurons Research Articles

Topographical Differences in Orexin Activation after Pain Assessments in Hb AA and HbSS Sickle Mice

The orexins are neuropeptides that are synthesized in the perifornical area (PFA), lateral hypothalamus (LH) and dorsomedial hypothalamus (DMH) and mediate arousal, sleep, energy balance, food and drug reward. The LH region is associated with reward, while the PFA and DMH are more associated with stress and arousal. Data suggest that orexin-A has an analgesic effect on inflammatory pain and it may affect mechanisms underlying the maintenance of neuropathic pain. To date, there have been no published studies to investigate this system as a potential target to treat neuropathic pain in a mouse model of sickle cell disease (SCD). Therefore, we seek to determine activational and topographical differences in neuronal activation of the orexin system in a mouse model of SCD by using an array of behavioral measures to characterize pain in transgenic mice expressing human hemoglobin.Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS) and age-/gender-matched controls (HbAA mice; n = 5/group, 20-30g) expressing normal human hemoglobin A are used in the study. All behavioral tests are performed in a quiet room maintained at a constant temperature of 23- 25°C. All mice are habituated to each test protocol and environment for at least three consecutive days before collecting baseline measurements and testing. Three measures are used to assess pain-related behaviors: thermal/heat hyperalgesia (test for heat sensitivity), cold hyperalgesia (for cold sensitivity), and mechanical hyperalgesia (sensitivity to mechanical stimulus). Ninety minutes after the pain assessment testing, brains from HbAA and HbSS mice are quick frozen and hypothalamic brain sections from the mice are processed to visualize orexin-A and c-Fos using double label immunohistochemistry.Preliminary analyses show that significant topographical differences are observed in LH versus PFA for HbAA and HbSS mice (p<0.05) and LH versus DMH for HbAA and HbSS mice (p<0.05). This indicates that the number of double labeled neurons in the LH is significantly lower than the number of double labeled neurons in the PFA and DMH of HbAA and HbSS mice. Additionally, a significant topographical difference is observed in LH versus DMH for only HbSS mice (p<0.05). These data demonstrate that the number of activated orexin neurons is greater after pain testing in the DMH (a region more associated with stress and arousal) versus LH in HbSS mice. There is a trend towards a significant increase in activated LH orexin neurons in HbAA versus HbSS mice. Additional studies are underway to determine whether fewer reward-associated, orexin neurons in the LH are activated in HbSS versus HbAA mice after pain testing, and whether this decrease in orexin activation may provide a possible explanation for the increased hyperalgesia in HbSS sickle mice, which contributes to neuropathic pain observed in SCD. DisclosuresGupta:Tautona Group: Consultancy, Honoraria; Fera Pharmaceuticals LLC: Consultancy, Honoraria.

Activity-based anorexia activates CRF immunoreactive neurons in female rats

Activity-based anorexia (ABA) is a well-established animal model mimicking the eating disorder anorexia nervosa (AN). Since the pathophysiology of AN is yet poorly understood and specific drug treatments are lacking so far, animal models might be useful to further understand this disease. ABA consists of time-restricted access to food for 1.5 h/day and the possibility to exercise in a running wheel for 24 h/day. This combination leads to robust body weight loss as observed in AN. Here, we investigated the activation of brain corticotropin-releasing factor (CRF) neurons, a transmitter involved in the response to stress, emotional processes and also food intake. After development of ABA, rat brains were processed for c-Fos and CRF double immunohistochemistry. ABA increased the number of c-Fos/CRF double labeled neurons in the paraventricular nucleus (PVN) and the dorsomedial hypothalamic nucleus (DMH) compared to the ad libitum (AL, ad libitum fed, no running wheel) and activity (AC, ad libitum fed, running wheel, p < 0.05) but not to the restricted feeding (RF, food for 1.5 h/day, no running wheel, p > 0.05) group. Also the number of CRF neurons was increased in the DMH of ABA rats compared to AL and AC (p < 0.05). In the Edinger-Westphal nucleus (EW) the number of c-Fos positive neurons was increased in ABA and RF compared to AC (p < 0.05), while the number of double labeled neurons was not different (p > 0.05). Taken together, brain CRF activated under conditions of ABA might play a role in the development and maintenance of this animal model and possibly also in human AN.

Unpredictable chronic mild stress exerts anxiogenic-like effects and activates neurons in the dorsal and caudal region and in the lateral wings of the dorsal raphe nucleus

In previous studies, we verified that exposure to unpredictable chronic mild stress (UCMS) facilitates avoidance responses in the elevated T-maze (ETM) and increased Fos-immunoreactivity in different brain structures involved in the regulation of anxiety, including the dorsal raphe (DR). Since, it has been shown that the DR is composed of distinct subpopulations of serotonergic and non-serotonergic neurons, the present study investigated the pattern of activation of these different subnuclei of the region in response to this stress protocol. Male Wistar rats were either unstressed or exposed to the UCMS procedure for two weeks and, subsequently, analyzed for Fos-immunoreactivity (Fos-ir) in serotonergic cells of the DR. To verify if the anxiogenic effects observed in the ETM could be generalized to other anxiety models, a group of animals was also tested in the light/dark transition test after UCMS exposure. Results showed that the UCMS procedure decreased the number of transitions and increased the number of stretched attend postures in the model, an anxiogenic effect. UCMS exposure also increased Fos-ir and the number of double-labeled neurons in the mid-rostral subdivision of the dorsal part of the DR and in the mid-caudal region of the lateral wings. In the caudal region of the DR there was a significant increase in the number of Fos-ir. No significant effects were found in the other DR subnuclei. These results corroborate the idea that neurons of specific subnuclei of the DR regulate anxiety responses and are differently activated by chronic stress exposure.

Comparison of commonly used retrograde tracers in rat spinal motor neurons.

The purpose of this study was to investigate the effect of four fluorescent dyes, True Blue (TB), Fluoro-Gold (FG), Fluoro-Ruby (FR), and 1,1’-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), in retrograde tracing of rat spinal motor neurons. We transected the muscle branch of the rat femoral nerve and applied each tracer to the proximal stump in single labeling experiments, or combinations of tracers (FG-DiI and TB-DiI) in double labeling experiments. In the single labeling experiments, significantly fewer labeled motor neurons were observed after FR labeling than after TB, FG, or DiI, 3 days after tracer application. By 1 week, there were no significant differences in the number of labeled neurons between the four groups. In the double-labeling experiment, the number of double-labeled neurons in the FG-DiI group was not significantly different from that in the TB-DiI group 1 week after tracer application. Our findings indicate that TB, FG, and DiI have similar labeling efficacies in the retrograde labeling of spinal motor neurons in the rat femoral nerve when used alone. Furthermore, combinations of DiI and TB or FG are similarly effective. Therefore, of the dyes studied, TB, FG and DiI, and combinations of DiI with TB or FG, are the most suitable for retrograde labeling studies of motor neurons in the rat femoral nerve.

Convergent nociceptive input to spinal dorsal horn neurons after peripheral nerve injury.

The number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the spinal dorsal horn evoked by noxious stimulation was previously shown to be increased following peripheral nerve injury, and this increase was proposed to reflect the neuropathic pain state. The aim of this study was to investigate whether anomalous convergent primary afferent input to spinal dorsal horn neurons contributed to nerve injury-induced c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input from different branches of the sciatic nerve after injury to the tibial nerve. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and also by electrical stimulation (ES) of the injured tibial nerve, respectively. The number of c-Fos-IR neurons was significantly decreased 3 days after the injury. However, the number of c-Fos-IR neurons returned to the control level 14 days after the injury. P-ERK immunoreactive (p-ERK-IR) neurons were induced in the central terminal field of the tibial nerve by ES of the tibial nerve. The topographic distribution pattern and number of such p-ERK-IR neurons remained unchanged after the nerve injury. The time course of changes in the number of double-labeled neurons, that presumably received convergent primary afferent input, showed a pattern similar to that of c-Fos-IR neurons after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves may contribute to c-Fos hyperinducibility in the spinal dorsal horn.

Possible involvement of convergent nociceptive input to medullary dorsal horn neurons in intraoral hyperalgesia following peripheral nerve injury.

Previous studies demonstrated that the number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the medullary dorsal horn (MDH) evoked by noxious stimulation was increased after peripheral nerve injury, and such increase has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the MDH for convergent collateral primary afferent input to second order neurons deafferented by peripheral nerve injury, and to explore a possibility of its contribution to the c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input. c-Fos expression and the phosphorylation of ERK were induced by the intraoral application of capsaicin and by electrical stimulation of the inferior alveolar nerve (IAN), respectively. The number of c-Fos-IR neurons in the MDH induced by the intraoral application of capsaicin was increased after IAN injury, whereas the number of p-ERK immunoreactive neurons remained unchanged. The number of double-labeled neurons, that presumably received convergent primary afferent input from the lingual nerve and the IAN, was significantly increased after IAN injury. These results indicated that convergent primary nociceptive input through neighboring intact nerves may contribute to the c-Fos hyperinducibility in the MDH and the pathogenesis of neuropathic pain following trigeminal nerve injury.

A Retrograde Tracing Study of Compensatory Corticospinal Projections in Rats with Neonatal Hemidecortication

To examine the compensatory mechanisms in rats that underwent left decortication at postnatal day 7 (P7), we injected the retrograde tracers fluorescein isothiocyanate-cholera toxin B subunit (FITC-CTB) and Fast Blue (FB) into the right and left upper cervical spinal cord, respectively, at postoperative weeks 2, 3, 4, and 5 and counted the number of retrogradely labeled corticospinal neurons in the right cerebral cortex compared with that in normally developed rats. Significantly more ipsilaterally projecting neurons were labeled with FITC-CTB in the decorticated rats compared with normal rats at all time points examined. The number of labeled neurons was similar to that at P7 in normal rats. There were also some FITC-CTB and FB double-labeled neurons in both decorticated and normal rats. The number of double-labeled neurons in the decorticated rats increased each week and was significantly greater than that in normal rats at postoperative weeks 4 and 5. The present results suggest that the elimination of ipsilaterally projecting axons observed in normal rats was prevented in the decorticated rats, so that the cerebral cortex neurons on the unlesioned side projected corticospinal tracts to the ipsilateral spinal cord. Furthermore, the collaterals of the corticospinal tracts originating from the cerebral cortex on the unlesioned side also project to the ipsilateral spinal cord. These compensatory mechanisms might underlie the acquisition of motor function in these animals.

Active projections to medullary raphe during conditioned fear in the rat

Psychological stress leads to sympathetic and behavioural responses that are mediated by the medullary raphe nuclei (MR). These nuclei receive direct projections from the hypothalamus, which drive the tachycardia, and the periaqueductal gray (PAG), which drives behaviour. To confirm that these projections are active during conditioned fear, we combined fear-induced Fos expression with cholera toxin B retrograde tracing from the MR. Rats were either placed for 30 min in a context where they had previously experienced footshocks (conditioned fear, N=5) or left undisturbed in their homecages (control, N=5) prior to sacrifice. Relative to the total number of double labelled neurons, most of these were found in the hypothalamus (24.5±6.8%) and PAG (34.2±8.8%). Within the PAG, double labelling was found in the dorsomedial (14.9±5.0%), lateral (9.9±2.4%) and ventrolateral columns (9.4±1.9%). The anterior hypothalamus had about the same number of double labelled neurons (5.6±2.1%) as the dorsomedial (5.8±1.9%), paraventricular (5.8±1.3%) and perifornical nuclei (4.4±2.0%). The prefrontal cortex was the third most labelled region (15.0±3.8%). Double labelling was also found in the midbrain tegmentum (11.3±4.6%), nucleus cuneiformis (5.3±2.4%) and A5 (2.0±0.7%) area. These results confirm that the most important active afferents to MR during conditioned fear are in the hypothalamus and PAG.

Regional differences in estradiol effects on numbers of HSD2-containing neurons in the nucleus of the solitary tract of rats

Estrogens affect body fluid balance, including sodium ingestion. Recent findings of a population of neurons in the hindbrain nucleus of the solitary tract (NTS) of rats that are activated during sodium need suggest a possible central substrate for this effect of estrogens. We used immunohistochemistry to label neurons in the NTS that express 11-β-hydroxysteroid dehydrogenase type 2 (HSD2), an enzyme that promotes aldosterone binding, in male rats, and in ovariectomized (OVX) rats given estradiol benzoate (EB) or oil vehicle (OIL). During baseline conditions, the number of HSD2 immunoreactive neurons in the NTS immediately rostral to the area postrema was greater in EB-treated OVX rats compared to those in OIL-treated OVX and male rats. A small number of HSD2 immunoreactive neurons was also labeled for dopamine-β-hydroxylase (DBH), an enzyme involved in norepinephrine biosynthesis. Double-labeled neurons in the NTS were located primarily in the more lateral portion of the HSD2 population, at the level of the area postrema in all three groups, with no sex or estrogen-mediated differences in the number of double-labeled neurons. These results suggest that two subpopulations of HSD2 neurons are present in the NTS. One subpopulation, which does not colocalize with DBH and is increased during conditions of elevated estradiol, may contribute to the effects of estrogens on sodium ingestion. The role of the other, smaller subpopulation, which colocalizes with DBH and is not affected by estradiol, remains to be determined, but one possibility is that these latter neurons are part of a larger network of catecholaminergic input to neuroendocrine neurons in the hypothalamus.

Efficacy of fluorescent tracers in retrograde labeling of cutaneous afferent neurons in the rat

Background In the present study, the labeling efficacy of tracers Fluoro-ruby (FR), Fluoro-emerald (FE), True Blue (TB), Fluoro-Gold (FG), Diamidino Yellow (DY) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) to retrogradely label the cutaneous afferent neurons in the rat was examined. Methods The proximal stump of the transected sural nerve was exposed for 1 hour either to one of the examined dyes (FR, FE, TB, FG, DY and DiI group) in single labeling experiments, or to mixtures of two dyes (TB-FG, FG-DiI, TB-DY and TB-DiI group) in double labeling experiments ( n = 5 for each group). After 10 days, dorsal root ganglia (DRGs) L3-S1 were harvested, cut to 20 μm thick longitudinal sections and all labeled neurons were counted. Results The average numbers of labeled DRG neurons in FR group (1063 ± 158; mean ± SD) and FE group (1067 ± 203) were statistically significantly lower than those in TB group (2831 ± 379), FG group (2802 ± 134), DY group (2888 ± 262) or DiI group (2900 ± 278) ( p < 0.05). In double labeling experiments, the average number of double labeled neurons in TB-DY group (2208 ± 207) was statistically significantly lower than those in TB-FG group (2775 ± 316), FG-DiI group (2921 ± 419), or TB-DiI group (2805 ± 179) ( p < 0.05). Conclusions Among examined tracers, TB, FG, DY and DiI, have the highest and similar labeling efficacy for retrograde labeling of cutaneous afferent neurons in the rat. The tracers TB, DiI and FG effectively label the same neuronal population in double labeling, therefore, their combinations are most suitable for double retrograde labeling studies of cutaneous afferent neurons in the rat.

Taste‐evoked Fos expression in nitrergic neurons in the nucleus of the solitary tract and reticular formation of the rat

The current investigation used double labeling for NADPHd and Fos-like immunoreactivity to define the relationship between nitric oxide synthase-containing neural elements and taste-activated neurons in the nucleus of the solitary tract (NST) and subjacent reticular formation (RF). Stimulation of awake rats with citric acid and quinine resulted in significant increases in the numbers of double-labeled neurons in both the NST and RF, suggesting that some medullary gustatory neurons utilize nitric oxide (NO) as a transmitter. Overall, double-labeled neurons were most numerous in the caudal reaches of the gustatory zone of the NST, where taste neurons receive inputs from the IXth nerve, suggesting a preferential role for NO neurons in processing gustatory inputs from the posterior oral cavity. However, double-labeled neurons also exhibited a preferential distribution depending on the gustatory stimulus. In the NST, double-labeled neurons were most numerous in the rostral central subnucleus after either stimulus but had a medial bias after quinine stimulation. In the RF, after citric acid stimulation, there was a cluster of double-labeled neurons with distinctive large soma in the parvicellular division of the lateral RF, subjacent to the rostral tip of NST. In contrast, in response to quinine, there was a cluster of double-labeled neurons with much smaller soma in the intermediate zone of the medial RF, a few hundred micrometers caudal to the citric acid cluster. These differential distributions of double-labeled neurons in the NST and RF suggest a role for NO in stimulus-specific gustatory autonomic and oromotor reflex circuits.

End-to-Side Nerve Grafts: Experimental Study in Rats

The effectiveness of the end-to-side nerve graft in comparison with the end-to-end cable-graft was determined in rat sciatic nerve transection models. Sixty Sprague-Dawley rats were randomly divided into four groups with different reconstructive methods for two branches of the sciatic nerve: Group I, median nerve graft with end-to-side neurorrhaphy; Group II, median and ulnar nerve grafts with end-to-end neurorrhaphy; Group III, no repair; and Group IV, sham operation. Between Groups I and II, there were no significant differences in the functional, electrophysiologic, and histologic evaluations. In retrograde tracing of the spinal cord and dorsal root ganglia, the number of double-labeled neurons was significantly higher in Group I. End-to-side nerve grafts show good functional recovery, require less graft, and are easy to perform. The authors find this method to be an effective alternative in facial nerve reconstruction and of great value in various kinds of peripheral nerve surgery.

Activation of adrenal preganglionic neurons during autonomic dysreflexia in the chronic spinal cord-injured rat

Autonomic dysreflexia (AD) occurs in a majority of high paraplegic and quadriplegic patients and is particularly characterized by a paroxysmal hypertension elicited by somatic or visceral stimuli. We have previously shown that plasma adrenaline and noradrenaline levels were significantly increased during episodes of AD in the 30-day spinal cord-injured (SCI) rats, suggesting the participation of adrenal catecholamines in the cardiovascular changes associated to AD. Thus, adrenal sympathetic preganglionic neurons (SPN) could be activated by visceral afferences leading to AD. The aim of this study was then to demonstrate whether visceral stimulation that induces AD activates adrenal SPN in chronic SCI rats. To this end, a retrograde tracer, the cholera toxin B subunit (CTB), was combined with the immunocytochemical detection of Fos protein after visceral stimulation. Chronic SCI rats received a CTB injection into the adrenal gland and, 3 days later, were stimulated by repetitive distension of the colon. Results showed that this stimulation elicited typical hypertensive episodes of AD and a significant increase in the number of double-labeled neurons (CTB/Fos immunoreactive neurons) in the thoracic spinal cord below the level of injury (T4 segment) when compared to the stimulated non-SCI rats. In conclusion, visceral stimulations in the chronic SCI rats activate adrenal SPN, which could induce release of catecholamines by the adrenal medulla. The present study brings new data on the spinal mechanisms of AD cardiovascular dysfunctions.

Activation of ventrolateral medullary neurons projecting to spinal autonomic areas after chemical stimulation of the central nucleus of amygdala: a neuroanatomical study in the rat

Several studies have shown that the central nucleus of amygdala is involved in cardiovascular regulation. The control of this function may be mediated by activation of the ventrolateral medulla neurons that project to preganglionic neurons located in the intermediolateral nucleus of the spinal cord. The aim of the present study was to examine whether stimulation of the central nucleus of amygdala activated ventrolateral medulla neurons projecting to the intermediolateral nucleus. For this purpose, the injection of a retrograde tracer, the cholera toxin b subunit (CTb), into the intermediolateral nucleus of the T2 segment was combined with immunohistochemical detection of Fos protein following chemical stimulation of the central nucleus of amygdala. Results showed that retrogradely labeled neurons were found throughout the ventrolateral medulla. Moreover, chemical stimulation of the central nucleus of amygdala induced: (1) a decrease of arterial blood pressure; (2) an expression of Fos protein mainly in sub-populations of neurons located in the intermediate and caudal parts of the ventrolateral medulla; (3) a significantly higher number of double labeled neurons (CTb-immunoreactive/Fos-immunoreactive) in the rostral part of the ventrolateral medulla than in the other parts of this region. These results show that the central nucleus of amygdala influences the activity of brainstem neurons projecting to the intermediolateral nucleus. Data were discussed in terms of descending amygdalofugal pathways involved in the hypotension.

Substance p receptor (neurokinin-1)-expressing neurons in lamina i of the spinal cord encode for the intensity of noxious stimulation: a c-fos study in rat

The substance Preceptor neurokinin-1 is expressed by a subset of neurons in the rat spinal cord. We have combined immunostaining for Fos, a marker of noxious peripheral stimulation, and neurokinin-1 to examine whether nociceptive signals from particular peripheral tissues (skin, muscle or knee joint) or activity generated by nerve injury or formalin-induced inflammation are preferentially modulated by substance P. Our results indicate that superficial and deep spinal neurokinin-l-positive neurons process nociceptive information in markedly different ways. In lamina I, the number of double-labelled neurons was positively correlated with the intensity of the stimulus (defined by the total Fos count) and was not directly related to any particular peripheral target. However, in the deeper layers of the spinal cord (V–X), there was no such correlation, and stimulation of joint nociceptors and formalin-induced inflammation produced the greatest proportion of Fos/neurokinin-1 co-localization, suggesting a particular role for substance P in the mediation of joint pain and inflammatory hyperalgesia. Thus, lamina I neurokinin-I receptor-bearing neurons appear to be involved in intensity discriminative aspects of pain, whereas the deep neurokinin-1 cells are involved in spatial localization or the detection of particular nociceptive submodalities.

Hypertensive rats exhibit heightened expression of corticotropin-releasing factor in activated central neurons in response to restraint stress

To test the hypothesis that chronically elevated sympathetic drive is associated with hyperreactiveness of autonomic centers in the brain to stress, adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats (Wistar Kyoto [WKY] and Sprague Dawley [SD] rats) were acutely exposed to restraint stress; controls from each strain were not stressed. Brain sections were prepared for Fos immunohistochemistry to identify activated neurons in the paraventricular nucleus of the hypothalamus, Barrington's nucleus of the pons, nucleus of the tractus solitarius, and ventrolateral medulla, or for combined Fos immunohistochemistry and corticotropin-releasing factor (CRF) in situ hybridization in the paraventricular nucleus and Barrington's nucleus. Restraint led to increased activation of neurons in all nuclei. Strain differences were found only in the caudal and rostral paraventricular nucleus where restraint resulted in greater numbers of activated neurons in SHRs compared to either normotensive strain. Levels of CRF mRNA in Barrington's nucleus of unrestrained rats were similar among strains. After restraint, mRNA levels and double labeled neurons were increased in Barrington's nucleus of SHRs. In unstressed rats, CRF mRNA levels were elevated in some regions of the paraventricular nucleus in SHRs. After restraint, mRNA levels increased throughout the paraventricular nucleus of SHRs. Significantly greater numbers of double labeled neurons were found in the dorsolateral medial and ventral medial parvocellular paraventricular nucleus of stressed SHRs compared to WKY or SD rats. These data show that chronic elevation in sympathetic activity, present in SHRs, is associated with hyperreactiveness of the paraventricular and Barrington's nucleus including recruitment of neurons to express CRF, and may have important implications for the response of the hypothalamo–pituitary–adrenal axis during stress.

A topographic re-evaluation of the nigrostriatal projections to the caudate nucleus in the cat with multiple retrograde tracers.

The anatomical organization of the cat nigrostriatal projections to the caudate nucleus was studied by retrograde tracer techniques. Horseradish peroxidase conjugated with wheat germ agglutinin and fluorescent retrograde tracers such as Fast Blue and Diamidino Yellow were injected concomitantly in different regions of the caudate nucleus. The distribution of single and double retrogradely labeled neurons was analysed in the substantia nigra pars compacta, substantia nigra pars lateralis, retrorubral area and adjacent ventral tegmental area. Adjacent sections processed for acetylcholinesterase were used as histochemical markers for the densocellular zone of the substantia nigra. The main findings of this study are: (1) The rostral caudate nucleus receives projections mainly from the caudal substantia nigra while the caudal caudate nucleus receives projections from all rostrocaudal levels of the substantia nigra. (2) The substantia nigra pars lateralis projects very specifically to the caudal caudate nucleus. (3) The ventral retrorubral area close to the medial lemniscus projects to all rostrocaudal levels of the caudate nucleus. (4) The rostral caudate nucleus receives projections mainly from the medial substantia nigra while more caudal sectors of the caudate nucleus receive projections from the medial and lateral substantia nigra. (5) A dorsoventral inversion of nigrostriatal projections from the medial substantia nigra pars compacta and the adjacent ventral tegmental area to the caudate nucleus was established. In contrast, we found zones within the retrorubral area projecting both to the dorsal and ventral caudate nucleus. (6) Distant injections of two different fluorescent tracers regarding both the dorsoventral and the rostrocaudal coordinates, yielded double-labeled neurons that were mainly located in the medial and caudal portions of the substantia nigra and in the ventral retrorubral area. However, the number of double-labeled neurons was higher after separated injections in the dorsoventral axis, suggesting that the collateralization to the caudate nucleus occurs mainly in the dorsoventral plane. (7) A clustering organization of nigrostriatal cells projecting to the caudate nucleus was detected mainly in the intermediate rostrocaudal part of the substantia nigra pars compacta and in the retrorubral area. The results of this comprehensive study on the cat nigrostriatal pathway to the caudate nucleus show novel findings on the anatomical organization of the nigrostriatal projections which might help the understanding of the complex architecture of nigral neurons projecting to the caudate nucleus in carnivores.

Colocalization of ionotropic glutamate receptor subunits with NADPH-diaphorase-containing neurons in the rat mesopontine tegmentum

Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration.

Organization of the vagal preganglionic neurons innervating the stomach in the Japanese quail (Conturinx japonica) double labeling fluorescent method.

Cytoarchitectural study indicated that the dorsal motor nucleus of the vagal nerve (nX) was formed by two subnuclei in a coronal section. One of them had large and round neurons, and occupied the dorsal part of the nX (Xd). The second one had small and oval neurons, and occupied the ventral part of the nX (Xv). The double labeling fluorescent method was performed to determine the organization of preganglionic neurons in the nX innervating different muscles of the quail stomach. Injections of nuclear yellow (NY) into the proventriculus, and of fast blue (FB) into the gizzard showed that there were no double-labeled neurons in the nX. NY- and FB-labeled neurons were found throughout the nX. After separate injections of NY and FB into both the cranioventral and caudodorsal thick muscles, and the craniodorsal and caudodorsal thin muscles, many NY- and FB-labeled neurons were distributed and intermingled in the middle two thirds of the nX. Considerable numbers of double-labeled neurons were also found at the middle level of the nX. There was no side-predominancy of the population of the preganglionic neurons innervating the quail stomach. These results show that, many neurons of both sides of the nX are separately innervating both the proventriculus and gizzard, and considerable numbers of single neurons of the nX also innervate a pair of thin or thick muscles with collateral axons.

Cholinergic projections to the anterior thalamic nuclei in the rat: a combined retrograde tracing and choline acetyl transferase immunohistochemical study.

Retrograde transport of horseradish peroxidase (HRP) was combined with choline acetyltransferase (ChAT) immunohistochemistry to study cholinergic projections to the anterior thalamic nuclei in the rat. Small iontophoretic injections of HRP placed into different subdivisions of the anterior thalamic nuclear complex resulted in distinct patterns of retrograde labelling in two major cholinergic cell groups of the mesopontine tegmentum, the laterodorsal tegmental nucleus (LDTg), in which a majority of the labelled cells was located, and the pedunculopontine tegmental nucleus (PPT). After injections into the posterior subdivision of the anteroventral thalamic nucleus (AVp), double-labelled neurons were present predominantly in the ipsilateral LDTg while a smaller number was found in the PPT. In the ipsilateral LDTg, 60-70% of ChAT-positive neurons were HRP-labelled, and 90-95% of the HRP-labelled neurons were ChAT-positive. In the contralateral LDTg, 30-40% of ChAT-positive neurons were HRP-labelled. After injections in the medial subdivision of the anteroventral thalamic nucleus (AVm), the pattern of labelling in LDTg was similar to that detected after injections in the AVp. The number of double-labelled neurons in the LDTg and PPT was much lower after injections into AVm than after injections into AVp. When injections were confined to the anterodorsal thalamic nucleus (AD), no HRP-labelled cells were present in the LDTg or PPT. These results show that the LDTg and PPT are the sources of the cholinergic input to the rat anterior thalamus. The major projection from LDTg and PPT is to the AVp, whereas there is a lighter cholinergic projection to the AVm. The AD does not receive a projection from cholinergic cells in the mesopontine tegmentum.