Morphometric and quantitative evaluation of the NADH-diaphorase positive myenteric neurons of the jejunum of streptozotocin-diabetic rats supplemented with acetyl-L-carnitine

In this study we investigated the effect of the acetyl-L-carnitine (ALC) supplementation on the myenteric neurons of the jejunum of rats made diabetic at the age of 105 days by streptozotocin (35 mg/kg body weight). Four groups were used: non-diabetic (C), non-diabetic supplemented with ALC (CC), diabetic (D), diabetic supplemented with ALC (DC). After 15 weeks of diabetes induction the blood was collected by cardiac puncture to evaluate glycaemia and glycated haemoglobin. Next the animals were killed and the jejunum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The neuronal counts were made in 80 microscopic fields, in tissue samples of five animals of each group. The profiles of the cell bodies of 1000 neurons per group were analysed. Diabetes induced a significant increase in the area of the cell body and decrease in the number of NADH-diaphorase positive myoenteric neurons. ALC suplementation to the diabetic group promoted smaller hypertrophic effects and less neuronal loss than in the myoenteric neurons of the diabetic rats, and in addition diminished the body weight decrease and reduced the fasting glycaemia. © 2005 Blackwell Verlag.

Direct and indirect connections between cochlear root neurons and facial motor neurons: Pathways underlying the acoustic pinna reflex in the albino rat

Cochlear root neurons (CRNs) are involved in the acoustic startle reflex, which is widely used in behavioral models of sensorimotor integration. A short-latency component of this reflex, the auricular reflex, promotes pinna movements in response to unexpected loud sounds. However, the pathway involved in the auricular component of the startle reflex is not well understood. We hypothesized that the auricular reflex is mediated by direct and indirect inputs from CRNs to the motoneurons responsible for pinna movement, which are located in the medial subnucleus of the facial motor nucleus (Mot7). To assess whether there is a direct connection between CRNs and auricular motoneurons in the rat, two neuronal tracers were used in conjunction: biotinylated dextran amine, which was injected into the cochlear nerve root, and Fluoro-Gold, which was injected into the levator auris longus muscle. Under light microscopy, close appositions were observed between axon terminals of CRNs and auricular motoneurons. The presence of direct synaptic contact was confirmed at the ultrastructural level. To confirm the indirect connection, biotinylated dextran amine was injected into the auditory-responsive portion of the caudal pontine reticular nucleus, which receives direct input from CRNs. The results confirm that the caudal pontine reticular nucleus also targets the Mot7 and that its terminals are concentrated in the medial subnucleus. Therefore, it is likely that CRNs innervate auricular motoneurons both directly and indirectly, suggesting that these connections participate in the rapid auricular reflex that accompanies the acoustic startle reflex. © 2008 Wiley-Liss, Inc.

C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

Control of breathing and blood pressure by parafacial neurons in conscious rats

New Findings: • What is the central question of this study? The main purpose of the present manuscript was to investigate the cardiorespiratory responses to hypoxia or hypercapnia in conscious rats submitted to neuronal blockade of the parafacial region. We clearly showed that the integrity of parafacial region is important for the respiratory responses elicited by peripheral and central chemoreflex activation in freely behavior rats. • What is the main finding and its importance? Since the parafacial region is part of the respiratory rhythm generator, they are essential for postnatal survival, which is probably due to their contribution to chemoreception in conscious rats. The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n= 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABAA agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min-1 kg-1 with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats. © 2012 The Authors. Experimental Physiology © 2012 The Physiological Society.

Alterations in the duodenum myenteric neurons of Wistar rats after ingesting of 2,4 dichlorophenoxyacetic acid

The 2,4 dichlorophenoxyacetic acid (2,4-D) is a systemic herbicide whose effects in animal organic systems have been examined in previous studies, being the neurotoxicity considered the predominant effect. However, the studies that detect the 2,4-D neurotoxicity have merely focused in the central nervous system, and therefore, little is known about the effect of this herbicide in the enteric nervous system. This study aimed to verifying the 2,4-D effects on the myenteric neurons in duodenum of Wistar rats. Ten 60-day-old male Wistar rats (Rattus norvegicus) were divided in two groups: control group (C) that did not receive 2,4-D and experimental group (E) that received 5.0 mg of 2,4-D/kg for 15 days. At the end of experimental period, the animal were euthanized, the duodenum was collected and processed for NADPH-diaphorase histochemical analysis in order to expose the nitrergic myenteric neurons (NADPH-dp). In the light microscopy analysis, the whole-mount preparation obtained from duodenum of each animal were image-captured in 120 and 40 fields, for quantitative and morphometric analyses of myenteric neurons, respectively. The neuronal density was not affected when comparing the two groups, but an increase (p > 0.05) of 8.5% was observed in the cell body area of neurons in the E group. In conclusion, the ingestion of 2,4-D at a dosage of 5.0 mg/kg body weight for 15 days does not change the neuronal density, but promotes the hypertrophy of NADPH-dp myenteric neurons in duodenum of the rats of this study.

Serotonergic neurons in the nucleus raphé obscurus are not involved in the ventilatory and thermoregulatory responses to hypoxia in adult rats

The medullary raphé is an important component of the central respiratory network, playing a key role in CO2 central chemoreception. However, its participation in hypoxic ventilatory responses is less understood. In the present study, we assessed the role of nucleus raphé obscurus (ROb), and specifically 5-HT neurons confined in the ROb, on ventilatory and thermoregulatory responses to hypoxia. Chemical lesions of the ROb were performed using either ibotenic acid (non-specific lesion; control animals received PBS) or anti-SERT-SAP (5-HT specific lesion; control animals received IgG-SAP). Ventilation (VE; whole body plethysmograph) and body temperature (Tb; data loggers) were measured during normoxia (21% O2, N2 balance) and hypoxia exposure (7% O2, N2 balance, 1h) in conscious adult rats. Ibotenic acid or anti-SERT-SAP-induced lesions did not affect baseline values of VE and Tb. Similarly, both lesion procedures did not alter the ventilatory or thermoregulatory responses to hypoxia. Although evidence in the literature suggests a role of the rostral medullary raphé in hypoxic ventilatory responses, under the present experimental conditions our data indicate that caudal medullary raphé (ROb) and its 5-HT neurons neither participate in the tonic maintenance of breathing nor in the ventilatory and thermal responses to hypoxia. © 2013 Elsevier B.V.

Effect of the bone marrow cell transplantation on elevated plus-maze performance in hippocampal-injured mice

Several reports have shown that the hippocampus plays an important role in different aspects of the emotional control. There is evidence that lesions in this structure cause behavioral disinhibition, with reduction of reactions expressing fear and anxiety. Thus, to portray the aptitude of cell therapy to abrogate injuries of hippocampal tissue, we examined the behavioral effects of bone marrow mononuclear cells (BMMCs) transplantation on C57BL/6 mice that had the hippocampus damaged by electrolytic lesion. For this purpose, mice received, seven days after bilateral electrolytic lesion in the dorsal hippocampus, culture medium or BMMCs expressing the enhanced green fluorescent protein (EGFP) transgene. One week after transplantation, animals were tested in the elevated plus-maze (EPM). On the whole, three assessment sessions in the EPM were carried out, with seven days separating each trial. Thirty-five days after the induction of injury, mice were sacrificed and their brains removed for immunohistochemistry. The behavioral evaluation showed that the hippocampal lesion caused disinhibition, an effect which was slightly lessened, from the second EPM test, in transplanted subjects. On the other hand, immunohistochemical data revealed an insignificant presence of EGFP+ cells inside the brains of injured mice. In view of such scenario, we hypothesized that the subtle rehabilitation of the altered behavior might be a result from a paracrine effect from the transplanted cells. This might have been caused by the release of bioactive factors capable of boosting endogenous recuperative mechanisms for a partial regaining of the hippocampal functions. © 2013 Elsevier B.V.

A1 Noradrenergic Neurons Lesions Reduce Natriuresis and Hypertensive Responses to Hypernatremia in Rats

Noradrenergic neurons in the caudal ventrolateral medulla (CVLM; A1 group) contribute to cardiovascular regulation. The present study assessed whether specific lesions in the A1 group altered the cardiovascular responses that were evoked by hypertonic saline (HS) infusion in non-anesthetized rats. Male Wistar rats (280-340 g) received nanoinjections of antidopamine-β-hydroxylase-saporin (A1 lesion, 0.105 ng.nL-1) or free saporin (sham, 0.021 ng.nL-1) into their CVLMs. Two weeks later, the rats were anesthetized (2% halothane in O2) and their femoral artery and vein were catheterized and led to exit subcutaneously between the scapulae. On the following day, the animals were submitted to HS infusion (3 M NaCl, 1.8 ml • kg-1, b.wt., for longer than 1 min). In the sham-group (n = 8), HS induced a sustained pressor response (ΔMAP: 35±3.6 and 11±1.8 mmHg, for 10 and 90 min after HS infusion, respectively; P<0.05 vs. baseline). Ten min after HS infusion, the pressor responses of the anti-DβH-saporin-treated rats (n = 11)were significantly smaller(ΔMAP: 18±1.4 mmHg; P<0.05 vs. baseline and vs. sham group), and at 90 min, their blood pressures reached baseline values (2±1.6 mmHg). Compared to the sham group, the natriuresis that was induced by HS was reduced in the lesioned group 60 min after the challenge (196±5.5 mM vs. 262±7.6 mM, respectively; P<0.05). In addition, A1-lesioned rats excreted only 47% of their sodium 90 min after HS infusion, while sham animals excreted 80% of their sodium. Immunohistochemical analysis confirmed a substantial destruction of the A1 cell group in the CVLM of rats that had been nanoinjected withanti-DβH-saporin. These results suggest that medullary noradrenergic A1 neurons are involved in the excitatory neural pathway that regulates hypertensive and natriuretic responses to acute changes in the composition of body fluid. © 2013 da Silva et al.

LESION OF THE COMMISSURAL NUCLEUS OF THE SOLITARY TRACT/A2 NORADRENERGIC NEURONS FACILITATES THE ACTIVATION OF ANGIOTENSINERGIC MECHANISMS IN RESPONSE TO HEMORRHAGE

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

IB4(+) and TRPV1(+) sensory neurons mediate pain but not proliferation in a mouse model of squamous cell carcinoma

Background: Cancer pain severely limits function and significantly reduces quality of life. Subtypes of sensory neurons involved in cancer pain and proliferation are not clear.Methods: We produced a cancer model by inoculating human oral squamous cell carcinoma (SCC) cells into the hind paw of athymic mice. We quantified mechanical and thermal nociception using the paw withdrawal assays. Neurotoxins isolectin B4-saporin (IB4-SAP), or capsaicin was injected intrathecally to selectively ablate IB4(+) neurons or TRPV1(+) neurons, respectively. JNJ-17203212, a TRPV1 antagonist, was also injected intrathecally. TRPV1 protein expression in the spinal cord was quantified with western blot. Paw volume was measured by a plethysmometer and was used as an index for tumor size. Ki-67 immunostaining in mouse paw sections was performed to evaluate cancer proliferation in situ.Results: We showed that mice with SCC exhibited both mechanical and thermal hypersensitivity. Selective ablation of IB4(+) neurons by IB4-SAP decreased mechanical allodynia in mice with SCC. Selective ablation of TRPV1(+) neurons by intrathecal capsaicin injection, or TRPV1 antagonism by JNJ-17203212 in the IB4-SAP treated mice completely reversed SCC-induced thermal hyperalgesia, without affecting mechanical allodynia. Furthermore, TRPV1 protein expression was increased in the spinal cord of SCC mice compared to normal mice. Neither removal of IB4(+) or TRPV1(+) neurons affected SCC proliferation.Conclusions: We show in a mouse model that IB4(+) neurons play an important role in cancer-induced mechanical allodynia, while TRPV1 mediates cancer-induced thermal hyperalgesia. Characterization of the sensory fiber subtypes responsible for cancer pain could lead to the development of targeted therapeutics.

THE RETINAL TARGETS OF CENTRIFUGAL NEURONS AND THE RETINAL NEURONS PROJECTING TO THE ACCESSORY OPTIC-SYSTEM

In birds, neurons of the isthmo-optic nucleus (ION), as well as ''ectopic'' neurons, send axons to the retina, where they synapse on cells in the inner nuclear layer (INL). Previous work has shown that centrifugal axons can be divided into two anatomically distinct types depending on their mode of termination: either ''convergent'' or ''divergent'' (Ramon y Cajal, 1889; Maturana and Frenk, 1965). We show that cytochrome-oxidase histochemistry specifically labels ''convergent'' centrifugal axons and target neurons which appear to be amacrine cells, as well as three ''types'' of ganglion cells: two types found in the INL (displaced ganglion cells) and one in the ganglion cell layer. Labeled target amacrine cells have distinct darkly labeled ''nests'' of boutons enveloping the somas, are associated with labeled centrifugal fibers, and are confined to central retina. Lesions of the isthmo-optic tract abolish the cytochrome-oxidase labeling in the centrifugal axons and in the target amacrine cells but not in the ganglion cells. Cytochromeoxidase-labeled ganglion cells in the INL are large; one type is oval and similar to the classical displaced ganglion cells of Dogiel, which have been reported to receive centrifugal input; the other type is rounder. Rhodamine beads injected into the accessory optic system results in retrograde label in both types of cells, showing that two distinct types of displaced ganglion cells project to the accessory optic system in chickens. The ganglion cells in the ganglion cell layer that label for cytochrome oxidase also project to the accessory optic system. These have proximal dendrites that ramify in the outer inner plexiform layer. Neither the target amacrine cells nor either of the displaced ganglion cells are immunoreactive for the inhibitory transmitter gamma aminobutyric acid. At least some of the target amacrine cells may, however, be cholinoceptive: we found that the antibody to the alpha-7 subunit of the nicotinic ACh receptor labels a population of cells in the INL that are similar in location, size, and the presence of labeled bouton-like structures to those we find labeled with cytochrome oxidase. This antibody also labels neurons in the ION proper but not ectopic cells. In conclusion, it appears that cytochrome oxidase may be a marker for ''convergent'' centrifugal axons and at least one of their target cells in the INL.