Adrenergic and muscarinic receptor subtypes functional regulation during diabetogenesis: Effect of curcumin and vitamin D3 pre-treatment

In the present study, the initial phase was directed to confirm the effects of curcumin and vitamin D3 in preventing or delaying diabetes onset by studying the blood glucose and insulin levels in the pre-treated and diabetic groups. Behavioural studies were conducted to evaluate the cognitive and motor function in experimental rats. The major focus of the study was to understand the cellular and neuronal mechanisms that ensure the prophylactic capability of curcumin and vitamin D3. To elucidate the mechanisms involved in conferring the antidiabetogenesis effect, we examined the DNA and protein profiles using radioactive incorporation studies for DNA synthesis, DNA methylation and protein synthesis. Furthermore the gene expression studies of Akt-1, Pax, Pdx-1, Neuro D1, insulin like growth factor-1 and NF-κB were done to monitor pancreatic beta cell proliferation and differentiation. The antioxidant and antiapoptotic actions of curcumin and vitamin D3 were examined by studying the expression of antioxidant enzymes - SOD and GPx, and apoptotic mediators like Bax, caspase 3, caspase 8 and TNF-α. In order to understand the signalling pathways involved in curcumin and vitamin D3 action, the second messengers, cAMP, cGMP and IP3 were studied along with the expression of vitamin D receptor in the pancreas. The neuronal regulation of pancreatic beta cell maintenance, proliferation and insulin release was studied by assessing the adrenergic and muscarinic receptor functional regulation in the pancreas, brain stem, hippocampus and hypothalamus. The receptor number and binding affinity of total muscarinic, muscarinic M1, muscarinic M3, total adrenergic, α adrenergic and β adrenergic receptor subtypes were studied in pancreas, brain stem and hippocampus of experimental rats. The mRNA expression of muscarinic and adrenergic receptor subtypes were determined using Real Time PCR. Immunohistochemistry studies using confocal microscope were carried out to confirm receptor density and gene expression results. Cell signalling alterations in the pancreas and brain regions associated with diabetogenesis and antidiabetogenesis were assessed by examining the gene expression profiles of vitamin D receptor, CREB, phospholipase C, insulin receptor and GLUT. This study will establish the anti-diabetogenesis activity of curcumin and vitamin D3 pre-treatment and will attempt to understand the cellular, molecular and neuronal control mechanism in the onset of diabetes.Administration of MLD-STZ to curcumin and vitamin D3 pre-treated rats induced only an incidental prediabetic condition. Curcumin and vitamin D3 pretreated groups injected with MLD-STZ exhibited improved circulating insulin levels and behavioural responses when compared to MLD-STZ induced diabetic group. Activation of beta cell compensatory response induces an increase in pancreatic insulin output and beta cell mass expansion in the pre-treated group. Cell signalling proteins that regulate pancreatic beta cell survival, insulin release, proliferation and differentiation showed a significant increase in curcumin and vitamin D3 pre-treated rats. Marked decline in α2 adrenergic receptor function in pancreas helps to relent sympathetic inhibition of insulin release. Neuronal stimulation of hyperglycemia induced beta cell compensatory response is mediated by escalated signalling through β adrenergic, muscarinic M1 and M3 receptors. Pre-treatment mediated functional regulation of adrenergic and cholinergic receptors, key cell signalling proteins and second messengers improves pancreatic glucose sensing, insulin gene expression, insulin secretion, cell survival and beta cell mass expansion in pancreas. Curcumin and vitamin D3 pre-treatment induced modulation of adrenergic and cholinergic signalling in brain stem, hippocampus and hypothalamus promotes insulin secretion, beta cell compensatory response, insulin sensitivity and energy balance to resist diabetogenesis. Pre-treatment improved second messenger levels and the gene expression of intracellular signalling molecules in brain stem, hippocampus and hypothalamus, to retain a functional neuronal response to hyperglycemia. Curcumin and vitamin D3 protect pancreas and brain regions from oxidative stress by their indigenous antioxidant properties and by their ability to stimulate cellular free radical defence system. The present study demonstrates the role of adrenergic and muscarinic receptor subtypes functional regulation in curcumin and vitamin D3 mediated anti-diabetogenesis. This will have immense clinical significance in developing effective strategies to delay or prevent the onset of diabetes.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Neuronal Expression of Cd36, Cd44, and Cd83 Antigen Transcripts Maps to Distinct and Specific Murine Brain Circuits

Cells recruited by the innate immune response rely on surface-expressed molecules in order to receive signals from the local environment and to perform phagocytosis, cell adhesion, and others processes linked to host defense. Hundreds of surface antigens designated through a cluster of differentiation (CD) number have been used to identify particular populations of leukocytes. Surprisingly, we verified that the genes that encode Cd36 and Cd83 are constitutively expressed in specific neuronal cells. For instance, Cd36 mRNA is expressed in some regions related to circuitry involved in pheromone responses and reproductive behavior. Cd44 expression, reanalyzed and detailed here, is associated with the laminar formation and midline thalamic nuclei in addition to striatum, extended amygdala, and a few hypothalamic, cortical, and hippocampal regions. A systemic immune challenge was able to increase Cd44 expression quickly in the area postrema and motor nucleus of the vagus but not in regions presenting expressive constitutive expression. In contrast to Cd36 and Cd44, Cd83 message was widely distributed from the olfactory bulb to the brain stem reticular formation, sparing the striatopallidum, olivary region, and cerebellum. Its pattern of expression nevertheless remained strongly associated with hypothalamic, thalamic, and hindbrain nuclei. Unlike the other transcripts, Cd83 mRNA was rapidly modulated by restraint stress. Our results indicate that these molecules might play a role in specific neural circuits and present functions other than those attributed to leukocyte biology. The data also suggest that these surface proteins, or their associated mRNA, could be used to label neurons in specific circuits/regions. J. Comp. Neurol. 517:906-924, 2009. (C) 2009 Wiley-Liss, Inc.

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats (Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 +/- 1 degrees C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia. respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem. (C) 2011 Elsevier B.V. All rights reserved.

INHIBITION OF THE CAUDAL PRESSOR AREA REDUCES CARDIORESPIRATORY CHEMOREFLEX RESPONSES

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague-Dawley rats (250-280 g, n=5-8/group) were used. In conscious rats, most of the commNTS neurons (66 +/- 11%) and part of the CPA neurons (36 +/- 7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9 +/- 4%) or the CPA (6 +/- 2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 mu g/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Contribution of excitatory amino acid receptors of the retrotrapezoid nucleus to the sympathetic chemoreflex in rats

In the present study, we evaluated the role of glutamatergic mechanisms in the retrotrapezoid nucleus (RTN) in changes of splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) elicited by central and peripheral chemoreceptor activation. Mean arterial pressure (MAP), sSND and PND were recorded in urethane-anaesthetized, vagotomized, sino-aortic denervated and artificially ventilated male Wistar rats. Hypercapnia (10% CO(2)) increased MAP by 32 +/- 4 mmHg, sSND by 104 +/- 4% and PND amplitude by 101 +/- 5%. Responses to hypercapnia were reduced after bilateral injection of the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate (AP-5; 100mm in 50 nl) in the RTN (MAP increased by 16 +/- 3 mmHg, sSNDby 82 +/- 3% and PND amplitudeby 63 +/- 7%). Bilateral injection of the non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione(DNQX; 100 mm in 50 nl) and the metabotropic receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100mm in 50 nl) in the RTN did not affect sympathoexcitatory responses induced by hypercapnia. Injection of DNQX reduced hypercapnia-induced phrenic activation, whereas MCPG did not. In animals with intact carotid chemoreceptors, bilateral injections of AP-5 and DNQX in the RTN reduced increases in MAP, sSND and PND amplitude produced by intravenous injection of NaCN (50 mu g kg(-1)). Injection of MCPG in the RTN did not change responses produced by NaCN. These data indicate that RTN ionotropic glutamatergic receptors are involved in the sympathetic and respiratory responses produced by central and peripheral chemoreceptor activation.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t) SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e. g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EHrats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

Central nitric oxide modulates hindquarter vasodilation elicited by AMPA receptor stimulation in the NTS of conscious rats

Microinjection of S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the nucleus of the solitary tract (NTS) of conscious rats causes hypertension, bradycardia, and vasoconstriction in the renal, mesenteric, and hindquarter vascular beds. In the hindquarter, the initial vasoconstriction is followed by vasodilation with AMPA doses >5 pmol/100 nl. To test the hypothesis that this vasodilation is caused by activation of a nitroxidergic pathway in the NTS, we examined the effect of pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 nmol/100 nl, microinjected into the NTS) on changes in mean arterial pressure, heart rate, and regional vascular conductance (VC) induced by microinjection of AMPA (10 pmol/100 nl in the NTS) in conscious rats. AMPA increased hindquarter VC by 18 ± 4%, but after pretreatment with L-NAME, AMPA reduced hindquarter VC by 16 ± 7% and 17 ± 9% (5 and 15 min after pretreatment, P < 0.05 compared with before pretreatment). Pretreatment with L-NAME reduced AMPA-induced bradycardia from 122 ± 40 to 92 ± 32 beats/min but did not alter the hypertension induced by AMPA (35 ± 5 mmHg before pretreatment, 43 ± 6 mmHg after pretreatment). Control injections with D-NAME did not affect resting values or the response to AMPA. The present study shows that stimulation of AMPA receptors in the NTS activates both vasodilatatory and vasoconstrictor mechanisms and that the vasodilatatory mechanism depends on production of nitric oxide in the NTS. Copyright © 2006 the American Physiological Society.

Inhibitory mechanism of the nucleus of the solitary tract involved in the control of cardiovascular, dipsogenic, hormonal, and renal responses to hyperosmolality

The nucleus of the solitary tract (NTS) is the primary site of visceral afferents to the central nervous system. In the present study, we investigated the effects of lesions in the commissural portion of the NTS (commNTS) on the activity of vasopressinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, plasma vasopressin, arterial pressure, water intake, and sodium excretion in rats with plasma hyperosmolality produced by intragastric 2 M NaCl (2 ml/rat). Male Holtzman rats with 15-20 days of sham or electrolytic lesion (1 mA; 10 s) of the commNTS were used. CommNTS lesions enhanced a 2 M NaCl intragastrically induced increase in the number of vasopressinergic neurons expressing c-Fos in the PVN (28 ± 1, vs. sham: 22 ± 2 c-Fos/AVP cells) and SON (26 ± 4, vs. sham: 11 ± 1 c-Fos/AVP cells), plasma vasopressin levels (21 ± 8, vs. sham: 6.6 ± 1.3 pg/ml), pressor responses (25 ± 7 mmHg, vs. sham: 7 ± 2 mmHg), water intake (17.5 ± 0.8, vs. sham: 11.2 ± 1.8 ml/2 h), and natriuresis (4.9 ± 0.8, vs. sham: 1.4 ± 0.3 meq/1 h). The pretreatment with vasopressin antagonist abolished the pressor response to intragastric 2 M NaCl in commNTS-lesioned rats (8 ± 2.4 mmHg at 10 min), suggesting that this response is dependent on vasopressin secretion. The results suggest that inhibitory mechanisms dependent on commNTS act to limit or counterbalance behavioral, hormonal, cardiovascular, and renal responses to an acute increase in plasma osmolality. © 2013 the American Physiological Society.

Segregated anatomical input to sub-regions of the rodent superior colliculus associated with approach and defense

The superior colliculus (SC) is responsible for sensorimotor transformations required to direct gaze toward or a way from unexpected, biologically salient events. Significant changes in the external world are signaled to SC through primary multisensory afferents, spatially organized according to a retinotopic topography. For animals, where anunexpected event could indicate the presence of either predator or prey, early decisions to approach or avoid are particularly important. Rodents' ecology dictates predators are most often detected initially as movements in upper visual field (mapped in medial SC), while appetitive stimuli are normally found in lower visual field (mapped in lateral SC). Our purpose was to exploit this functional segregation to reveal neural sites that can bias or modulate initial approach or avoidance responses. Small injections of Fluoro-Gold were made into medial or lateral sub-regions of intermediate and deep layers of SC (SCm/SCl). A remarkable segregation of input to these two functionally defined areas was found. (i) There were structures that projected only to SCm (e.g., specific cortical areas, lateral geniculate and suprageniculate thalamic nuclei, ventromedial and premammillary hypothalamic nuclei, and several brain-stem areas) or SCl (e.g., primary somatosensory cortex representing upper body parts and vibrissae and parvicellular reticular nucleus in the brainstem). (ii) Other structures projected to both SCm and SCl but from topographically segregated populations of neurons (e.g., zona incerta and substantia nigra pars reticulata). (iii) There were a few brainstem areas in which retrogradely labeled neurons were spatially overlapping (e.g., pedunculopontine nucleus and locus coeruleus). These results indicate significantly more structures across the rat neuraxis are in a position to modulate defense responses evoked from SCm, and that neural mechanisms modulating SC-mediated defense or appetitive behavior are almost entirely segregated.

Chronic Intermittent Hypoxia Depresses Afferent Neurotransmission in NTS Neurons by a Reduction in the Number of Active Synapses

Long-term synaptic plasticity has been recently described in brainstem areas associated to visceral afferent sensory integration. Chronic intermittent hypoxia (CIH), an animal model for studying obstructive sleep apnea in humans, depresses the afferent neurotransmission in nucleus tractus solitarii (NTS) neurons, which affect respiratory and autonomic regulation. Here we identified the synaptic mechanisms of CIH-induced depression of the afferent neurotransmission in NTS neurons in juvenile rats. We verified that CIH reduced the amplitude of both NMDA and non-NMDA glutamatergic excitatory currents (eEPSCs) evoked by tractus solitarii stimulation (TS-eEPSC) of second-order neurons in the NTS. No changes were observed in release probability, evidenced by absence of any CIH-elicited effects on short-term depression and failures in EPSCs evoked in low calcium. CIH also produced no changes in TS-eEPSC quantal size, since the amplitudes of both low calcium-evoked EPSCs and asynchronous TS-eEPSCs (evoked in the presence of Sr2+) were unchanged. Using single TS afferent fiber stimulation in slices from control and CIH rats we clearly show that CIH reduced the quantal content of the TS-eEPSCs without affecting the quantal size or release probability, suggesting a reduction in the number of active synapses as the mechanism of CIH induced TS-eEPSC depression. In accordance with this concept, the input-output relationship of stimulus intensity and TS-eEPSC amplitude shows an early saturation in CIH animals. These findings open new perspectives for a better understanding of the mechanisms underlying the synaptic plasticity in the brainstem sensory neurons under challenges such as those produced by CIH in experimental and pathological conditions.

Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats. (C) 2012 Elsevier B.V. All rights reserved.

Near-Term Fetal Hypoxia-Ischemia in Rabbits MRI Can Predict Muscle Tone Abnormalities and Deep Brain Injury

Background and Purpose-The pattern of antenatal brain injury varies with gestational age at the time of insult. Deep brain nuclei are often injured at older gestational ages. Having previously shown postnatal hypertonia after preterm fetal rabbit hypoxia-ischemia, the objective of this study was to investigate the causal relationship between the dynamic regional pattern of brain injury on MRI and the evolution of muscle tone in the near-term rabbit fetus. Methods-Serial MRI was performed on New Zealand white rabbit fetuses to determine equipotency of fetal hypoxia-ischemia during uterine ischemia comparing 29 days gestation (E29, 92% gestation) with E22 and E25. E29 postnatal kits at 4, 24, and 72 hours after hypoxia-ischemia underwent T2- and diffusion-weighted imaging. Quantitative assessments of tone were made serially using a torque apparatus in addition to clinical assessments. Results-Based on the brain apparent diffusion coefficient, 32 minutes of uterine ischemia was selected for E29 fetuses. At E30, 58% of the survivors manifested hind limb hypotonia. By E32, 71% of the hypotonic kits developed dystonic hypertonia. Marked and persistent apparent diffusion coefficient reduction in the basal ganglia, thalamus, and brain stem was predictive of these motor deficits. Conclusions-MRI observation of deep brain injury 6 to 24 hours after near-term hypoxia-ischemia predicts dystonic hypertonia postnatally. Torque-displacement measurements indicate that motor deficits in rabbits progressed from initial hypotonia to hypertonia, similar to human cerebral palsy, but in a compressed timeframe. The presence of deep brain injury and quantitative shift from hypo-to hypertonia may identify patients at risk for developing cerebral palsy. (Stroke. 2012;43:2757-2763.)

Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats

Costa-Silva JH, Zoccal DB, Machado BH. Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats. Am J Physiol Regul Integr Comp Physiol 302: R785-R793, 2012. First published December 28, 2011; doi:10.1152/ajpregu.00363.2011.-Sympathetic overactivity and altered respiratory control are commonly observed after chronic intermittent hypoxia (CIH) exposure. However, the central mechanisms underlying such neurovegetative dysfunctions remain unclear. Herein, we hypothesized that CIH (6% O-2 every 9 min, 8 h/day, 10 days) in juvenile rats alters glutamatergic transmission in the commissural nucleus tractus solitarius (cNTS), a pivotal site for integration of peripheral chemoreceptor inputs. Using an in situ working heart-brain stem preparation, we found that L-glutamate microinjections (1, 3, and 10 mM) into the cNTS of control rats (n = 8) evoked increases in thoracic sympathetic nerve (tSN) and central vagus nerve (cVN) activities combined with inhibition of phrenic nerve (PN) activity. Besides, the ionotropic glutamatergic receptor antagonism with kynurenic acid (KYN; 250 mM) in the cNTS of control group (n = 7) increased PN burst duration and frequency. In the CIH group (n = 10), the magnitude of L-glutamate-induced cVN excitation was smaller, and the PN inhibitory response was blunted (P < 0.05). In addition, KYN microinjections into the cNTS of CIH rats (n = 9) did not alter PN burst duration and produced smaller increases in its frequency compared with controls. Moreover, KYN microinjections into the cNTS attenuated the sympathoexcitatory response to peripheral chemoreflex activation in control but not in CIH rats (P < 0.05). These functional CIH-induced alterations were accompanied by a significant 10% increase of N-methyl-D-aspartate receptor 1 (NMDAR1) and glutamate receptor 2/3 (GluR2/3) receptor subunit density in the cNTS (n = 3-8, P < 0.05), evaluated by Western blot analysis. These data indicate that glutamatergic transmission is altered in the cNTS of CIH rats and may contribute to the sympathetic and respiratory changes observed in this experimental model.

Contribution of the retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats

Moraes DJ, Dias MB, Cavalcanti-Kwiatkoski R, Machado BH, Zoccal DB. Contribution of retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats. J Neurophysiol 108: 882-890, 2012. First published May 16, 2012; doi:10.1152/jn.00193.2012.-Central mechanisms of coupling between respiratory and sympathetic systems are essential for the entrainment between the enhanced respiratory drive and sympathoexcitation in response to hypoxia. However, the brainstem nuclei and neuronal network involved in these respiratory-sympathetic interactions remain unclear. Here, we evaluated whether the increase in expiratory activity and expiratory-modulated sympathoexcitation produced by the peripheral chemoreflex activation involves the retrotrapezoid nucleus/parafacial respiratory region (RTN/pFRG). Using decerebrated arterially perfused in situ rat preparations (60-80 g), we recorded the activities of thoracic sympathetic (tSN), phrenic (PN), and abdominal nerves (AbN) as well as the extracellular activity of RTN/pFRG expiratory neurons, and reflex responses to chemoreflex activation were evaluated before and after inactivation of the RTN/pFRG region with muscimol (1 mM). In the RTN/pFRG, we identified late-expiratory (late-E) neurons (n = 5) that were silent at resting but fired coincidently with the emergence of late-E bursts in AbN after peripheral chemoreceptor activation. Bilateral muscimol microinjections into the RTN/pFRG region (n = 6) significantly reduced basal PN frequency, mean AbN activity, and the amplitude of respiratory modulation of tSN (P < 0.05). With respect to peripheral chemoreflex responses, muscimol microinjections in the RTN/pFRG enhanced the PN inspiratory response, abolished the evoked late-E activity of AbN, but did not alter either the magnitude or pattern of the tSN reflex response. These findings indicate that the RTN/pFRG region is critically involved in the processing of the active expiratory response but not of the expiratory-modulated sympathetic response to peripheral chemoreflex activation of rat in situ preparations.