Unanesthetized Decerebrate Rats Research Articles

Mechanisms contributing to the genesis of hypoglossal preinspiratory discharge.

Preinspiratory discharge manifests in the neuronal recordings of the pre-Bötzinger complex, parafacial respiratory group, retrotrapezoid nucleus, and Kölliker-Fuse nucleus, as well as the efferent neural discharge of respiratory-related nerves innervating upper airway musculature. This neural component of triphasic eupnea contemporaneously contributes to the genesis of native and originate respiratory rhythmic activity, as well as the preinspiratory component of efferent neural respiratory discharges. In the course of our investigations evaluating hypoglossal discharge in response to asphyxia, we noted a curious pattern of neural respiratory recovery following postasphyxia resuscitation in hypoglossal, vagal, and phrenic neurograms in unanesthetized decerebrate rats. Specifically, we observed a gradual return of a pseudobiphasic eupnea characterized by initial transition bursts followed by robust eupneic bursts with dynamics inclusive of a gradually and progressively increasing duration of the hypoglossal eupneic bursts and duration and amplitude of the preinspiratory component of these bursts, as well as progressively lengthening expiratory interval between these bursts in the phrenic nerve discharge. This was followed by conversion to regular triphasic eupnea. We discuss our extrapolations based on these findings regarding eupneic respiratory central pattern generation and mechanisms contributing to the genesis of preinspiratory activity in hypoglossal discharge.

The crossed phrenic phenomenon.

The cervical spine is the most common site of traumatic vertebral column injuries. Respiratory insufficiency constitutes a significant proportion of the morbidity burden and is the most common cause of mortality in these patients. In seeking to enhance our capacity to treat specifically the respiratory dysfunction following spinal cord injury, investigators have studied the “crossed phrenic phenomenon”, wherein contraction of a hemidiaphragm paralyzed by a complete hemisection of the ipsilateral cervical spinal cord above the phrenic nucleus can be induced by respiratory stressors and recovers spontaneously over time. Strengthening of latent contralateral projections to the phrenic nucleus and sprouting of new descending axons have been proposed as mechanisms contributing to the observed recovery. We have recently demonstrated recovery of spontaneous crossed phrenic activity occurring over minutes to hours in C1-hemisected unanesthetized decerebrate rats. The specific neurochemical and molecular pathways underlying crossed phrenic activity following injury require further clarification. A thorough understanding of these is necessary in order to develop targeted therapies for respiratory neurorehabilitation following spinal trauma. Animal studies provide preliminary evidence for the utility of neuropharmacological manipulation of serotonergic and adenosinergic pathways, nerve grafts, olfactory ensheathing cells, intraspinal microstimulation and a possible role for dorsal rhizotomy in recovering phrenic activity following spinal cord injury

Dynamic changes in phrenic motor output following high cervical hemisection in the decerebrate rat

Hemisection of the spinal cord at C2 eliminates ipsilateral descending drive to the phrenic nucleus and causes hemidiaphragmatic paralysis in rats. Phrenic nerve (PhN) or diaphragmatic activity ipsilateral to hemisection can occasionally be induced acutely following hemisection by respiratory stressors (i.e., hypercapnia, asphyxia, contralateral phrenicotomy) and becomes spontaneously active days-to-weeks later. These investigations, however, are potentially confounded by the use of anesthesia, which may suppress spontaneously-active crossed phrenic pathways. Experiments were performed on vecuronium-paralyzed, unanesthetized, decerebrate adult male rats and whole PhN activity recorded continuously before, during, and after high cervical hemisection at the C1 spinal level. Crossed phrenic activity recovered spontaneously over minutes-to-hours with maximal recovery of 11.8±3.1% (m±SE) in the PhN ipsilateral to hemisection. Additionally, there was a significant increase in PhN activity contralateral to hemisection of 221.0±40.4% (m±SE); since animals were artificially-ventilated, these changes likely represent an increase in central respiratory drive. These results underscore the state-dependence of crossed bulbophrenic projections and suggest that unanesthetized models may be more sensitive in detecting acute recovery of respiratory output following spinal cord injury (SCI). Additionally, our results may suggest an important role for a group of C1–C2 neurons exhibiting respiratory-related activity, spared by the higher level of hemisection. These units may function as relays of polysynaptic bulbophrenic pathways and/or provide excitatory drive to phrenic motoneurons. Our findings provide a new model for investigating acute respiratory recovery following cervical SCI, the high C1-hemisected unanesthetized decerebrate rat and suggest a centrally-mediated increase in central respiratory drive in response to high cervical SCI.

Excitatory convergence of periaqueductal gray and somatic afferents in the solitary tract nucleus: role for neurokinin 1 receptors.

Our previous studies (Boscan P, Kasparov S, and Paton JF. Eur J Neurosci 16: 907-920, 2002) showed that activation of somatic afferents attenuated the baroreceptor reflex via neurokinin type 1 (NK(1)) and GABA(A) receptors within the nucleus of the solitary tract (NTS). The periaqueductal gray matter (PAG) can also depress baroreceptor reflex function and project to the NTS. In the present study, we have tested the possibility that the dorsolateral (dl)-PAG projects to the NTS neurons that also respond to somatic afferent input. In an in situ, arterially perfused, unanesthetized decerebrate rat preparation, somatic afferents (brachial plexus), cervical spinal cord, and dl-PAG were stimulated electrically, whereas NTS neurons were recorded extracellularly. From 45 NTS neurons excited by either brachial plexus or dl-PAG stimulation, 41 received convergence excitatory inputs from both afferents. Onset latency and evoked peak discharge frequency from brachial plexus afferents were 39.4 +/- 4.7 ms and 10.7 +/- 1.1 Hz, whereas this was 43.9 +/- 6.4 ms and 7.9 +/- 1 Hz, respectively, following dl-PAG stimulation. As revealed by using a paired pulse stimulation protocol, monosynaptic connections were found in 9 of 36 neurons tested from both spinal cord and dl-PAG. We tested NK(1)-receptor sensitivity in 38 neurons that received convergent inputs from brachial plexus/PAG. Fifteen neurons were sensitive to selective antagonism of NK(1) receptors. CP-99994, the NK(1) antagonist, failed to alter ongoing firing activity but reduced the evoked peak discharge frequency following stimulation of both brachial plexus (from 12.3 +/- 1.8 to 7.2 +/- 1.3 Hz; P < 0.01) and PAG (from 7.8 +/- 1.5 to 4.5 +/- 1 Hz; P < 0.01). We conclude that 1) somatic brachial and PAG afferents can converge onto single NTS neurons; 2) this convergence occurs via either direct or indirect pathways; and 3) NK(1) receptors are activated by some of these inputs.

Cardiovascular responses to microinjections of glutamate into the nucleus tractus solitarii of unanesthetized supracollicular decerebrate rats

In anesthetized rats, microinjections of excitatory amino acids (EAAs) into the nucleus tractus solitarii (nTS), in a region located immediately rostral to the calamus scriptorius (CS), have been generally reported to elicit depressor and bradycardic responses. On the other hand, in conscious freely moving rats, similar microinjections have been reported to elicit pressor and bradycardic responses. These divergent results have been attributed to the effect of anesthetics. A reinvestigation of the effects of EAAs into the nTS in unanesthetized animals became necessary in order to resolve this controversy. The microinjection technique used in freely moving conscious rats suffers from several technical limitations; for example, microinjections cannot be delivered stereotaxically. In order to avoid these limitations, the present experiments were carried out in unanesthetized supracollicular decerebrate rats. A systematic mapping of nTS in these rats, using microinjections of the solutions of EAAs in artificial cerebrospinal (aCSF) fluid, confirmed that depressor and bradycardic responses are elicited from all the sites in the nTS extending from the CS to a level about 1 mm rostral to it. Pressor responses were elicited by microinjections of l-glutamate ( l-Glu) only from the chemoreceptor projection site (a region of the commissural subnucleus, 0.1–0.5 mm caudal to the CS, 0–0.5 mm lateral to the midline and 0.4–0.5 mm deep from the medullary surface). The pressor responses elicited from the aforementioned site were accompanied with bradycardia; this response may be due to diffusion of l-Glu to the dorsal motor nucleus of vagus because the bradycardia disappeared when the depth of the microinjection was reduced to 0.3, instead of 0.5 mm, from the dorsal medullary surface. When urethane was administered intravenously in unanesthetized decerebrate rats, the responses to microinjections of l-Glu remained unchanged, i.e., depressor and bradycardic responses were elicited from all the sites in the nTS extending from the CS to a level about 1 mm rostral to it and pressor and tachycardic responses were elicited from the chemoreceptor projection site. These observations indicated that there is no anesthetic-induced qualitative alteration of the cardiovascular responses to microinjections of EAAs into the nTS.

Interactions between NMDA and AMPA/kainate receptors in the control of micturition in the rat

In unanesthetized decerebrate rats, GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5 H-2,3-benzodiazepine hydrochloride), an AMPA/kainate receptor antagonist, and MK-801 (dizocilpine), an NMDA receptor antagonist, acted synergistically to depress the micturition reflex. MK-801 (1 mg/kg i.v.) and GYKI 52466 (4 mg/kg i.v.) administered separately had no or only a small depressant effect on reflex bladder contractions but markedly depressed external urethral sphincter activity. However, in MK-801-treated rats, GYKI 52466 decreased the amplitude, frequency and duration of reflex bladder contractions. These results suggest that both AMPA/kainate and NMDA glutamate receptors are important in the micturition reflex pathway and that these receptors may be activated in parallel at some site in the pathway so that excitatory transmission via only one receptor type is sufficient to mediate reflex activation of the bladder.

Effects of GYKI 52466 and CNQX, AMPA/kainate receptor antagonists, on the micturition reflex in the rat

GYKI 52466, a non-competitive AMPA/kainate glutamate receptor antagonist, administered in graded doses (0.5–8 mg/kg, i.v.) at 10 min intervals, decreased the amplitude and duration of reflex bladder contractions (maximal inhibition = 63%), the intercontraction interval (maximal inhibition = 83%) and external urethral spincter activity (maximal inhibition = 91%) in urethane-anesthetized (1.2 g/kg, s.c.) intact rats during continuous transurethral cystometrograms. On the other hand, in unanesthetized decebrate rats, the drug did not alter reflex bladder activity but did produce a significant depression of sphincter activity (maximal inhibition = 59%). The depressant effects of single doses of GYKI 52466 (4 mg/kg, i.v.) on external urethral sphincter EMG activity occurred with similar time courses in both urethane-anesthetized (1.2 g/kg, s.c.) intact and unanesthetized decerebrate rats during continuous transurethral cystometrograms. In urethane-treated (0.6 g/kg, i.p.) decerebrate rats, GYKI 52466 (0.5-4 mg/kg, i.v.) depressed bladder contraction amplitude and sphincter EMG activity, similar to the effects in urethane-anesthetized (1.2 g/kg, s.c.) intact rats. CNQX (0.01-1 mg/kg, i.v.), a competitive AMPA/kainate receptor antagonists, administered to urethane-anesthetized (1.2 g/kg. s.c.) intact or unanesthetized decerebrate rats did not alter the bladder or the external urethral sphincter activity during continuous transurethral cystometrograms, possibly due to the inability of the drug to readily cross the blood-brain barrier. The present results indicate that glutamatergic excitatory transmission mediated by AMPA/kainate receptors is essential for the activation of external urethral sphincter activity during micturition in anesthetized and unanesthetized preparation. However, the depressant effect of GYKI 52466 on reflex bladder activity is only unmasked by urethane anesthesia, raising the possibility that urethane interacts with AMPA/kainate glutamate receptors in the spinobulbospinal micturition reflex pathway.

Sympathetic responses to stimulation of area postrema in decerebrate and anesthetized rats

The effects of electrical and chemical stimulation of the area postrema (AP) on mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were examined in urethan- and pentobarbital sodium-anesthetized rats and in unanesthetized decerebrate rats. The AP was electrically stimulated over a range of frequencies (10-100 Hz) and intensities (10-80 microA) with a pulse duration of 0.2 or 1.0 ms. The excitatory amino acid L-glutamate (100 or 200 mM) was microinjected into the AP to preferentially stimulate neuronal cell bodies. In urethan-anesthetized rats, electrical stimulation of the AP decreased MAP and RSNA. In pentobarbital sodium-anesthetized rats, MAP and RSNA were markedly increased by AP stimulation. In unanesthetized decerebrate rats, increases in MAP and RSNA were also observed during electrical AP stimulation. Microinjection of L-glutamate had no effect on MAP and RSNA in anesthetized or in unanesthetized rats. These results indicate that electrical AP stimulation increases sympathetic output in the unanesthetized decerebrate rat and that anesthesia modifies this sympathetic response. The findings also suggest that peripheral responses to L-glutamate and electrical stimulation of the AP are mediated over different central pathways.

Alteration by urethane of glutamatergic control of micturition.

The i.v. administration of MK-801 (0.001-3 mg/kg), a non-competitive NMDA receptor antagonist, did not alter reflex bladder activity in unanesthetized decerebrate rat recorded during fast infusion (0.21 ml/min) cystometry or under isovolumetric conditions, but did depress reflex bladder contractions in doses between 0.1 and 3 mg/kg i.v. in the urethane-anesthetized (1.2 g/kg s.c.) intact rat during fast infusion cystometry. The ED50 and the dose to produce maximal inhibition in urethane-anesthetized intact rats were 0.25 mg/kg and 3 mg/kg i.v., respectively. During slow infusion (0.04 ml/min) cystometry, in unanesthetized decerebrate rats, MK-801 (0.1-1 mg/kg i.v. or 6-60 micrograms i.t.) decreased by 12-44% the micturition volume threshold (VT) but did not change the amplitude and duration of the bladder contractions. The administration of a larger i.t. dose (60 micrograms) of MK-801 produced no further decrease in VT but decreased the amplitude of bladder contractions by 24%. External urethral sphincter electromyogram activity was reduced or abolished by MK-801 (0.01-3 mg/kg i.v.) in both unanesthetized decerebrate and urethane-anesthetized intact rats with ED50 of 0.12 mg/kg and 0.05 mg/kg, respectively. These results indicate that NMDA receptors play an important role in both facilitatory and inhibitory central neural control of voiding function and that there is a significant interaction between urethane anesthesia and NMDA glutamatergic transmission. Thus, even though urethane anesthesia has been useful for studying the physiological characteristics of the micturition reflex, it seems inappropriate for analyzing the normal transmitter role of glutamic acid in reflex voiding.

Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat.

In a previous study we investigated the intraspecific defensive reactions evoked by excitation of neurons in the intermediate third of the midbrain periaqueductal gray matter (PAG) of the rat. Experiments revealed that activation of neurons in this region of the PAG mediated: (i) backward defensive behavior, characterized by upright postures and backward movements, and (ii) reactive immobility ("freezing"), in which the rat remained immobile, but reacted with backward defensive behavior to investigative, non-aggressive contact initiated by the partner. In the present study, we aimed to extend our understanding of PAG mediation of defensive behavior by observing: (i) in a non-aggressive social interaction test, the behavioral effects of microinjections of low doses of kainic acid (40 pmol in 200 nl) made in the caudal third of the PAG; and (ii) the behavioral and cardiovascular effects of microinjections of D,L-homocysteic acid (5-10 nmol in 50-100 nl) made in the PAG of the unanesthetized decerebrate rat. Kainic acid injections into the area lateral to the midbrain aqueduct in the caudal third of the PAG evoked: (i) forward avoidance behavior, characterized by forward locomotion and occasional hop/jumps; (ii) reactive immobility ("freezing"), in which the rat remained immobile, but reacted with forward avoidance behavior to investigative, non-aggressive contact initiated by the partner; and (iii) 22-28 kHz ultrasonic vocalizations. These injections also evoked a dramatic increase in defensive responsiveness to tactile stimuli on the half of the body contralateral, but not ipsilateral, to the site of injection. Electroencephalographic measurements indicated that none of these effects were secondary to seizure activity. In the decerebrate rat, D,L-homocysteic acid injections in the caudal third of the PAG evoked forward running movements along with increased blood pressure and heart rate, the strongest effects being evoked from the region lateral to the midbrain aqueduct. More rostrally, sites in the intermediate PAG evoked backward "defensive" movements, which were also associated with increased blood pressure and heart rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Sciatic nerve blood flow response to carbon dioxide

Sciatic nerve blood flow (NBF) during hypercarbia was examined in unanesthetized decerebrate rats by means of laser Doppler flowmetry (LDF). During inspiration of gas mixtures containing no CO 2, followed by either 5, 10 or 20% CO 2, arterial pCO 2 increased by 13, 18 and 68 mm Hg, respectively. Blood pressure (BP) and the LDF signal, which were measured continuously, increased for 30–40 s following the start of inhalation of CO 2 and then decreased. Three min after the start of inhalation of 5 or 10% CO 2, BP had returned to the baseline and the LDF signal was increased by 14 and 15%, respectively, compared with preinhalation values. In rats inspiring 20% CO 2, systemic BP remained elevated 12% above the baseline and NBF was increased by 18%. The results indicate that dilatation of the vasa nervorum during hypercarbia is less than that at cerebral blood vessels. The nerve vasculature dilates maximally in response to 5% CO 2, with a rise in NBF of about 1.1% per mm Hg increase in p aCO 2.

Blood flow in rat sciatic nerve during hypotension

Laser Doppler flowmetry was utilized to examine blood flow in sciatic nerves of barbiturate-anesthetized and of unanesthetized decerebrate rats, in response to hypotension induced by graded exsanguination or by graded clamping of the descending aorta. Continuous laser Doppler flowmetry signals decreased linearly with decreasing arterial blood pressure. In anesthetized as well as in unanesthetized rats, the signal approached zero at a systemic blood pressure of 14 mm Hg or less. The results do not demonstrate autoregulation of blood flow in the rat sciatic nerve during systemic hypotension.