Short-term plasticity of descending synaptic input to phrenic motoneurons in rats.

Respiratory afferent stimulation can elicit increases in respiratory motor output that outlast the period of stimulation by seconds to minutes [short-term potentiation (STP)]. This study examined the potential contribution of spinal mechanisms to STP in anesthetized, vagotomized, paralyzed rats. After C(1) spinal cord transection, stimulus trains (100 Hz, 5-60 s) of the C(1)-C(2) lateral funiculus elicited STP of phrenic nerve activity that peaked several seconds poststimulation. Intracellular recording revealed that individual phrenic motoneurons exhibited one of three different responses to stimulation: 1) depolarization that peaked several seconds poststimulation, 2) depolarization during stimulation and then exponential repolarization after stimulation, and 3) bistable behavior in which motoneurons depolarized to a new, relatively stable level that was maintained after stimulus termination. During the STP, excitatory postsynaptic potentials elicited by single-stimulus pulses were larger and longer. In conclusion, repetitive activation of the descending inputs to phrenic motoneurons causes a short-lasting depolarization of phrenic motoneurons, and augmentation of excitatory postsynaptic potentials, consistent with a contribution to STP.

Substance P in the hypoglossal nucleus of the rat.

The distribution of substance P (SP)-containing synaptic terminals in the hypoglossal nucleus (XII) of adult rats was examined by retrograde peroxidase labelling and immunocytochemistry. From the location of peroxidase injections into the tongue and of labelled neurones in the ventral lamina of XII, motor neurones that supply intrinsic vertical, longitudinal and transverse fibres as well as the extrinsic muscle genioglossus appear to have been labelled. SP-containing terminals were found making contact, and sometimes dual synapses, with unlabelled neuronal dendrites but not with retrogradely labelled somata or dendrites. These findings suggest that SP terminals may contact dendrites of interneurones or of neurones supplying other extrinsic muscles located in the anterior part of the tongue. Dual SP-containing synapses between XII motor neurones may be the means by which tongue muscle fibres are recruited and their function synchronized.

Fetuin in neurons of the retina and cerebellum during fetal and postnatal development of the rat.

Although long known to be a liver-derived fetal plasma glycoprotein, fetuin has more recently been shown to be present in sub-populations of neurons in the developing nervous system of a number of mammalian species. We have extended these observations to examine the fetuin immunoreactivity (IR) in developing rat retina and cerebellum. Fetuin-IR was first seen in the retina on embryonic day (E)19 in a sub-population of cells in the retinal ganglion cell layer and a small proportion of cells in the neuroblastic layer. The proportion of cells in the ganglion layer exhibiting fetuin-IR increased until postnatal day (P)10 when all cells in this layer were strongly immunoreactive. From P14 onwards fetuin-IR was absent or very weak and restricted to a small proportion of ganglion cells. In the developing cerebellum, the outer and inner granule cell layers, the deep nuclei and cells in the sub-cortical white matter exhibited fetuin-IR from E19 to P10. There was little fetuin-IR in the cerebellum at ages P14 and older, and Purkinje cells did not exhibit fetuin-IR at any time. The results show that fetuin appears in many neurons in the retina and cerebellum that are differentiating during the period from E19 to P10. The concentration of fetuin in cerebrospinal fluid is at its highest in this same period which suggests that some sub-populations of neurons could obtain fetuin from extracellular fluid during this period; however, the lack of fetuin-IR in other neuronal populations suggests that fetuin uptake is not a general property of developing neurons.

Hypoxia attenuates the respiratory response to injection of substance P into the nucleus of the solitary tract of the rat.

Prolonged or repetitive bouts of hypoxia may desensitize the brain stem respiratory centres leading to reduced stimulation of ventilation. We investigated the possible involvement of changes in the sensitivity of the commissural nucleus of the solitary tract (cNTS) to the tachykinin peptide, substance P (SP). Urethane-anaesthetised rats were allowed to breath room air (normoxic) or subjected to four, 30 s bouts of hypoxia (10% O2/90% N2) prior to the injection of SP (750 pmol) into the cNTS. In normoxic rats (n = 5), SP produced a fall in frequency (f, 88+/-4% control) after 4 min and a maximum rise in tidal volume (VT) after 6 min (138+/-10% control) leading to an overall increase in minute ventilation (VE, maximum, 127+/-12% control after 2 min). In rats (n = 5) exposed to four bouts of hypoxia and allowed to recover for 10 min, injection of SP produced a similar fall in f but a delayed and significantly (P < 0.001) reduced VT (maximum after 10 min, 110+/-1% control) and hence, VE response (104+/-3% control). Sixty min after hypoxia, the f, VT and VE responses to SP were identical to those of normoxic rats. These data suggest that hypoxia desensitizes SP receptors in the cNTS and this may partly explain why the respiratory response to hypoxia declines over time.

Ventilatory and metabolic responses to cold and hypoxia in conscious rats with discrete hypothalamic lesions.

We tested the hypothesis that hypothalamic nuclei involved in thermoregulatory control could represent a site of integration of the metabolic and ventilatory response to cold and hypoxia. Electrolytic lesions were performed bilaterally under stereotaxic guide, either within the anterior or posterior hypothalamic areas of adult rats. One week later, oxygen consumption (VO2) and ventilation (VE) were measured in the conscious animals during warm (27 degrees C) or cold (12 degrees C) conditions, in normoxia (21% O2) or hypoxia (10% O2), and compared to measurements obtained in control rats, which were either intact or sham-operated. VO2, VE, and body temperature did not differ between lesioned and control rats during warm normoxia. In cold and hypoxia, singly or combined, VE/VO2 was higher in the lesioned rats, because of higher VE. The differences in the cold were mostly confined to rats with anterior lesions, whereas differences in hypoxia were mostly in rats with posterior lesions. We conclude that the integrity of the anterior and posterior hypothalamic areas is important for the proper coupling of metabolism and ventilation during cold or hypoxic stimuli.

Substance P receptors in brain stem respiratory centers of the rat: regulation of NK1 receptors by hypoxia.

Substance P (SP) is a key neurotransmitter involved in the brain stem integration of carotid body chemoreceptor reflexes. In this study, the characteristics and location of SP receptors in the rat brain stem and their regulation by hypoxia were investigated using homogenate radioligand binding and quantitative autoradiography. Specific binding of [125I] Bolton-Hunter SP (BHSP) to brain stem homogenates was saturable (approximately 0.3 nM) and to a single class of high-affinity sites (K(d), 0.16 nM; maximum density of binding sites, 0.43 fmol/mg wet weight tissue). The order of potency of agonists for inhibition of BHSP binding was SP > [Sar9Met(O2)11]SP >> neurokinin A > septide > neurokinin B >> [Nle10]-neurokinin A(4-10) = senktide, and for nonpeptide antagonists, RP 67580 > CP-96,345 >> RP 68651 = CP-96,344, consistent with binding to NK1 receptors. The effect of single and multiple, 5-min bouts of hypoxia (8.5% O2/91.5% N2) on BHSP binding was investigated using quantitative autoradiography. Binding sites were localized to the lateral, medial and commissural nucleus of the solitary tract (NTS), the hypoglossal nucleus, central gray and the spinal trigeminal tract and nucleus (Sp5 and nSp5, respectively). Five min after a single bout of hypoxia, the density of BHSP binding sites had decreased significantly (P < .05) in the medial NTS (-33%) and lateral NTS (-24%) when compared to normoxic controls. However, the normal receptor complement was restored within 60 min of the hypoxic challenge. In the Sp5, a significant decrease (P < .05) in binding was observed 5 min after hypoxia which was still apparent after 60 min. In contrast, the density of BHSP binding sites in the hypoglossal nucleus decreased slowly and was significantly lower (P < .05) than normoxic controls 60 min after hypoxia. Five min after repetitive hypoxia (3 x 5 min bouts), BHSP binding in the NTS was reduced by more than 40%. Studies in homogenates showed that the affinity of SP for BHSP binding sites was not affected by repetitive hypoxia (K(d)s, normoxic, 0.27 nM; hypoxic, 0.24 nM). These data suggest that afferent input from carotid body chemoreceptors may dynamically regulate NK1 receptors in several brain stem nuclei that are intimately involved in stimulating ventilation during hypoxia, and that the time-course of receptor turnover may differ from region to region in the brain stem. The temporary loss of NK1 receptors in the NTS may partly explain why adequate ventilation is often not maintained during hypoxia.

Modulation of the synaptic drive to respiratory premotor and motor neurons.

The characteristics of GABAergic inhibitory modulation of respiratory bulbospinal neuronal activity and short-term potentiation (STP) of phrenic motoneuronal activity were studied. Extracellular unit recording and picoejection techniques in anesthetized dogs showed that both the spontaneous rhythmic and reflexly induced discharge patterns of inspiratory (I) and expiratory (E) premotor neurons were proportionately amplified by the localized application of picomole amounts of bicuculline (Bic), a competitive GABAA antagonist. Intracellular recording and paired-pulse stimulation techniques in anesthetized rats demonstrated an STP of phrenic motor output that appears to be mediated by NMDA receptors and is associated with facilitation of EPSPs and prolonged depolarization of individual phrenic motoneurons. We speculate that both GABAergic gain modulation of premotor neuronal activity and NMDA-mediated STP of phrenic activity may be neural substrates which are involved with the optimization of respiratory and non-respiratory behaviors, via adaptive and/or differential control of breathing.

Respiratory responses to combined hypoxia and hypothermia in rats after posterior hypothalamic lesions.

Urethane-anaesthetised rats were exposed to hypoxia (7% O2 in N2) for 5 min periods while body core temperature (Tbc) was maintained within the normal range (37-38 degrees C) using an abdominal heat exchanger. Animals were exposed to hypoxia and after placement of electrolytic lesions in either the anterior (n = 6) or posterior hypothalamus (n = 6). Neither lesion altered respiration while rats breathed air at either Tbc. At normal Tbc, rats responded to hypoxia with increased ventilation throughout the exposure period. This response was unchanged by lesions in either location. At reduced Tbc rats responded to hypoxia with an initial increase in ventilation followed by depression to below air-breathing levels. This depressive response was unchanged after anterior hypothalamic lesions but eliminated after posterior hypothalamic lesions. It is concluded that neurons either originating in the posterior hypothalamus, or passing through it, play a role in the interaction between cold and hypoxia which leads to inhibition of respiration.

How thick should a retina be? A comparative study of mammalian species with and without intraretinal vasculature.

In this study we describe a measurement method which closely approximates in vivo retinal thickness. Using this method we examined the laminar organisation of vascular and avascular retinae from placental and marsupial mammals. Thickness measurements on retinal wholemounts show that the avascular retinae of the placental guinea pig (140 microns) and the marsupial brushtail possum (170 microns) are thinner and show less centroperipheral taper than do the vascular retinae of placental cats (250 microns), rats (220 microns) and marsupial quolls (220 microns). In general, limitation in thickness of avascular retinae is borne by most retinal layers, but most particularly by the inner plexiform layer, the synaptic region farthest removed from the choroidal blood supply. Except for the absence of blood vessels, the histological organisation of the brushtail possum's retina resembles closely that of its fellow marsupial, the quoll's. In contrast, intraretinal organisation differs amongst the two avascular retinal species with the guinea pig displaying a much coarser photoreceptor grain.

An appraisal of some lesioning methods applied to the posterior hypothalamus in rats.

The behavioral changes and histological damage to the brain of rats were examined following lesions of the midlateral posterior hypothalamic area (MLPHA) made by passing electrical current through a metal electrode or through a glass pipette containing hydrogen ion (HCl) or kainic acid. Control experiments included placement of pipettes containing saline and imposing the same current as used in kainic-acid-containing electrodes (sham). Subcutaneous injections of kainic acid assessed damage that may be attributed to general neuronal cytotoxicity or limbic seizures. The effect of diazepam on alleviating behavioral changes and structural damage in kainic-acid-injected rats was examined. The current used to inject kainic acid produced significant damage to both neurons and axons of passage at the injection site. Degeneration of nerve terminals following kainic acid injections in the MLPHA was widespread, not predictably reduced by diazepam premedication and differed in only minor details from degeneration induced by subcutaneous injections.

The nature of posterior hypothalamic projections to cardiorespiratory centers in the brainstem.

Focal electrical stimulation of the midlateral posterior hypothalamus in the rat produces rapid shallow respiration accompanied by a rise in arterial blood pressure. Stimulation of presumably intrinsic neurons only by glutamate induces slower deeper respiration associated with a fall in blood pressure.

Projections of the midlateral posterior hypothalamic area influencing cardiorespiratory function in rats.

The influence of the midlateral posterior hypothalamic area (MLPHA) on arterial blood pressure and respiration was examined in the rats. Electrical stimulation produced a rapid transient increase in blood pressure and rapid shallow respiration. Glutamate, which stimulates neurons and not fibers of passage, however, caused a fall in blood pressure and slowing of respiration. Projections of MLPHA traced by horseradish peroxidase (HRP) and 3H leucine showed reciprocal connections with limbic forebrain centers but only indirect connections with brainstem cardiorespiratory centers. It is concluded that MLPHA may be a relay through which the limbic system can gain access to and depress cardiorespiratory function.

Respiratory roles of genioglossus, sternothyroid, and sternohyoid muscles during sleep.

We examined the respiratory activity of the genioglossus, sternothyroid, and sternohyoid muscles of the rat during nonrapid eye movement (non-REM) and REM sleep. Each animal carried implanted electrodes for recording the integrated EMG activity of respiratory muscles, the postural tone (EMG), and electrocortical activity (polygraphic identification of sleep-waking states). The three upper airway muscles exhibited inspiratory activity during non-REM sleep while rats breathed ambient air. Curled up postures promoted inspiratory activity of genioglossus and sternothyroid muscles, an effect enhanced by CO2 breathing but reduced by hypoxic breathing. During REM sleep, genioglossus and sternothyroid muscles lost their activity but the sternohyoid muscles retained their inspiratory activity. We conclude that the genioglossus and sternothyroid muscles contribute to upper airway patency during non-REM sleep, an effect CO2 augments but hypoxia reduces. The sternohyoid muscles have at least two functions during both sleep states: they contribute to maintenance of upper airway patency and to rib cage fixation, thereby optimizing the ventilatory action of the diaphragm.

The facial nucleus of the rat: representation of facial muscles revealed by retrograde transport of horseradish peroxidase.

The representation of facial muscle groups in the facial nucleus of rat was examined by retrograde transport of HRP. Motoneurons supplying muscle groups are arranged in longitudinal columns. Those supplying nasolabial muscles are located in the lateral and ventral intermediate segments, posterior auricular muscles in a medial column, platysma in an intermediate column; the lower lip and ocular muscles are in the ventral and dorsal segments respectively of the intermediate column. The posterior belly of the digastric muscle is supplied by motoneurons extending from the dorsal aspect of the facial nucleus to the caudal pole of the trigeminal motor nucleus.

Brain stem projections to the facial nucleus of the rat.

Horseradish peroxidase was injected into the medial and lateral columns of the facial nucleus of the rat. Following medial injections, cells were labelled by retrograde transport in the ipsilateral spinal trigeminal nucleus (caudalis) both medial vestibular nuclei, contralateral midbrain reticular formation and nucleus of the lateral lemniscus. The periaqueductal grey, interstitial nucleus and nucleus of Darkschewitch were also labelled ipsilaterally. Injections into the lateral column of the facial nucleus labelled the spinal trigeminal nucleus (oralis) and parabrachial nuclei ipsilaterally and the Darkschewitch and red nuclei contralaterally.

The size of motor units in laryngeal muscles of the rat.

The size of motor units in rat laryngeal muscles was determined by correlating the number of neurons labeled by i.m. injections of horseradish peroxidase with the number of motor end plates stained for acetylcholinesterase. The cricothyroid has a motor unit size of 8 muscle fibers per motor neuron and the posterior cricoarytenoid 4-5 muscle fibers per motor neuron.

Slice preparation of rat medulla and pons maintained for five hours in vitro.

Slices of rat medulla and pons were incubated in bicarbonate-buffered medium and their electrical activity was monitored for five hours with microelectrodes. The morphology of these slices was compared with that of the same region of rats of the same age using prior perfusion or immersion in fixatives before incubation. Many neurons in incubated slices show shrinkage necrosis (apoptosis) but not dilatation of the endoplasmic reticulum seen in most neurons fixed immediately after slicing. In incubated slices, some processes but not somata of glia appeared swollen: to a lesser extent some dendritic and axonal processes were swollen. Glia showed no cytoplasmic reaction after five hours to indicate that they might phagocytose damaged tissue components. Synapses appeared morphologically normal after the period of incubation and there was an apparent increase in numbers of profiles resembling growth cones.

Monoaminergic innervation of the carotid sinus in mammals: a histochemical study.

The bifurcations of the common carotid arteries in the rabbit, cat, pig, rat and guinea pig were examined for the comparative structure and adrenergic innervation of the carotid sinus. The carotid sinus was poorly innervated in the cat, pig and guinea pig but densely innervated in the rabbit. No adrenergic innervation could be detected in the carotid arteries or carotid sinus of the rat. The relationship of adrenergic innervation to the tunica media also varies between species, penetration being pronounced in the pig, occasional in the cat and rabbit and very slight in the guinea pig. The arrangement of adrenergic nerve fibres is circular in the cat and pig, circular and plexiform in the guinea pig and spiral in the rabbit where an additional longitudinal plexus is present. The functional implications of species variations in innervation of the carotid sinus region in relation to cardiovascular control is discussed.