The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat

Neuroscience Letters 232 (1997) 63–66

The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat Edwin R. Yeh a , c, Bernadette Erokwu b, Joseph C. LaManna a ,*, Musa A. Haxhiu b a

Department of Neurology, School of Medicine (BRB), Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4938, USA b Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA Received 15 April 1997; received in revised form 25 July 1997; accepted 25 July 1997

Abstract In this study we sought to determine the role of the paraventricular nucleus of the hypothalamus (PVN) in modulating respiratory output. Experiments were performed in urethane anesthetized, vagotomized and mechanically ventilated Wistar rats. Electromyographic activity of the diaphragm (DEMG) was recorded and used to define the respiratory effects of PVN stimulation. The ventilation rate and volume were pre-adjusted so that baseline activity was 30% of the activity observed upon addition of 7% CO2 in O2. Microinjection of l-glutamate (4 nmol, 100 nl) into the PVN produced an increase in peak DEMG, and an increase in frequency of DEMG discharge. Changes in respiratory timing were mainly due to shortening of expiratory time (0.66 ± 0.06 s vs. 0.90 ± 0.10 s; mean ± SEM; P , 0.05), while inspiratory time was less affected (0.48 ± 0.04 vs. 0.51 ± 0.04 s; P . 005). The rate of rise of DEMG increased by 101 ± 28% from the baseline (P , 0.05). In addition, neuroanatomical tracing studies suggest the presence of direct connection between PVN and phrenic motoneurons. The results indicate that PVN neurons participate in regulation of breathing activity and in coordination of cardiovascular and respiratory functions.  1997 Elsevier Science Ireland Ltd. Keywords: Control of breathing; Electromyographic activity of the diaphragm; Paraventricular nucleus; Hypothalamus; Rat; Respiratory timing; Inspiratory time; Expiratory time; Retrograde tracing

The paraventricular nucleus (PVN), a cell group located bilaterally along the third ventricle, regulates neuroendocrine and cardiovascular functions, and modulates sympathetic outflow [1,9,10,12,14]. Because many stimuli that influence sympathetic activity also affect breathing, it might be expected that stimulation of the PVN would change ventilation. Hence, the aim of this study was, first to examine whether activation of PVN neurons results in changes in respiratory output and pattern of breathing, and second to determine the connections between PVN and phrenic motoneurons through which PVN neurons might influence respiration. Physiological experiments were performed in urethane anesthetized (1.2 g/kg) Wistar rats (n = 10). Animals were instrumented, as earlier described [11]. Following bilateral cervical vagotomy, rats were ventilated with 100% oxygen * Corresponding author. Tel.: +1 216 3681112; fax: +1 216 3681144; e-mail: [email protected]

(O2). The rate and the volume of the ventilator were adjusted to provide an end tidal CO2 at which the diaphragm electromyographic activity (DEMG) was 30% of that obtained with 7% CO2 in O2. At this level of respiratory drive, arterial PCO2 is 33 ± 3 mmHg, with a pH of 7.42 ± 0.02, and arterial PO2 above 350 mmHg. l-Glutamate (4 nmol, 100 nl) was microinjected into the PVN (2.1 mm caudal to the bregma, 0.4 mm lateral to midline, 8 mm from the surface of the skull) [15]. The microinjections were made through pairs of glass micropipettes pulled from capillary tubing and broken back to diameter of 40–60 mm, which were mounted in a custom-made device that allowed precise adjustment of the distance of the micropipettes from the midline. A thin metal bar connected within the device was used to identify the midline point along the bregma-calamus scriptorius axis. This device enabled simultaneous bilateral microinjection of the vehicle or glutamate [6]. The DEMG recorded by twisted-pair insulated tungsten

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0579-X

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wire was full wave rectified, amplified and integrated by a moving averager with a time constant of 100 ms before being digitally sampled, double buffered and stored by the computer in real time. The data were then processed by an event detector composed of peak and valley detectors to obtain quantitative measures of ventilatory output such as the inspiratory time (Ti), the expiratory time (Te), the peak DEMG amplitude, and the respiratory drive (Peak DEMG/Ti). Statistical analysis using paired t-tests was done to compare control values with the changes that occurred with microinjections of vehicle or l-glutamate. Activation of the PVN by the microinjection of l-glutamate caused a significant increase in DEMG amplitude (Fig. 1A). There was also a corresponding increase in frequency which was mainly attributed to the shortening of the expiratory time (Te). The expiratory time decreased by 26%, from 0.90 ± 0.10 to 0.66 ± 0.06 s (mean ± SEM; P , 0.05; Fig. 1B), while inspiratory time was less affected (0.51 ± 0.04 vs. 0.48 ± 0.04; P . 0.05; Fig. 1C). The mean duration of the respiratory period (Ti + Te) decreased from 1.40 ± 0.13 to 1.14 ± 0.08 s (P , 0.05). A doubling of the normalized peak DEMG amplitude was observed (P , 0.05; Fig. 1D). Finally, the respiratory drive, estimated from the ratio of peak DEMG amplitude over the inspiratory time (peak DEMG/Ti) increased by 101 ± 28% from the baseline (P , 0.05). To eliminate the possibility that the activation was caused

by diffusion of the injected chemicals into nearby regions such as the lateral hypothalamus (LHA), we microinjected the same amount of l-glutamate 4 mm above, 2 mm below and 2 mm lateral to PVN, respectively and there were no detectable changes (n = 3). However, when the microinjection was made in the LHA, there was a decrease in mean arterial blood pressure from 92 to 72 mmHg and a transient increase in phrenic activity, as expected [19]. This observation provides a means of differentiating the two regions. Hence, it is unlikely that the respiratory effects of l-glutamate administration into the PVN could be due to diffusion of the chemical into the LHA because with the respiratory effects there was an increased blood pressure while direct administration of l-glutamate into LHA elicits a fall in blood pressure. To define whether PVN neurons project to phrenic motoneurons, neuroanatomical tracing studies were performed in two groups of rats. In the first group of animals (n = 3), cholera toxin b subunit (CTb; List. Biol. Lab., Campbell, CA 95008), a retrograde tracer, was injected into the ventral horns of cervical spinal cord, extending from the C3 to C5 (100 nl of 0.1% per site). Injections were made with a glass micropipette (40–60 mm diameter), 0.7 mm from the midline and 1.4 mm from the dorsal surface of the spinal cord [5]. Five to seven days following CTb injections, the rats were anesthetized, perfused, the brains were removed, postfixed, cryoprotected, cut at 50 mm in the transverse plane,

Fig. 1. (A) An example of the effects of bilateral l-glutamate microinjection into PVN (1 and 4 nmol/site, 100 nl) on DEMG and respiratory timing. Activation of PVN neurons induced an increase the peak DEMG activity, and in breathing frequency. Similar responses were observed in all studied rats (n = 10). Average results of changes in (B) expiratory (Te) and (C) inspiratory duration (Ti) and (D) peak DEMG amplitude induced by activation of PVN neurons. *Statistically significant differences.

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Fig. 2. Photomicrograph showing retrogradely labeled PVN neurons following microinjection of CTb into the phrenic nucleus. PVN, paraventricular nucleus; LHA, lateral hypothalamic nucleus; AHA, anterior hypothalamic nucleus; VMH, ventral medial hypothalamus; III, the third ventricle.

and stained for CTb [7]. To define whether labeling in the PVN following injections into the phrenic nucleus was related to selective projection from the PVN to the phrenic nucleus, in the second group of rats, we injected two tracers, one in the phrenic nucleus, and another in the upper thoracic intermediolateral cell column (IML). In each of three animals, 100 nl/site of 4% fluorogold was injected unilaterally into the ventral horns as described, and 100 nl/site of undiluted rhodamine-labeled microspheres was administered into the intermediolateral cell column (0.5 mm lateral to midline, and 0.9 mm ventral), at the level of the third and the fourth thoracic spinal cord. In two other animals, undiluted rhodamine-labeled microspheres were injected into the phrenic motoneurons, while green-labeled microspheres were injected into IML. After 5–7 days of survival, animals were deeply anesthetized, perfused with saline, followed by 4% paraformaldehyde in phosphate buffer saline (pH 7.4). The brains were removed, processed and examined with bright field or fluorescent microscopy, for the presence of tracers in the PVN. Following injection of the CTb into the ventral horns of cervical spinal cord, labeled cells were found in the PVN (Fig. 2). No labeling of IML neurons at upper thoracic segments was observed, indicating that a tracer was not taken up in a sufficient amount by PVN axon terminals synapsing on sympathetic preganglionic neurons in these segments. The same pattern of labeling in the PVN was observed

with two other tracers injected into phrenic motoneurons. Injection of a retrograde tracer into IML also was associated with labeling of PVN cells, but the number of labeled PVN neurons was much less than following injections into ventral horns of cervical spinal cords. Double labeling was observed in about 10% of PVN cells that project to cervical spinal cord. Conceivably, in experiments using fluorogold as a tracer, double labeling could be partly due to a transport of tracer through fibers of passage when injected into ventral horns [2], but this is unlikely to happen following latex microsphere injections [18], or CTb injections. The present study indicates that PVN can exert excitatory influences on respiratory drive. This effect can be mediated through PVN-brainstem and/or PVN-spinal cord pathways. In a previous study using the retrograde transneuronal marker, pseudorabies virus (PRV), we had shown that PVN was consistently labeled when the PRV was injected into the right hemidiaphragm of C8–T1 spinalectomized rats after ipsilateral cervical vagotomy, or in phrenic nerve [4]. Because, PRV infects also the second order neurons, the results of the present study indicate the presence of direct projection from PVN to the phrenic motoneurons, and extend earlier studies related to connectivity of PVN with brainstem and spinal cord autonomic nuclei [8,13,16, 17,20,21]. Physiologically, the present study demonstrated for the first time that activation of the PVN significantly influenced

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[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10] Fig. 3. Schematic diagram of the pathways involved in mediation of changes induced in breathing activity and respiratory timing by PVN stimulation.

the magnitude of DEMG and the respiratory timing, which could be mediated by two pathways: (1) through the brainstem bulbospinal and, (2) probably via the direct connection with phrenic nucleus (Fig. 3). Although there are few physiological studies relating activation of PVN and respiration, Ferguson et al. [3] have indirectly inferred through electrical stimulation of the subfornical organ that the changes in the respiratory pattern observed were mediated through the medullary center via the PVN. The relatively long changes in respiratory pattern observed following the injection of l-glutamate into PVN could be attributed to PVN’s projection (possibly through vasopressin and oxytocin [12]), to brainstem respiratory related cells, and to phrenic motoneurons. In summary, the results of this study indicate that the changes in breathing activity associated with activation of the PVN neurons are part of complex responses as a consequence of activation of a network controlling autonomic, neuroendocrine, cardiovascular, and respiratory functions.

[11]

[12]

[13]

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