INTRODUCTION

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The role of second messenger systems in neuronal populations underlying generation and maintenance of eucapnic ventilation in mammals is mostly unknown. Protein kinase C (PKC) is a prominent second messenger system in mammalian cells which is ubiquitously expressed in neural tissue (1), and has been implicated in the transduction of multiple extracellular signals into the neuronal cell (2). The PKC family consists of 12 major isoforms divided into three major subgroups, classic (, 1, 2, and ), novel (,  , , , and µ), and atypical PKC (, , and ) based on their structural characteristics and activation requirements (3, 4). In cortical regions, expression of certain PKC isoforms is developmentally regulated with increasing levels of expression occurring with advancing postnatal age (5, 6). Furthermore, most of the known PKC isoforms are expressed within the dorsocaudal brainstem nuclei of the rat (7, 8), where they play a significant role in hypoxic chemotransduction. In addition, a previous study from our laboratory showed that modification of endogenous PKC activity by a blood-brain barrier permeable PKC inhibitor (Ro 32-0432) prolonged expiratory duration (TE) and reduced normoxic ventilation in conscious, freely behaving adult rats (9). Thus, we hypothesized that developmental changes in PKC expression and endogenous PKC activity may underlie important components of ventilatory patterning in the postnatal rat.

	METHODS

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The experimental protocols were approved by the Institutional Animal Use and Care Committee. Time-pregnant Sprague-Dawley rats were obtained from a commercial breeder (Charles River, Wilmington, MA) and delivery times recorded. For all experiments, pups were randomly selected from every litter at postnatal ages 2-3 d, 5-6 d, 10- 12 d, and 20-21 d. These ages have been previously shown as being representative of important maturational changes in ventilation (10).

Protocol

Ventilatory measurements were initially performed for approximately 30 min before and after intraperitoneal administration of 0.2 ml normal saline (control). Animals were then allowed to recover with their mother for at least 60 min, were returned to the barometric chamber, and a new baseline was determined. Animals were then injected with the systemically active, blood-brain barrier permeable, nonisoform-selective PKC inhibitor Ro 32-0432 (100 mg/kg intraperitoneally in 0.2 ml normal saline; Roche Products, Welwyn Garden City, UK; 11-13), and ventilation was monitored for 2 to 6 h. Ro 32-0432 is a bisindolylmaleimide derivative in which, in addition to a straight-chain alkyl side-chain bearing a cationic substitute, the position of the amine substituent was correctly conformationally restricted (12). In a subset of animals, recordings were repeated after a second dose of normal saline to ascertain reproducibility and variability of ventilatory patterning. The adequacy of Ro 32-0432 inhibitor dosages and experimental time frames has been previously validated (9). Because ventilatory recordings using the methods described subsequently are sensitive to body temperature changes, we measured changes in core body temperature in three 13-d-old rat pups after receiving Ro 32-0432 injections, and confirmed that no significant changes in body temperature occurred.

Ventilatory Recordings

Respiratory measures were continuously acquired in the freely behaving, unrestrained animal placed in a previously calibrated 0.5-L barometric chamber (Buxco Electronics, Troy, NY), using the methods described by Bartlett and Tenney (14), and Pappenheimer (15). To minimize the long-term effect of signal drift caused by temperature and pressure changes outside the chamber, a reference chamber of equal size in which temperature was measured using a T type thermocouple was used. In addition, as previously recommended by Epstein and colleagues, a correction factor was incorporated into the software routine to account for inspiratory and expiratory barometric asymmetries (16). Environmental temperature was maintained within 29 to 32° C, which corresponds to usual temperatures recorded in the dam. A calibration volume of 0.5 ml of air was repeatedly introduced into the chamber prior to, and upon completion of recordings. At least 30 min before the start of each protocol, animals were allowed to acclimate to the chamber, in which humidified air (90% relative humidity) warmed at 30° C was passed through at a rate of 2 L · min¹, using a precision flow pump-reservoir system. Pressure changes in the chamber caused by the inspiratory and expiratory temperature changes (17) were measured using a high gain differential pressure transducer (Model MP45-1; Validyne, Northridge, CA). Analog signals were continuously digitized, and analyzed on-line by a microcomputer software program (Buxco Electronics). A rejection algorithm was included in the breath-by-breath analysis routine and allowed for accurate rejection of motion-induced artifacts. Tidal volume (VT), inspiratory duration (TI), TE, respiratory frequency (f), and minute ventilation (E) were computed and stored for subsequent off-line analysis.

Immunoblot Analysis

Rat pups at postnatal ages 2 d, 5 d, 10 d, and 15 d, and adult male rats were killed with a pentobarbital overdose. The skull was rapidly opened, the brain was extracted, immediately placed on dry ice, and dissected under surgical microscopy. The obex was visually identified, and a coronal section 1.5 mm caudal to 1.5 mm rostral to the obex was performed. The dorsal half of this brainstem section (DB) as well as a portion of parietofrontal cortex (Cx) were carefully removed. Tissues corresponding to the DB or Cx from 4 to 10 animals were pooled and homogenized at 0° C with a tissue blender in 20 mM tris (hydroxymethyl) aminomethane (Tris)/HCl buffer pH 7.5, containing 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethyleneglycol-bis- (-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), 25 µg/ml leupeptin, 25 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged for 10 min at 1,000 × g at 4° C to remove cell debris. Protein content was measured in each soluble fraction using the Bradford method (DC-Biorad protein assay, Hercules, CA), and samples frozen at 70° C until analysis. Proteins (75 µg/sample) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% acrylamide gel), and transferred on a 0.2 µM nitrocellulose membrane. Membranes were blocked for 1 h in a 5% nonfat dry milk solution in Tris-buffered saline (TBS)- Tween. After overnight incubations with antibodies to , , , , , and µ PKC isoforms (Transduction Laboratories, Lexington, KY), membranes were washed and incubated for 1 h with a horseradish peroxidase (HRP)-labeled goat anti-mouse (1:30,000; Kirkegaard & Perry Laboratories, Gaithersburg, MD). In all experiments, a control lysate provided by Transduction Laboratories for each PKC isoform was included. The concentrations of the PKC isoform antibodies were as follows: PKC  (1:2,000), PKC  (1:250), PKC  (1:500), PKC  (1:250), PKC  (1:250), and PKC µ (1:800). Proteins were visualized by enhanced chemiluminescence (ECL; Amersham), and semiquantitative analysis of PKC isoform bands was performed by scanning densitometry. At least five different experiments each consisting of pooled tissue obtained from 4 to 10 animals were conducted for each postnatal age. To normalize data across experiments, densitometric values for each postnatal age were expressed as a ratio in which the control lysate densitometric readings served as the denominator.

Data Analysis

Values are reported as mean ± SEM unless indicated otherwise. Baseline ventilation was defined as the average of the 3 min immediately preceding each intraperitoneal injection. Post-treatment ventilatory measurements were defined as the average of three consecutive 1-min bins corresponding to the maximal ventilatory changes. Differences in data among the various age groups or within each age group for the saline and Ro 32-0432 treatments were compared by analysis of variance (ANOVA; two-way ANOVA for repeated measures) and the Newman-Keuls test (18). A p value of < 0.05 was considered statistically significant.

	RESULTS

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Ventilatory Measurements

In all animals, following Ro 32-0432 administration E decreases occurred within 10 to 15 min after injection, had peaked in all animals within 20 min, and remained within 90% of peak changes for at least 4 to 6 h (Table 1). The ventilatory reduction induced by Ro 32-0432 was largest in the youngest animals, and gradually decreased with advancing postnatal age (Table 1; Figure 1; p < 0.002, two-way ANOVA), such that at 20 to 21 d postnatal age E decreases were similar to those previously found in adult rats (9). At all postnatal ages, E decreases were primarily associated with TE prolongation. However, in the 2-3-d and 5-6-d-old groups, mild increases in TI also developed. VT changes were minimal and did not reach statistical significance at any postnatal age (Table 1; Figure 1). Concordantly, VT/TI was markedly reduced in youngest pups (from 0.73 ± 0.13 ml/s in control conditions to 0.37 ± 0.03 ml/s after Ro 32-0432, i.e., 39.7 ± 6.1%) whereas smaller decreases in VT/TI occurred in 5-6-d (21.7 ± 3.4%), 10-12-d (22.1 ± 4.4%), and 20-21-d-old pups (10.2 ± 5.6%). PKC inhibition with Ro 32-0432 exhibited significant age dependencies for TE, f, and E (p < 0.002, two-way ANOVA; Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Mean (± SEM) changes (expressed as percentage change from corresponding baseline) in TI, TE, f, VT, and E after intraperitoneal injection of either vehicle (open squares) or 100 mg/kg Ro 32-0432 (closed circles) in 2-3-d, 5-6-d, 10-12-d, and 20-21-d-old rat pups. Significant differences between vehicle and Ro 32-0432 were present at all postnatal ages for TE, f, and E (p < 0.01), whereas such differences were restricted only to the younger animals for TI (p < 0.01). VT changes with Ro 32-0432 were not significantly different at any postnatal age. A prominent age-dependent effect of Ro 32-0432 was found for TE, f, and E (p < 0.002, two-way ANOVA).

PKC Expression in Dorsal Brainstem and Cortex

Western blots of protein equivalents from tissue homogenates derived from dorsal brainstem and cortical tissue at various postnatal ages revealed markedly different changes in PKC expression within DB and Cx with maturation (Figures 2 and 3). PKC  increased in both DB (p < 0.04) and Cx (p < 0.01) over time. However, while significant increases in PKC  occurred in Cx with advancing postnatal age (p < 0.02), significantly decreased rather than increased expression emerged with maturation in DB (p < 0.02; Figures 2 and 3). In contrast, no developmental changes occurred in DB for PKC  whereas increased postnatal expression of PKC  in Cx was observed (p < 0.02; Figures 2 and 3). Interestingly, expression of PKC  was lower in DB compared with Cx (p < 0.001; Figures 2 and 3). PKC  expression levels were highest in younger animals in both DB and Cx and diminished with age (p < 0.02). In DB, expression of PKC µ was highest in 2-d-old pups and decreased with age (p < 0.02; Figures 2 and 3), whereas in Cx PKC µ increased with time reaching peak levels in adult animals (p < 0.02).

View larger version (80K): [in this window] [in a new window]   Figure 2.   Representative immunoblots for PKC , , , , , and µ of dorsal brainstem (DB) and neocortex (Cx) tissue lysates harvested from 2-d, 5-d, 10-d, and 15-d-old rat pups and adult rats.

View larger version (31K): [in this window] [in a new window]   Figure 3.   Mean (± SEM) PKC expression in DB and Cx for isoforms , , , , , and µ at 2 d, 5 d, 10 d, and 15 d postnatally and in adult rats (n = 5). To normalize across experiments, individual results were expressed as tissue lysate:control lysate densitometry ratios (see METHODS). In the Cx, increased expression occurred for all PKC isoforms except PKC  with maturation (p < 0.02). For PKC , decreases rather increases occurred with advancing postnatal age (p < 0.02). In the DB, no significant changes with age occurred for PKC , and , whereas increased expression of PKC  emerged (p < 0.04). In contrast, significant age-dependent decreases in PKC , , and µ were present in the DB (p < 0.02).

	DISCUSSION

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We have shown that in the developing rat pup endogenous PKC activity is a major determinant of normoxic ventilatory patterning. During the initial postnatal days, PKC inhibition was associated with marked disruption of mechanisms underlying respiratory rhythm. With advanced maturation, TE prolongation, which emerged as the major respiratory component affected by Ro 32-0432, resulted in progressively diminished effects. Thus, youngest animals exhibited increased dependency on PKC-mediated second messenger pathways mediating normoxic ventilation, and such role abated with increasing postnatal age.

PKC is very abundant in neuronal tissue; for example, PKC  is exclusively found in the central nervous system (3). Immunocytochemical localization studies for PKC  and  isoforms in adult rat brain reveal heterotopic expression of these isoforms in various neural structures suggestive of important functional implications (19, 20). Indeed, a substantial body of evidence points to a critical role for PKC in both pre- and postsynaptic modulation of neuronal activity (2). Thus, the relative abundance of PKC in neural tissue and the heterogeneity of distribution for the various isoforms within the central nervous system is strongly suggestive that PKC may underlie components of central ventilatory output. In support of such hypothesis, Haji and colleagues showed that PKC activity modulates tonic activity and excitability of expiratory neurons within the ventral respiratory group in the cat (21). Because the excitatory synaptic drive of these expiratory neurons relies on glutamate via N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/quisqualate receptors (22), excitatory cationic inward currents in these expiratory neurons appear to be under PKC-mediated tonic modulatory control (21). In addition, we have previously shown that endogenous PKC activity mediates both tonic and chemosensitive excitatory respiratory drives during both normoxia and hypoxia in freely behaving adult rats (7). Thus, current evidence supports the contention that PKC plays a prominent role in neuronal pathways responsible for maintaining spontaneous ventilatory output in general, and more specifically in the regulation of TE (9).

Current results further extend such findings to the developing animal which demonstrates increased dependency on PKC-mediated signal transduction pathways for generation and maintenance of respiratory timing mechanisms. As the rat pup matures, PKC dependency of f is reduced and reaches adult levels by 20 to 21 d of age. Indeed, the magnitude of TE prolongation and consequent ventilatory reduction were strikingly similar to those previously found in adult rats (9). Furthermore, decreases in VT/TI as an indicator of respiratory drive occurred following Ro 32-0432 in all animals in the absence of significant body temperature changes. The VT/TI changes were however more pronounced in the youngest pups, lending further support to the concept of a developmentally regulated PKC dependency of respiratory drives. It should be stressed that the current study precludes any definitive inferences on the potential neural sites whose activity was modulated by administration of the PKC inhibitor. However, we postulate that signal transduction pathways of glycinergic or GABA-ergic receptors mediating neuronal excitability of expiratory neurons within the Bötzinger complex could be candidate loci for the observed effect on expiratory duration induced by Ro 32-0432 (23). Indeed, this brainstem region which encompasses the rostral portion of the nucleus ambiguus just caudal to the facial nucleus, and an area located in the nucleus retroambiguus at the level of the caudal medulla displays a high density of neurons with rhythmical changes in membrane potential characterized by a depolarization in the intervals between phrenic bursts, thereby indicative of expiratory activity (24, 25). Alternatively, endogenous PKC activity could play a modulatory role on cellular mechanisms involved in presynaptic neurotransmitter release or affect postsynaptic receptor activation in one or more brainstem regions mediating respiratory timing.

Additional points regarding the developmental changes in normoxic ventilation after PKC inhibition deserve some comment. First, we can not exclude the possibility that Ro 32-0432 may cross the blood-brain barrier more readily in younger rat pups. However, we selected the dose of Ro 32-0432 based on a dose-response curve in adult animals whereby increases in dosage beyond 100 mg/kg were not associated with additional changes in ventilation (9). Thus, it is unlikely that differences in drug transport across the blood-brain barrier account for the changes in ventilation. Second, it is possible that PKC inhibition could have affected carotid body afferent input either via a direct effect on glomus cell tonic activity or by modification of synaptic relays in the petrosal ganglion. However, we found no evidence that peripheral chemoreceptor afferent input was modified in adult rats based on the absence of any difference in the ventilatory responses when experiments were conducted in normoxia and hyperoxia (9). Thus, if PKC plays a functional role at the carotid body level we postulate that such role will be of minor consequences to normoxic ventilatory patterning.

It was not our specific aim to identify at this stage which of the PKC isoforms mediate the developmentally regulated respiratory functions affected by inhibition of endogenous PKC activity. However, we attempted to gain more insights into this issue by examining postnatal patterns of PKC expression in the dorsal brainstem and neocortex. We targeted the dorsal brainstem rather than the whole medulla because multiple nuclei with defined respiratory roles are located in this region, and such assessment would extend on previous information from similar studies in the adult rat (7). It is clear that more detailed mapping of PKC isoform expression in the brainstem will be necessary, and that studies assessing the effect of PKC inhibition on c-fos expression as a marker of neuronal recruitment during application of respiratory stimuli may provide valuable information regarding expression-function relationships (26). Our findings in the neocortex for PKC , PKC , and PKC  are in close agreement with previous studies showing increased levels of expression with maturation (5, 6, 27- 29). Such temporally mediated increases have been postulated to correlate with ongoing synaptogenesis in various brain regions such as the visual cortex, hippocampus, and cerebellum (27, 30). In the current study, significantly different postnatal changes in DB compared Cx occurred for the three calcium-dependent PKC isoforms. This was not surprising because marked differences in time-course of PKC postnatal expression have been previously reported for subregions of the hippocampus (31). Indeed, PKC - transcripts showed a gene-specific expression pattern, and significant differences in expression were observed between the neurons of CA1, CA3, and fascia dentata within the hippocampus (31). Considering the developmental changes in the ventilatory responses to administration of Ro 32-0432, the more likely PKC candidates underlying such responses would be those PKC isoforms displaying progressive decreases in expression within the dorsal brainstem over time, i.e., PKC , PKC , and PKC µ. Obviously, verification of such hypothetical framework will have to await future development of isoform-selective PKC inhibitors or creation of transgenic animal models with site-specific targeted disruptions in a particular PKC isoform gene.

In summary, we have shown that developing rat pups display enhanced dependency on PKC-mediated pathways for respiratory rhythm generation and preservation of ventilatory output. Such maturational changes may reflect dynamic postnatal alterations in the relative contributions of second messenger systems to the central pattern generator neural network.