The effects of diazepam dependence and withdrawal on morphine-induced antinociception and changes in locomotion in male and female rats.

Male and female rats were exposed for 3 weeks to diazepam (DZ)-filled or empty capsules (CTR) prior to the daily administration of morphine (MOR, 5 mg/kg, IP) for 5 days. Thereafter, capsules were removed and 48 h later MOR was injected for the next 5 days. The tail-flick latency (TFL) was measured prior to and 15, 30, and 60 min after MOR assessed analgesia. Locomotion (LOC) was determined before and 15 min after injection. Prior to MOR injection (baseline), male rats were more sensitive to the thermal stimulus and were less active than female rats. Daily MOR injections neither affected the baseline TFL nor LOC. Regardless of gender, MOR produced greater analgesia in DZ-dependent and withdrawn rats than in CTR. MOR analgesia was greater in DZ-dependent male than in female rats. Gender differences in MOR analgesia were not of statistical significance in DZ-withdrawn rats. The first dose of MOR produced more depression of LOC in DZ-dependent female than in male rats. Across the time of MOR injections, female DZ-dependent and withdrawn rats were less active than CTR. LOC increased with repeated administration of MOR in all groups of rats. In summary, DZ dependence and withdrawal enhanced MOR analgesia in rats of both sexes. Regardless of chronic treatment, MOR produced more analgesia and less depression of LOC in male than in female rats. It is suggested that a decrease in the function of the GABAergic system plays a role in alteration of MOR analgesia.

The pharmacodynamics of PK 11195 in diazepam-dependent male and female rats.

These studies were undertaken to 1) determine whether repeated dosing with the peripheral benzodiazepine antagonist PK 11195 alters its ability to precipitate withdrawal abstinence in diazepam-dependent rats; 2) whether the administration of PK 11195 and the central benzodiazepine antagonist, flumazenil, 3 days apart to the same rat produces an ordering effect in the intensity of withdrawal abstinence; 3) whether there are gender differences in these effects. Age-matched male and female Sprague Dawley rats had capsules implanted weekly that contained approximately equal (mg/kg) doses of diazepam (120 and 90 mg, respectively) or empty capsules (controls). After 5 implants, the maximum precipitated withdrawal score (PAS(MAX)) induced by PK 11195 and/or flumazenil (10 mg/kg/IV, respectively) was measured. Repeated administration of PK 11195 (1x/day for 5 days) induced tolerance with regard to the intensity of the PAS(MAX) and with gender-related differences. When PK 11195 was administered weekly (5 weeks), rather than daily, tolerance did not develop in either sex. The PK 11195- and flumazenil-induced PAS(MAX) was not changed by the order in which they were administered. There were gender differences in that females had a higher PAS(MAX) after flumazenil than after PK 11195 and vocalized more after all treatments than males.

Precipitated withdrawal in the substantia nigra in diazepam-dependent female rats.

Female rats were exposed to diazepam (DZ) implants (90 mg/week) or to empty capsules (controls) for 5 weeks. Rats were focally injected (1 microl) into the substantia nigra (SNR) with the central (CBR) and peripheral (PBR) benzodiazepine receptor antagonists, flumazenil [(FLU) 6.25, 12.5, or 25 microg], and PK 11195 [(PK) 3.125, 6.25, 12.5, or 25 microg], respectively. Rats were observed for behavioral and EEG manifestation of withdrawal syndrome. In female rats, both FLU and PK induced a dose-related precipitated abstinence score (PAS), tachypnea, and head bobbing. Twitches and jerks tended to increase with increasing dose of both FLU and PK. Furthermore, FLU evoked dose-related turning and head and body tremors. The FLU- and the PK-induced PAS were accompanied by an increase in total power of the EEG in the SNR. The involvement of the CBR and PBR in physical dependence on DZ in the SNR is suggested. The present data in female rats are discussed with regard to similarities and differences with previous studies in male rats.

Diazepam-treated female rats: flumazenil- and PK 11195-induced withdrawal in the hippocampus CA1.

Six female rats had a loading dose of 180 mg of diazepam (DZ) contained in two Silastic capsules implanted in their backs. Thereafter, a single 90-mg capsule was implanted weekly for 4 weeks prior to weekly microinjections of 1 microl of flumazenil (6.25, 12.5, or 25 microg) and PK 11195 (3.125, 6.25, or 12.5 microg) or vehicle into the CA1. Three control rats had empty capsules implanted but received only the high dose of flumazenil after 5 weeks. The time of DZ exposure spanned 8 weeks. Mean steady-state plasma levels of DZ were 1.06 +/- 0.11, and the mean total (DZ + metabolites) was 2.46 microg/ml +/- 0.37. Flumazenil elicited a dose-related precipitated withdrawal score (PAS) in DZ-treated rats (but not in controls) characterized by dose-related increases in convulsive (twitches and jerks), motor and autonomic signs, dose-related increases in the percent of total power in the low frequency (1-4 Hz), and decreases in the high-frequency (18-26 Hz) bands of the EEG recorded from the dentate and the amygdala. PK 11195 produced a dose-related increase in the 4-12 Hz band of the EEG recorded from the CA1, whereas the PAS was mild and not dose-related. However, the 6.25 and 12.5-microg doses elicited a significant PAS that tended to increase with dose. These data indicate that chronic DZ produces dependence, and that in the CA1 it involves the participation of central and possibly peripheral benzodiazepine (BZ) receptors located within this structure.

Effect of activation of 5-HT1A receptors at the ventral medulla on phrenic nerve activity.

The purpose of this study was to determine the effects of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OH-DPAT), on respiratory activity (phrenic nerve activity) following application to the ventral medullary surface in the cat. In addition, in order to determine if the action of 8-OH-DPAT was localized to structures at the ventral medulla, we examined the distribution of [3H]8-OH-DPAT to other brain regions and to the peripheral circulation. 8-OH-DPAT (0.0625-8 micrograms) produced a dose-related increase in respiratory rate when applied at either the intermediate or caudal areas on the ventral surface of the medulla. The maximal change in respiratory rate was 9 +/- 2 and 8 +/- 1 breaths/min at the intermediate and caudal areas, respectively. Integrated phrenic nerve amplitude was not significantly affected at these sites except at the 8 micrograms dose where it was decreased. No change in phrenic nerve activity was observed with 8-OH-DPAT application at the rostral area. [3H]8-OH-DPAT was not found to distribute to other brain regions or to the peripheral circulation following application to the ventral medullary surface. The results of this study suggest that 8-OH-DPAT causes changes in respiratory activity, primarily respiratory rate, by acting on neuronal structures at the ventral surface of the medulla.

Substance P immunoreactive projections to the ventral respiratory group in the rat.

Retrograde tracing (rhodamine beads) combined with immunohistochemistry was used to determine the origin of neurons containing substance P that project to the rostral ventral respiratory group in the rat. Double-labeled neurons (rhodamine beads plus substance P immunoreactivity) were found in the midline caudal raphe nuclei (raphe obscurus, raphe pallidus, and raphe magnus) and in the ventrolateral medulla in the parapyramidal region. The findings of this study suggest that substance P-containing neurons in the caudal raphe nuclei and parapyramidal region project to inspiratory neurons in the rostral ventral respiratory group and may therefore influence their activity.

Analgesic actions of local anesthetics and cobalt chloride in the rat brain stem.

A low-intensity thermally evoked tail avoidance reflex (LITETAR) was used to study changes in nociceptive response produced by local anesthetics and cobalt chloride microinjected into the dorsal posterior mesencephalic tegmentum (DPMT) of conscious rats. Dose-related prolongation of the LITETAR (e.g., analgesia) was observed when lidocaine, cocaine, and bupivacaine were administered into the DPMT. Analgesic actions were also demonstrated when cobalt chloride was microinjected into the DPMT. The analgesic actions of these different neuronal suppressants provide support for the hypothesis that there exists tonic activity of hyperalgesic processes in the rat brain stem.

Immunohistochemistry of central respiratory neurotransmitters.

Significant progress has been made in identifying the nuclei and pathways involved in central control of respiration in mammals. This knowledge provides a framework from which to focus on the question of which putative neurotransmitters may be involved in central respiratory control. Application of chemical neuroanatomical methods, particularly immunohistochemistry, is a powerful approach to this important question. This commentary discusses how immunohistochemical approaches have been used so far to extend our knowledge of central respiratory neurotransmitters and also how they can be further applied to gain new information. The results of immunohistochemical studies provide a rationale for subsequent physiological and pharmacological studies of respiratory neurons where specific actions of putative neurotransmitters can be evaluated.

Ultrastructural evidence for serotonin-immunoreactive terminals contacting phrenic motoneurons in the cat.

The innervation of the phrenic motor nucleus in the cat by serotonin-containing neurons has been studied using retrograde tracing combined with immunohistochemistry at the electron microscope level. It was found that phrenic motoneuron cell bodies and dendrites are contacted by serotonin-immunoreactive synaptic terminals. This finding suggests that the activity of phrenic motoneurons is directly affected by serotonergic neurons.

Characterization of the effects of activation of ventral medullary serotonin receptor subtypes on cardiovascular activity and respiratory motor outflow to the diaphragm and larynx.

The purpose of the present study was to characterize the cardiorespiratory effects of activation of 5-HT1A, 5-HT1B and 5-HT2 receptor subtypes at the intermediate area of the ventral surface of the medulla. The agonists (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OH-DPAT), 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP) and (+/-)-1-(2,5-di-methoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) were used to activate these receptor subtypes, respectively. Application of each drug to the intermediate area produced a different profile of cardiorespiratory effects. 8-OH-DPAT (0.0625-4.0 micrograms) produced dose-dependent hypotension and bradycardia, which were antagonized by the 5-HT1A antagonists spiperone and spiroxatrine. The bradycardia was blocked by bilateral vagotomy. No significant changes in respiratory motor outflow to the larynx or diaphragm were observed after application of 8-OH-DPAT. TFMPP (10-1000 micrograms) also produced a dose-dependent hypotension and bradycardia. However, these effects were not antagonized by spiperone or blocked by vagotomy. A decrease in the amplitude of the recurrent laryngeal and phrenic nerve signals was observed after application of the highest dose of TFMPP. In contrast to the effects of 8-OH-DPAT and TFMPP, DOI (0.3-100 micrograms) produced an increase in blood pressure without any change in heart rate. Recurrent laryngeal and phrenic nerve activities were depressed, as was respiratory rate, after application of DOI. Both the cardiovascular and respiratory effects of DOI were blocked by the 5-HT2 antagonist ketanserin. These results indicate that activation of ventral medullary 5-HT receptor subtypes produces unique effects on cardiorespiratory activity.

Origin of serotonin-containing projections to the ventral respiratory group in the rat.

The major purpose of the present study was to determine the origin of the serotonin-containing neurons which project to the rostral ventral respiratory group in the rat. This was accomplished by using the technique of retrograde tracing with rhodamine-labeled latex microspheres (beads) combined with immunochemistry. The rhodamine-labeled beads were microinjected into electrophysiologically identified groups of inspiratory neurons in the rostral ventral respiratory group to retrogradely label neurons projecting to this site. Immunohistochemical processing of the tissue was then done to determine if serotonin was present in the retrogradely-labeled neurons. Serotonin-containing neurons projecting to the rostral ventral respiratory group were found in the raphe magnus, raphe obscurus, raphe pallidus and in the paraolivary region extending to the ventral medullary surface. No serotonin-containing neurons in more rostrally located raphe nuclei were found to project to the rostral ventral respiratory group. The findings suggest that caudal raphe serotonergic projections may affect the activity of respiratory neurons in the rostral ventral respiratory group. Projections to the rostral ventral respiratory group from other pontomedullary nuclei were also identified. Rhodamine-labeled neurons were found in the area of the Kölliker-Fuse nucleus, lateral and medial parabrachial nuclei, retrofacial nucleus, nucleus ambiguus/retroambigualis, nucleus tractus solitarius, A5 region, nucleus paragigantocellularis lateralis, retrotrapezoid nucleus, area postrema and spinal trigeminal nucleus. The projections to the rostral ventral respiratory group in the rat are similar to those previously described in the cat and suggest a common circuitry for the CNS control of breathing.

Effect of an alkylating analog of prazosin on alpha-1 adrenoceptor subtypes and arterial blood pressure.

Previous studies have shown that a chemically reactive analog of prazosin, SZL-49, reduces the alpha-1 adrenoceptor population by a maximum of 60% (Kusiak et al., 1989). These data support the idea that alpha-1 receptor subtypes exist and only one is sensitive to alkylation by SZL-49. In the present study male rats were injected (i.p.) with SZL-49 (0.5-30 mg/kg) and the effects on [3H]prazosin binding, systemic arterial blood pressure and the pressor response to phenylephrine were assessed 24 hr later. SZL-49 treatment decreased the number of [3H]prazosin sites without affecting receptor affinity. The maximal reduction in binding sites was 60% (32 fmol/mg). At doses of 0.5-, 1-, 5- and 10-mg/kg SZL-49 reduced the receptor number to the same level (83 fmol/mg control; 47-60 fmol/mg treated). Injection of SZL-49 had no effect on resting blood pressure. However, drug treatment (0.5 mg/kg and greater) shifted the phenylephrine dose-response curve to the right. The maximal 1-, 5- and 10-mg/kg SZL-49, the ED50 for phenylephrine was approximately the same. Therefore, over a 20-fold range of SZL-49 concentrations no further reduction in receptor number or phenylephrine ED50 was observed. These data support the idea that S2L-49-sensitive and resistant alpha-1 receptors exist in vivo. The alkylation resistant receptor is capable of maintaining resting blood pressure. Furthermore, both receptors appear to be involved in mediating the increase in blood pressure observed with phenylephrine.

gamma-Aminobutyric acid receptors at the ventral surface of the medulla inhibit respiratory motor outflow to the laryngeal musculature.

The effect of activating gamma-aminobutyric acid (GABA) receptors at the ventral surface of the medulla on the activity of the recurrent laryngeal nerve and phrenic nerve was assessed in the cat. Characteristics of the effects of GABA on the activity of the recurrent laryngeal nerve were compared with those on that of the phrenic nerve which has previously been shown to be inhibited by the application of GABA to the ventral surface of the medulla. Application of GABA (0.017-4.05 mg) to the intermediate area produced a dose-related inhibition of respiratory activity in the recurrent laryngeal nerve, as well as the phrenic nerve, that culminated in apnea. The inhibition in each nerve was seen as a decrease in amplitude of nerve activity with no change in respiratory rate. The onset time, peak time and recovery time from GABA-induced inhibition of activity in the recurrent laryngeal and phrenic nerves were not significantly different. The ED50 value for GABA and its 95% confidence interval for inhibition of the activities of the recurrent laryngeal and phrenic nerves were 0.26 mg (0.19-0.36 mg) and 0.27 mg (0.20-0.37 mg), respectively. Therefore, the potency of GABA for the inhibition of the activity of these nerves was not significantly different. The GABA receptor antagonist, bicuculline (10 micrograms), reversed the inhibition of the activities of both the recurrent laryngeal and the phrenic nerves. The time for return of phasic activity in each nerve after bicuculline was not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of substance P immunoreactivity in phrenic primary afferent neurons.

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to localize the putative neurotransmitter, substance P, in phrenic primary afferent neurons. Fast Blue was injected into the diaphragm and was found to label phrenic primary afferent neurons in sections from the fifth and sixth cervical dorsal root ganglia. The same sections were then treated with antiserum to substance P. A total of 11.4% of labelled phrenic primary afferent neurons contained substance P immunoreactivity. The diameters of the neurons ranged between 17 to 45 microns with a mean size of 29.7 +/- 0.7 microns (N = 81). The results suggest that substance P could be involved in mediating the transmission of sensory information from the diaphragm to the CNS.

Regulation of respiratory motor outflow to the larynx and diaphragm by GABA receptors.

Effects of intracisternal administration of the gamma-aminobutyric acid receptor agonist, muscimol, on recurrent laryngeal and phrenic nerve activities were assessed in the cat. Muscimol (0.1-2.4 micrograms) produced a dose-related inhibition of the respiratory activity in both of these nerves. The inhibition was seen as a decrease in the amplitude of nerve activity with no significant change in respiratory rate. The ED50 values for muscimol and its 95% confidence interval for inhibition of the inspiratory activity in the recurrent laryngeal and phrenic nerves were 0.22 micrograms (0.17-0.29) and 0.27 micrograms (0.19-0.38), respectively. The potency of muscimol for inhibition of these nerve activities was not significantly different. Expiratory activity in the recurrent laryngeal nerve was completely inhibited prior to either the inspiratory activity in this nerve or in the phrenic nerve. Intracisternal administration of the gamma-aminobutyric acid receptor antagonist, bicuculline, rapidly reversed the inhibition in both nerves. Reversal times for inhibition of the inspiratory activities in the recurrent laryngeal and phrenic nerves were not significantly different. Return of inspiratory activity in each nerve always preceded the return of expiratory activity in the recurrent laryngeal nerve. These results demonstrate that CNS gamma-aminobutyric acid receptors are involved in the regulation of respiratory motor outflow to the larynx as well as the diaphragm.

Immunohistochemical localization of serotonin- and substance P-containing fibers around respiratory muscle motoneurons in the nucleus ambiguus of the cat.

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to determine if the putative neurotransmitters, serotonin and substance P, are present around posterior cricoarytenoid muscle motoneurons. Fast Blue was injected into the posterior cricoarytenoid muscle of the larynx. Following a 14-21 day survival time to allow for transport of the dye, the animals were perfusion fixed and the brainstem was removed for analysis under the fluorescence microscope. Retrogradely labeled cell bodies containing Fast Blue were found within the nucleus ambiguus from 0.5 to 3.0 mm rostral to obex. These motoneurons ranged in size from 23 to 38 micron. The same tissue sections containing labeled posterior cricoarytenoid muscle motoneurons were then used to determine the distribution of serotonin and substance P around these motoneurons using the indirect immunofluorescence technique. A dense network of serotonin-containing immunoreactive fibers was found around posterior cricoarytenoid muscle motoneurons. The fibers contained varicosities which were in close proximity, actually appearing to surround these motoneurons. Substance P immunoreactive fibers and varicosities were also found around posterior cricoarytenoid muscle motoneurons. The density and pattern of distribution of the substance P immunoreactivity was similar to that of the serotonin immunoreactivity. These results suggest that these putative neurotransmitters may be involved in influencing the activity of posterior cricoarytenoid muscle motoneurons. Serotonin and substance P are also present around other respiratory motoneurons such as phrenic motoneurons. Therefore, these two neurotransmitters may have a more general role in influencing respiratory motor outflow.

Serotonin-mediated excitation of recurrent laryngeal and phrenic motoneurons evoked by stimulation of the raphe obscurus.

Short-latency averaged responses in the recurrent laryngeal nerve (RLN) and C5 phrenic nerve to electrical stimulation (2.5-80 microA; 2.5-160 Hz; 150 microseconds pulse duration) of the medullary nucleus raphe obscurus (RO) were investigated in anesthetized, paralyzed and artificially ventilated cats. The response evoked in RLN by stimulation within RO was excitatory and consisted of a single peak. Characteristics of this response in RLN were compared with those of the delayed excitatory response in C5 phrenic nerve, which we previously showed to be elicited by stimulation within RO. Mean latency to onset for the excitatory response in RLN was 5.7 +/- 0.3 ms, while the delayed excitatory response in C5 phrenic nerve occurred at 7.0 +/- 0.3 ms. The excitatory response in both nerves could be evoked when stimulation was applied during inspiration as well as during expiration. The stimulus threshold varied between 2.5 and 5 microA for evoking the inspiratory-phase response in each nerve. The magnitude of this response in RLN and in C5 phrenic nerve was directly related to current intensity and was dependent upon stimulus frequency. Intravenous administration of the serotonin receptor antagonist, methysergide (0.1-2.4 mg/kg) caused significant dose-related reductions in the response in each nerve. In summary, characteristics of the evoked response in RLN and phrenic nerve are similar in several ways. Both responses are: (1) excitatory in nature, (2) elicited at small stimulus currents, (3) affected similarly by increasing stimulus current and frequency, (4) elicited by stimulation during inspiration and expiration, and (5) mediated at least in part by activation of pathways using serotonin as a neurotransmitter.

Involvement of serotonin in the excitation of phrenic motoneurons evoked by stimulation of the raphe obscurus.

Short-latency averaged responses in the C5 phrenic nerves to electrical stimulation (2.5-80 microA; 5-80 Hz; 150 microseconds pulse duration) of raphe pallidus (RP) and raphe obscurus (RO) were investigated in anesthetized, paralyzed, and artificially ventilated cats. The responses to stimulation of RO were excitatory, whereas a mixture of inhibitory and excitatory responses of lesser magnitude were observed after stimulating in RP. The maximal response was obtained from the ventral part of RO and consisted of early and delayed excitatory responses that were of equal magnitude in both left and right C5 phrenic nerve roots. The mean latency for the early response was 2.5 +/- 0.1 msec and for the delayed response was 7.0 +/- 0.2 msec. Both responses were elicited during inspiratory phase stimulation, but only the delayed response was present during expiratory phase stimulation. The stimulus threshold of the early response was 5 microA; the delayed response was elicited at currents as small as 2.5 microA. Early and delayed responses were affected in different ways by increasing stimulus current and by increasing stimulus frequency. Intravenous administration of serotonin receptor antagonists methysergide (0.1-0.7 mg/kg), metergoline (33-244 micrograms/kg), and cinanserin (1.5-9.0 micrograms/kg) caused significant dose-related reductions in the magnitude of the delayed response, but did not significantly affect the early response. These data suggest that the early and delayed excitatory responses are mediated by different neuronal pathways. The early response does not involve serotonin release, while the later response is mediated at least in part by activation of a serotonergic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Central respiratory stimulation produced by thyrotropin-releasing hormone in the cat.

Thyrotropin-releasing hormone (TRH) was administered intracerebroventricularly and it's effects on respiration were evaluated in the alpha-chloralose anesthetized cat. Respiratory activity was measured using a Fleisch pneumotachograph to monitor tracheal airflow. TRH (0.28-28 nmol) caused an elevation in respiratory minute volume which was due to an increase in respiratory rate with no effect on tidal volume. The site of TRH-induced tachypnea was in the hindbrain as both injections into the cisterna magna and the fourth ventricle produced similar effects. No changes in respiratory activity were seen when TRH injection was restricted to the lateral and third ventricles (forebrain). Furthermore, systemic administration of TRH (28 nmol) produced no significant respiratory effects. The active analogue, [3-Me-His2]-TRH (2.7 nmol) produced the same respiratory effects as TRH. The inactive analogue, TRH free acid (28-280 nmol), caused no significant change in respiratory activity. The data suggest that TRH interacts with a specific receptor in the hindbrain of the cat to affect respiration.

Effect of electrical and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat.

The effect of electrical and chemical (L-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18-144 microA; 5-40 Hz; 100 microseconds pulse duration) resulted in significant (P less than 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were 47 +/- 17% and 146 +/- 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage, L-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P less than 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of 44 +/- 13% at 100 nmol and 80 +/- 13% at 200 nmol L-glutamate. No significant increase in respiratory frequency was observed with L-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.