Recent Abstracts

Hengen KB, Johnson SM, Carey HV, Behan M. Neural control of cardiorespiratory function in ground squirrels during hibernation. The FASEB Journal, Abstract #965.15, 2007.

Torpor and hibernation are among the most extreme examples of energy conservation and CNS plasticity in endotherms. During torpor in 13-lined ground squirrels (Spermophilus tridecemlineatus), respiratory rate decreases 98% and heart rate drops to ~ 1% of the aroused state. Despite the overall suppression of neuronal activity during torpor, cardiorespiratory function is carefully maintained and restored to normal levels during interbout arousals (IBA). Little is known of the central mechanisms that regulate autonomic function in hibernators. Thus, we are investigating neuronal populations in the brainstem that control cardiorespiratory function across the hibernation cycle. We used immunocytochemical detection of FOS, a marker of neuronal activity to identify and neurochemically characterize cardiorespiratory control centers in brainstem from summer squirrels and in specific hibernation states. Preliminary results indicate greater FOS activity in caudal raphe of torpid vs. summer squirrels; FOS was expressed in parabrachial nuclei in IBA squirrels, and some FOS-positive neurons in hibernators expressed 5-HT immunoreactivity. We speculate that cardiorespiratory nuclei remain active during torpor and demonstrate state-associated changes in neuronal activity patterns as animals cycle into and out of torpor.

 

Sladky KK, Paul-Murphy J, Miletic V, Kinney M, Klauer J, Johnson SM. Antinociceptive efficacy and respiratory effects of butorphanol and morphine in three reptile species. Proceedings, Association of Reptilian and Amphibian Veterinarians Annual Conference, 2007, pp. 51-52.

Managing pain in reptile species remains anecdotal, as few systematic studies evaluating analgesic efficacy, pharmacodynamics, and physiologic effects have been published. Butorphanol tartrate, a mixed opioid agonist/antagonist with k-agonist activity, is the most widely used analgesic drug in reptile medicine.5 However, there are no clinical data to substantiate its analgesic effect in reptiles. In contrast, morphine, an opioid with µ-agonist activity, attenuates behavioral responses to noxious thermal stimuli in anole lizards and crocodiles.2,3 Opioid drugs can cause profound respiratory depression in many species.4 For reptiles, µ-opioid receptor activation abolishes respiratory motor output in isolated turtle brainstems.1 The objectives of this study were to: 1) determine the effects of morphine sulfate and butorphanol tartrate on nociceptive behaviors in adult, red-eared slider turtles (Trachemys scripta), bearded dragons (Pogona vitticeps) and corn snakes (Elaphe guttata) using a thermal hind limb withdrawal latency test; and 2) evaluate effects of morphine and butorphanol on respiration in these three reptile species.

Infrared heat stimuli were applied to the plantar surface of turtle and bearded dragon hindlimbs, and to the ventral tail surface of corn snakes. Thermal withdrawal latencies were measured before and after subcutaneous administration of physiologic saline, butorphanol tartrate (2.8 or 28 mg/kg), or morphine sulfate (1.5, 6.5 or 20 mg/kg). Ventilation was measured in freely swimming turtles, restrained bearded dragons and corn snakes, before and after subcutaneous administration of physiologic saline, butorphanol tartrate, or morphine sulfate. Thermal withdrawal latencies sampled at 1, 2, 4, 8, and 24 hr post-injection were no different in reptiles receiving saline or either dose of butorphanol. However, hind limb thermal withdrawal latencies increased in all three reptile species after administration of morphine sulfate, indicating that morphine provided analgesia in these species. Ventilation was measured in freely swimming turtles, bearded dragons, and corn snakes, before and after subcutaneous administration of physiologic saline, butorphanol tartrate or morphine sulfate. Preliminary results suggest that saline had no significant effect on ventilation, while both butorphanol and morphine depressed ventilation. Butorphanol tartrate, the most widely used analgesic in reptiles, may not provide adequate analgesia in red-eared slider turtles, bearded dragons and corn snakes. However, morphine sulfate appears to be an effective analgesic in all three reptile species.

ACKNOWLEDGEMENTS

Supported by grants from the Morris Animal Foundation, Englewood, CO, 80112, and the American College of Laboratory Animal Medicine Foundation. The authors acknowledge Robert Creighton for his excellent technical assistance, and Claudia Hirsch and the animal care staff at the Charmany Research Facility for animal care and logistical assistance.

LITERATURE CITED

1. Johnson, S.M., J.E.R. Wilkerson, M.R. Wenninger, D.R. Henderson, and G.S. Mitchell. 2002. Role of synaptic inhibition in turtle respiratory rhythm generation. J Physiol (Lond). 544: 253-265.

2. Kanui, T.I. and K. Hole. 1992. Morphine and pethidine antinociception in the crocodile. J Vet Pharmaol Therap. 15: 101-103.

3. Mauk, M.D., R.D. Olson, G.J. LaHoste, and G.A. Olson. 1981. Tonic immobility produces hyperalgesia and antagonizes morphine analgesia. Science. 213: 353-354.

4. Pascoe, P,J. 2000. Opioid analgesics, In Matthews, K.A. (ed.), The Veterinary Clinics of North America, Small Animal Practice: Management of Pain, Vol. 30. WB Saunders Co, Philadelphia, Pp. 757-772.

5. Read, M.R. 2004. Evaluation of the use of anesthesia and analgesia in reptiles. J Amer Vet Med Assoc. 224 (4): 547-552.

 

Johnson SM. Reptilian respiratory rhythm generation: insights from in vitro and in vivo studies on turtles. The Physiologist, 49: C1-13, 2006.

Since reptiles represent a phylogenetic intermediate between amphibians and mammals, our goal is to understand how the turtle respiratory central pattern generator (CPG) produces rhythmic motor activity and identify mechanisms that may be conserved in vertebrates. Currently, we are testing whether pacemaker properties are required for rhythm generation, and whether the CPG is composed of coupled oscillatory neural networks. In adult turtle brainstems in vitro, rhythmic hypoglossal motor output persists during synaptic inhibition blockade, suggesting that pacemaker neurons or neural networks with pacemaker-like properties generate the rhythm. Although pacemaker neurons or networks have not yet been identified, pacemaker currents such as Ca-activated cation currents (rather than persistent Na currents) appear to be required for rhythmic activity. Since adjacent transverse slices of turtle brainstems produce rhythmic activity that is abolished by opioids and high bath pH (respiratory depressants), the turtle respiratory CPG appears to be composed of coupled oscillatory networks. However, in awake, freely swimming turtles, mu-opioid receptor activation decreases breathing frequency without any evidence yet for uncoupling of expiratory and inspiratory oscillatory networks (which occurs in mammals). Thus, our data are consistent with the hypothesis that the turtle respiratory CPG is composed of coupled oscillatory networks that contain neurons or networks with pacemaker properties. (NSF IOB-0517302)

REFERENCES:

Feldman JL, Del Negro CA. Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7:232-42, 2006.

Evaluates current hypotheses for respiratory rhythm generation.

Milsom WK, Chatburn J, Zimmer MB. Pontine influences on respiratory control in ectothermic and heterothermic vertebrates. Respir Physiol Neurobiol 143:263-280, 2004.

Reviews organization and evolution of respiratory CPG in non-mammalian vertebrates.

Johnson SM, Wilkerson JER, Wenninger MR, Henderson DR, Mitchell GS. Role of synaptic inhibition in turtle respiratory rhythm generation. J Physiol (Lond) 544: 253-265, 2002.

Rhythmic motor activity persists during synaptic inhibition blockade.

 

Majewski, DJ, Isaacson JS, Kinney ME, Johnson SM. Frequency plasticity is expressed in turtle hemibrainstems in vitro. The FASEB Journal, 20: A370-A371, 2006.

Thick transverse slices of turtle brainstems in vitro produce rhythmic motor activity that is abolished by DAMGO (µ-opiate receptor agonist) or high pH conditions, suggesting that the turtle respiratory network is composed of oscillatory networks distributed rostrocaudally in the brainstem. Our goal was to test whether hemibrainstems produce respiratory-related motor activity, and whether hemibrainstems express frequency plasticity. Brainstems of adult turtles (Pseudemys) were isolated and suction electrodes attached to XII cranial nerve roots to record respiratory-related motor bursts. Brainstems were completely cut along the midline to produce two hemibrainstems. Burst frequency in hemibrainstems (n=9) was 0.43 ± 0.09 and 0.48 ± 0.09 bursts/min at 2 hr and 6 hr post-hemisection, respectively. In different hemi-brainstems, DAMGO (1 µM; n=4) or high pH (pH=7.8; n=2) reversibly abolished the bursts. During synaptic inhibition blockade with strychnine and bicuculline (50 µM each; n=6), rhythmic bursts persisted for >2 hr. Since the respiratory rhythm of intact turtle brainstems responds almost identically to these perturbations, this suggests that the hemibrainstem rhythm is respiratory-related. When phenylbiguanide (5-HT3 agonist; 20 µM; 60-min) was applied to hemibrainstems (n=6), burst frequency acutely increased by 160 ± 35% above baseline (0.38 ± 0.06 bursts/min) and was 47 ± 10% above baseline after a 2-hr washout period, thereby demonstrating frequency plasticity. Thus, a respiratory-related neural network sufficient to express frequency plasticity is contained within the turtle hemibrainstem.

(Supported by NSF grant # IOB-0517302)