Wilma A. Spurrier

Further studies on opioids and hibernation: Delta opioid receptor ligand selectively induced hibernation in summer-active ground squirrels

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu, kappa, and delta opioid receptors into summer-active ground squirrels (Citellus tridecemlineatus). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala2-D-Leu5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occurring mu and kappa opioids may play an important role in the arousal state of hibernation.

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks.

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5, 000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient ex...Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.

Several blood and circulatory changes in the hibernation of the 13-lined ground squirrel, Citellus tridecemlineatus

A monitoring device for use in measuring earth potentials, including a hub carried by a handle unit and having a plurality of spokes extending therefrom. Each spoke has an electrolytic solution cell on its outer end, into which a spoke-mounted electrode extends. The outer end of the cell has a porous tip mounted thereon, which is wetted by the solution and engages the earth. The spokes and the hub are made of conductive material, and a brush-type electrical pickup on the handle unit engages the hub for transmitting signals detected by the wetted porous tips as they progressively engage the earth when the device is rolled along.

The blood-borne “trigger” for natural mammalian hibernation in the 13-lined ground squirrel and the woodchuck☆

A blood-borne trigger for natural mammalian hibernation is described and details of the conditions necessary for its successful utilization are enumerated: 1. 1. It is present in serum and cells of hibernation blood both in ground squirrels (Citellus tridecemlineatus) and woodchicks (Marmota monax). 2. 2. It acts intra and interspecifically insofar as appropriate hibernation serum of 13-lined ground squirrel when transfused can trigger hibernation in other ground squirrels of the same species, and appropriate hibernation serum of woodchucks can similarly trigger hibernation in both woodchucks and in 13-lined ground squirrels. Withdrawal, preparation, and transfusions follow a careful technique outlined in References 1 and 2. 3. 3. The whole blood, serum, and cells retain the hibernation-inducing power for at least 6 months whether kept at freezer temperature (−15 °C) or liquid nitrogen temperature (−196 °C). 4. 4. The trigger is effective whether used as serum or as dialyzate of that serum (prepared as described). The nondialyzed residue was not effective, and evidence has been found which indicates that an antitrigger may be present in that residue. 5. 5. Cold adaptation may be contraindicated for trigger effectiveness. 6. 6. The titer of trigger in a donors blood evidently increases as a bout of hibernation lengthens. This is noted by the fact that trigger taken from a donor long in hibernation induces hibernation more rapidly in recipients than does trigger from a donor in a short bout of hibernation. 7. 7. Trigger manifests its effect in winter whether a recipient animal is placed in a colddark or a warm window-lit room. (Trigger effect in a warm window-lit room in a nonhibernating season has not as yet been studied.) 8. 8. Trigger has not been found either in the blood of active or of aroused animals. Thus, effective material is not circumscribed by the expression “winter blood,” but rather by the expression “hibernation blood.” 9. 9. Female animals in all cases are more effective recipients than males, whereas neither sex is a better donor. 10. 10. Recipient animals receiving a trigger and maintained in cold-dark rooms will not show seasonal reversal, rather, have been observed to show bouts of hibernation intermittently for as long as 2 years. They seemingly do not return to the active state. A theoretical approach to hibernation induction is advanced which holds that the trigger is a small molecular material (SM) found in blood taken in hibernation. It is excreted, complexed, and/or metabolized in the arousing process. It may complex with another blood-borne trigger-inhibitor (LM) to form a complex (CM). The entire circannual cycle of hibernation can be related to relative concentrations of SM, LM, and CM. Speculation is further set forth as to hibernation of tissue as contrasted to hibernation of animals. This notion was presaged by others, notably by Lyman, Kayser, and Suomalainen. As Lyman stated (7) “… entrance into hibernation is more than the abandonment of the warm-blooded state.”

Summer hibernation of infant (six week old) 13-lined ground squirrels, Citellus tridecemlineatus

Abstract In an endeavor to more clearly define the physiological action of hibernation trigger (previously identified by the authors) an experiment was devised using infant ground squirrels. In one case, pregnant ground squirrel mothers were transfused intravenously with cold dialysate of serum of hibernating woodchuck, and in another case, infant (1 week to 3 week) squirrels were transfused intraperitoneally with the same material. When isolated and placed in a cold room (15 °C) at 6 weeks post partum , both the infant born of the transfused mothers, as well as the infant which has been transfused directly, hibernated in the summer. This was in contrast to controls of several kind, i.e., animals not injected, or receiving active or arousing woodchuck serum or heat-treated hibernation serum. Some details for a theory, which had been advanced in a previous article (4), were essentially substantiated by this experiment. It is our belief that this represents the first time mammalian hibernation has been observed in infant hibernators.

A circannual cycle in pinealocyte synaptic ribbons in the hibernating and seasonally reproductive 13-lined ground squirrel (Spermophilus tridecemlineatus)

Pinealocyte synaptic ribbons (SR) in the 13-lined ground squirrel were quantified at monthly intervals over a one-year period spanning the hibernating and reproductive cycles of these animals. SR numbers were high (70-90/20,000 microns2) during periods of activity and reproductive quiescence (May-October). With the onset of hibernation (November-December) there was a rapid 6 to 7-fold reduction in SR frequency. During arousal (January-February) and sexual maturation (March-April) there was a gradual increase in SR frequency. The winter decline in SR frequency is consistent with the decline in pineal melatonin during hibernation and supports the hypothesis that pinealocyte SR play an important role in the neurotransduction of melatonin biosynthesis.

Electrical and mechanical sustained automaticity of the isolated heart of the 13-lined ground squirrel: Both circannually and arousing from hibernation

Abstract 1. 1. A unique method is described for monitoring the electrical and mechanical performance of the isolated heart at 5° C. The cardiac tissue alone provides energy for sustained activity without perfusion or oxygenation. 2. 2. Isolated hearts from 239 hibernating and active ground squirrels demonstrated: 1. (a) The resistance to the cold of the isolated myocardium (resulting in longer sustained automaticity) increases as a circannual event. 2. (b) Hearts isolated from animals hibernating naturally in winter, or induced to hibernate in summer, consistently performed electrically for 8 or more hours in the cold, whereas the hearts of active animals stopped after 1 2 –3 hr depending on the season.

Hibernation “trigger” from hibernating woodchucks (Marmota monax) induces physiological alterations and opiate-like responses in the primate (Macacca mulatta)

Abstract 1. 1. Hibernation “trigger” (HT) from the blood of hibernating wood-chucks infused peripherally, or into the cerebral ventricle of the conscious monkey induces hypotherma, aphagia, bradycardia, reduction of creatine clearance, lower urine flow, and opiate -like behavioural modification. 2. 2. Naloxone and naltrexone, opiate antagonists, either reverse or retard these alterations. 3. 3. The “trigger” (HT) appears to be an endogenous opioid-like peptide unique to the hibernator and capable of inducing hibernation. 4. 4. Receptor sites in the brain of the primate (a non-hibernator) do respond to the “trigger” molecule and as a result, depression of physiology and behaviour occur.

Regional distribution of catecholamine histofluorescence in the heart of the 13-lined ground squirrel: Hibernation vs activity

1. In a total of fifty ground squirrel (Spermophilus tridecemlineauts) hearts studied, there was a significant increase in adrenergic catecholamine histofluorescnece in the winter hibernating state compared to the winter non-hibernating state. 2. There was a ssignificant increase in total cardiac noreppinephrine content in winter hibernation 0.44 ± 0.08 μg/g compared to winter non-hibernating 0.27 ±0.06 μg/g, P = <0.001. 3. The cardiac adrenergic to histofuorescence intensity was similar for natural hibernation and summer induced hibernation. 4. The variations in specific distribution and intensity of adrenergic histofluorescence throughout all areas of the heart is described for natural winter hibernation and non-hibernation, as well as summer induced hibernation and activity.