State-dependent changes of brain endogenous opioids in mammalian hibernation

Endogenous opioids belonging to three opioid families were measured in different states of the hibernation cycle in brain of the Columbian ground squirrels. Using high-performance liquid chromatography-EC detection, the hypothalamic and septal concentrations of met-enkephalin were found to be significantly higher (p > 0.05) in the hibernating state than that in the nonhibernating state. In contrast, met-enkephalin content in the medulla decreased significantly during hibernation. Leu-enkephalin content was only increased in the hypothalamus of hibernating animals. Using radioimmunoassay, dynorphin A-like immunoreactivity was observed to increase in the claustrum and striatum, whereas β-endorphin-like peptides showed a significant increase in the hypothalamus during hibernation. It is evident that the changes in endogenous opioids in brain during hibernation are state dependent, type specific and region specific. These specific alterations of various endogenous opioids may imply their different roles in hibernation.     

Autoradiographic determination of changes in opioid receptor binding in the limbic system of the Columbian ground squirrel at different hibernation states

To localize and quantify the state-dependent changes in various opioid receptor subtypes in the limbic system of non-hibernating and hibernating Columbian ground squirrels, quantitative receptor-binding autoradiography was used. Compared to the non-hibernating animals, the binding density of [³H]-[d-Pen^2,5]-enkephalin (DPDPE) to the δ receptor in the lateral septum, CA3, and the hippocampal fissure of the hippocampal formation was significantly decreased in the hibernating ground squirrels. A significant reduction in the binding density of [³H]-[d-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAGO) to μ receptor was also observed in the medial septum and the CA3 region of the hippocampus of the hibernating animals. In contrast, a decrease in [³H]ethylketocyclazocine (EKC) binding to the κ receptor was only observed in the claustrum and CA3 of the hippocampus during hibernation. The differential changes in binding to various opioid receptors suggest that different opioid subtypes may exert different physiological roles in regulating the specific states (entrance, maintenance and arousal) of a hibernation bout.     

Determination of striatal extracellular γ-aminobutyric acid in non-hibernating and hibernating Arctic ground squirrels using quantitative microdialysis

This study determined extracellular concentrations of gamma-aminobutyric acid ([GABA](ecf)) in striatum of non-hibernating and hibernating arctic ground squirrels to test the hypothesis that an increase in [GABA](ecf) was associated with profound CNS depression during hibernation. Quantitative microdialysis procedures were employed to circumvent the effects of low temperature on the relative recovery of the analyte across the dialysis membrane and yielded for the first time quantitative in vivo estimates of [GABA](ecf) in any brain region or any species. Laboratory housed, wild caught Arctic ground squirrels (Spermophilus parryii) were implanted intraperitoneally with radio transmitters that enabled the telemetric monitoring of activity and core body temperature (T(b)) and bilaterally implanted with cranial guide tubes that enabled the implantation of microdialysis probes into the striatum. Striatal [GABA](ecf) was determined in unrestrained, non-hibernating ground squirrels (T(b) range 34.7-38.9 degrees C) and hibernating ground squirrels (T(b) range 2.9-3.9 degrees C) using extrapolation to zero flow and very slow flow microdialysis techniques. The results show that [GABA](ecf) in non-hibernating squirrels was 73 nM and this level was decreased by approximately 50% during hibernation thereby suggesting that an increase in [GABA](ecf) does not play a major role in CNS depression during hibernation. The reduction of [GABA](ecf) parallels a decrease in plasma and CSF [glucose] and may be related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.     

14C]2-deoxyglucose uptake in ground squirrel brain during hibernation

Autoradiographic patterns of [14C]2-deoxyglucose uptake are described throughout the brains of hibernating and euthermic ground squirrels. Autoradiographs of the brains of hibernating animals are generally homogeneous in comparison to euthermic animals; hence, the relative 2-deoxyglucose uptake (R2DGU) of gray to white matter for the majority of the 85 neural structures examined decreases during hibernation. Two categories of structures are identified as potentially important in hibernation: (1) structures that have the highest R2DGU during hibernation (cochlear nucleus, paratrigeminal nucleus, and superior colliculus) and (2) structures that undergo the least reduction in R2DGU in the transition from euthermia to hibernation (suprachiasmatic nucleus and lateral septal nucleus). The percentage of reduction in R2DGU that a structure undergoes in the transition from euthermia to hibernation is proportional to the R2DGU of that structure during euthermia. The suprachiasmatic, paratrigeminal, and cochlear nuclei undergo less of a reduction than would be predicted from this relationship and may be particularly important during hibernation. Sensory nuclei that receive primary afferent projections are among the structures with the highest R2DGU during hibernation. These metabolically active structures may be responsible for the sensitivity of the hibernator to environmental stimuli.     

In vivo microdialysis study on changes in septal dynorphin and β-endorphin activities in active and hibernating Columbian ground squirrels

State-dependent changes in extracellular concentration of endogenous opioids in the septum of Columbian ground squirrels were examined in the hibernating and euthermic states using in vivo microdialysis. The order of estimated extracellular concentration was found to be: hibernating > interbout euthermia > non-hibernating euthermia for dynorphin A and interbout euthermia > hibernating > non-hibernating euthermia for β-endorphin. The apparent turnover rates of dynorphin A during hibernation was 15 times greater than that during euthermic non-hibernation phase and that of β-endorphin was 8-fold greater. These results demonstrate that subfamilies of endogenous opioids may vary differentially in their activities at different stages of an annual hibernation cycle and may reflect their different roles in the regulation of hibernation.     

Elevated arylalkylamine-N-acetyltransferase (AA-NAT) gene expression in medial habenular and suprachiasmatic nuclei of hibernating ground squirrels

Hibernation, an adaptive response for energy conservation in mammals, involves a variety of physiological changes. Melatonin is linked with the regulation of core body temperature and intervenes in generating circadian cycles; its role in seasonal (circannual) rhythms of hibernation is explored here. Melatonin is primarily produced in the pineal gland. Since arylalkylamine-N-acetyltransferase (AA-NAT) is the rate-limiting enzyme for synthesizing melatonin, AA-NAT gene expression was investigated to assess the possible role of melatonin in hibernation. The findings presented here utilized combined in situ hybridization and immunohistochemistry methodologies to evaluate the AA-NAT mRNA expression in brains of both hibernating and non-hibernating ground squirrels. Brains were examined for the expression of AA-NAT mRNA using a oligonucleotide AA-NAT probe; antibody against neurofilament-70 (NF-70) was used as a neuronal marker. All hibernating animals expressed significantly (P<0.01) elevated levels of AA-NAT mRNA in both the epithalamic medial habenular nuclei (MHb) area and the hypothalamic suprachiasmatic nuclei (SCN), which is also known as the master biologic clock. These findings represent the first demonstration of the expression of mRNA encoding for AA-NAT in the extra-pineal (i.e. SCN and MHb) sites of thirteen-lined ground squirrels and indicate that the habenular nucleus may be an important supplementary location for melatonin biosynthesis. The data presented here indicate that AA-NAT gene is one of the few specific genes up-regulated during hibernation and suggest that elevation of its expression in SCN and MHb may play an essential role in the generation and maintenance of hibernation.     

Evaluation of regeneration of nerve and reinnervation of skeletal muscle in the hibernating ground squirrel

The retrograde transport of HRP was used to determine the status of axonal transport in the peroneal and sciatic nerves of hibernating and nonhibernating ground squirrels following crush of the peroneal nerve at 10 to 12 mm (SNS) or sciatic nerve at 33 to 35 mm (LNS) from its entrance into the extensor muscle. The ability of the proximal segment to reestablish axonal continuity and thus neuromuscular transmission was also studied. Two weeks to 3 months after nerve crush the extensor muscles were injected with HRP. We found that during hibernation no axonal transport across the site of crush was seen even after 3 months and that regeneration of the nerve during this period was minimal. Evidence of slight regeneration seen at 90 days could be due to periods of awaking of the animals during their natural hibernation cycle. In these animals HRP deposits were seen only in the nerve distal to crush, i.e., between crush site and muscle. In the nonhibernating squirrels, axoplasmic flow was reestablished at the site of injury as early as 2 weeks after crush, and HRP could be detected in the spinal cord in motoneurons of the ipsilateral ventral horn at spinal levels L₃ to L₅. In one hibernating animal the peroneal nerve was crushed at the distal site (SNS) and also the spinal cord was injured by dropping a weight. After nerve crush and the spinal cord injury the hibernating state could not be maintained and the animal stayed awake 22 days. The time course of regeneration of the nerve in that animal was similar to that seen in nonhibernating squirrels. After nerve crush in nonhibernating animals, reaction product was also found in sensory cell bodies of dorsal root ganglia as well as in terminals in the substantia gelatinosa of the spinal cord at the same levels. Thus, the axonal transport occurs in hibernating and non-hibernating squirrels in both sensory and motor nerve fibers. The extensor muscle fibers of the hibernating squirrels showed substantial membrane depolarization 90 days after crush. Action potentials from these fibers could be obtained from 15 to 35 days only through stimulating the nerve segment distal to the crush. Stimulation of the proximal nerve segment did not evoke muscle activity. These results demonstrate that nerve regeneration was nearly abolished during hibernation and that blockade of axonal transport continued across a region of nerve crush for the duration of the hibernating period.     

Hippocampal and cortical opioid receptor binding: Changes related to the hibernation state

In vitro opioid receptor binding in the dorsal hippocampal formation and parietal cortex was surveyed in ground squirrels (Citellus lateralis) in the contrasting physiological states of hibernation and euthermia (i.e. not hibernating). Computer-assisted autoradiographic analysis of coronal sections incubated with [³H]dihydromorphine (DMH; 4 nM) revealed statistically significant reductions in specific opioid binding associated with hibernation. In the dorsal hippocampal formation of hibernating animals, binding in the striatum radiatum of CA3, hilus of the dentate gyrus and molecular layer of the dentate gyrus exhibited decreases up to 34% compared to euthermic animals. The stratum radiatum of CA3 exhibited the smallest decrease overall. DHM binding in parietal cortex displayed significant hibernation-related reductions, although they were not uniformly observed across all lamine at the 3 different brain levels examined. These experiments present evidence of changes in brain opioid binding related to the mammalian state of hibernation. The results suggest that changes in opioid receptor binding during hibernation may contribute to the earlier reported⁵ apparent failure of morphine physical dependence to develop during hibernation.     

Sciatic nerve response to injury in hibernating and non-hibernating ground squirrels

At 21 and 36 postcrush days, regenerating axonal sprouts were observed and counted in the distal stumps of sciatic nerves from non-hibernating ground squirrels (T_b= 37 °C). More importantly, regenerating axonal sprouts were present in the distal segments of sciatic nerves from hibernating ground squirrels (T_b= 4–10 °C), but the total number was significantly less than the number noted in non-hibernating animals.     

Seasonal and state-dependent changes in brain TRH receptors in hibernating ground squirrels.

Quantitative autoradiography was used to localize and quantify thyrotropin-releasing hormone (TRH) receptors in the brain of hibernating (H), winter euthermic (WE), and summer euthermic (SE) animals to further explore the state-dependent physiological and behavioral effects of TRH in ground squirrels. The pattern of [3H]MeTRH binding (Kd 6.7 +/- 0.1 nM) was heterogeneous and highly concentrated in structures primarily associated with the limbic forebrain. Statistically significant seasonal changes (SE vs. WE) were reflected by decreases in TRH receptor binding in the arcuate nucleus, dorsomedial nucleus, and ventral pallidum of WE animals. Increased binding in WE animals was evident in the suprachiasmatic nucleus and choroid plexus of the lateral ventricles. Significant state-dependent changes (WE vs. H) were characterized by decreases in TRH receptor binding in the hypothalamic paraventricular nucleus, medial preoptic area, ventral tegmental area, and choroid plexus of the lateral ventricles of H animals. Increased binding occurred in the anterior cortical nucleus of the amygdala in H animals. The results suggest that naturally occurring changes in central TRH systems may be important in the mediation of physiological and behavioral processes that undergo seasonal and state-dependent adjustments in hibernating mammals.     

In vivo analysis of dopamine and its metabolites in the caudate nucleus during euthermia and hibernation

Golden-mantled ground squirrels (Citellus lateralis) were chronically implanted with a unilateral push-pull cannula in the caudate nucleus. Perfusates obtained in these unanesthetized, unrestrained animals during the euthermic (non-hibernating) and hibernating states were analyzed for dopamine (DA) and its metabolites (homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 3-methoxy-4-hydroxyphenethanol (MOPET) using high performance liquid chromatography with electrochemical detection. The data revealed clear differences in the performance of the caudate DA system in the two states. During the euthermic state, DA metabolism was indicative of a constant and high turnover rate. Free DA was not detectable in the majority of samples, HVA was detected at consistently high levels, and DOPAC and conjugated DA were present at low levels. By contrast, DA metabolism was sharply altered during hibernation. Free DA was present at high concentrations and HVA concentrations were low. DOPAC was not detected in any sample whereas MOPET was present in all samples. Conjugated DA was present at high concentrations during the second half of the hibernation bout. The shift in the post-release disposition of DA could enhance the stability of DA receptors (i.e. prevent supersensitivity) during the prolonged periods of reduced neural activity typical of hibernation.     

Morphine distribution following infusion into lateral ventricle during hibernation and euthermia

The intracerebral pattern of diffusion of 3H-morphine was studied autoradiographically following continuous infusion (4, 9, and 18 hr; 1 microliter/hr) into the lateral ventricle during hibernation and euthermia (i.e., not hibernating) in ground squirrels (Citellus lateralis). Morphine diffusion into the parenchyma during both states was extensive, resulting in increased autoradiographic optical density of 34 structures examined. The zone of radiolabeled tissue was primarily ipsilateral, and it expanded with increasing duration of infusion. Diffusion into contralateral regions was more evident in hibernation, although the total area of radiolabeled tissue was not significantly greater than that of euthermic animals. The average optical densities of autoradiographs from hibernating brains were significantly greater than those from euthermic animals, suggesting greater accumulation of labeled material during hibernation. These data suggest that neuroactive compounds released into the ventricular space can achieve widespread distribution within the brain during hibernation (in which all physiological parameters are profoundly depressed) as well as during euthermia. Thus, the apparent lack of development of physical dependence to morphine during hibernation is not due to a restricted distribution of morphine in the hibernating brain.     

Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous system as determined by sandwich-enzyme immunoassay

We investigated endogenous levels of a novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the rat central nervous system. The amount of PACAP was measured by means of highly specific and sensitive sandwich-enzyme immunoassay. This assay system following HPLC analysis revealed that PACAP38 was a major portion of the total PACAP immunoreactivity and PACAP27 levels were negligibly low in the brain. Therefore, we measured the amount of PACAP38 in 62 regions punched out from frozen tissue sections. High amounts of PACAP38 were found in the lateral septal nucleus (intermediate part), diagonal band, central amygdaloid nucleus, several parts of the hypothalamus (suprachiasmatic, supraoptic, periventricular and arcuate nuclei), central gray, interpeduncular nucleus and dorsal raphe. The suprachiasmatic, paraventricular and periventricular hypothalamic nuclei showed the highest levels. A moderate amount of the peptide was observed in the lateral septal nucleus (dorsal part), medial septal nucleus, medial amygdaloid nucleus, thalamus (paraventricular, paratenial, central medial, ventromedial, reuniens and rhomboid nuclei), hypothalamus (lateral hypothalamic area and mammillary body), ventral tegmental area, interfascicular nucleus and in the locus coeruleus. Such a distribution of endogenous PACAP38 did not parallel the localization of PACAP binding sites which we had demonstrated recently. Moreover, the topographical distribution of PACAP38 observed in the present study differed from that of VIP which had been previously reported. The present results suggest that PACAP38 may have a neurotransmitter/neuromodulator role which is different from that of VIP in the central nervous system.     

Major changes in the brain histamine system of the ground squirrel Citellus lateralis during hibernation.

Hibernation in mammals such as the rodent hibernator Citellus lateralis is a physiological state in which CNS activity is endogenously maintained at a very low, but functionally responsive, level. The neurotransmitter histamine is involved in the regulation of diurnal rhythms and body temperature in nonhibernators and, therefore, could likely play an important role in maintaining the hibernating state. In this study, we show that histamine neuronal systems undergo major changes during hibernation that are consistent with such a role. Immunohistochemical mapping of histaminergic fibers in the brains of hibernating and nonhibernating golden-mantled ground squirrels (C. lateralis) showed a clear increase in fiber density during the hibernating state. The tissue levels of histamine and its first metabolite tele-methylhistamine were also elevated throughout the brain of hibernating animals, suggesting an increase in histamine turnover during hibernation, which occurs without an increase in histidine decarboxylase mRNA expression. This hibernation-related apparent augmentation of histaminergic neurotransmission was particularly evident in the hypothalamus and hippocampus, areas of importance to the control of the hibernating state, in which tele-methylhistamine levels were increased more than threefold. These changes in the histamine neuronal system differ from those reported for the metabolic pattern in other monoaminergic systems during hibernation, which generally indicate a decrease in turnover. Our results suggest that the influence of histamine neuronal systems may be important in controlling CNS activity during hibernation.     

Distribution of tyrosine-hydroxylase (TH)-immunoreactive neurons in the diencephalon of the pigeon (Columba livia domestica)

The distribution of tyrosine-hydroxylase (TH)-immunoreactive cell bodies and fibers in the diencephalon has been investigated with immunohistological techniques in the pigeon. The results suggest that TH is present in a number of morphologically distinct neuronal systems. Preoptic and hypothalamic TH neurons were subdivided into a medial periventricular and a lateral group. The medial group starts with a rostral collection of small cells in the preoptic region. A significantly larger collection of TH neurons occupies the paraventricular nucleus (PVN) (stratum cellulare internum) and mainly consists of large multipolar cells. Further caudally, the main concentration of cells is in the hypothalamic posteromedial and the periventricular regions of the tuberoinfundibular (arcuate) nucleus. No TH neuron was found in the ventral and lateral parts of the tuberoinfundibular region, suggesting that the prominent tuberoinfundibular dopaminergic system described in mammals is absent in the pigeon. This further substantiated by the relative scarcity of TH immunoreactive fibers and varicosities in the neurohemal zone of the median eminence (ME). The caudalmost components of the medial group appear to be continuous with the large population of TH neurons distributed in the midline of the mesencephalon. Tyrosine-hydroxylase-immunopositive cells have not been found in the paraventricular organ. The lateral group consists of TH neurons loosely arranged in the lateral hypothalamus, including regions of the supraoptic nucleus and hypothalamic posterolateral nucleus. Tyrosine-hydroxylase containing neurons vary widely in size, shape, and dendritic arborization in each diencephalic region. However, it is possible to distinguish two main cell types. Small bipolar neurons with two simple arborizing dendrites were concentrated in the medial periventricular system. The second type of cell is large, multipolar with four to five branching dendrites. This latter cell type occurs mainly in the lateral system and in the PVN. Major fiber bundles containing TH immunoreactivity were identified in the lateral and periventricular hypothalamus. The paraventricular organ and the organum vasculosum laminae terminalis contained the densest arborization of fibers and varicosities. In the ME, dense innervation was found in the subependymal layer. Dense arborizations of TH positive fibers and varicosities were located in the septal nuclei and the paleostriatum augmentatum.     

Temperature sensitivity of axonal transport in hibernating and nonhibernating rodents.

The temperature sensitivity of fast axonal transport of labeled protein in peripheral sensory axons was investigated in rats and hibernating and non-hibernating ground squirrels (Spermophilus r. richardsonii), using an in vitro technique which permitted incubation of the sciatic nerve for various periods of time at different temperatures. The relationship between velocity and temperature was exponential between 13 and 38°C, but below 13°C transport ceased or became too slow to detect. There were no significant differences in the relationship between temperature and velocity for the three classes of nerve studied. It is concluded that the peripheral nerves of hibernating ground squirrels are not modified to permit axonal transport to continue at the low body temperatures characteristic of the hibernating state.     

In vitro measurements of cholinergic activity in brain regions of hibernating ground squirrels

High affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity were measured in synaptosomal P2 fractions from four brain regions in a mammalian hibernator, the golden-mantled ground squirrel. The 14CO2 evolution from [6-14C]glucose was also measured. Comparisons were made across the euthermic (not hibernating) and hibernation state in synaptosomes from cortex (CTX), preoptic area and hypothalamus (POA/HYP), olfactory apparatus (OA), and hippocampus (HPC). HACU was significantly increased in the CTX, from hibernating ground squirrels compared to euthermic animals. ChAT activity was significantly increased in the CTX and OA from hibernating animals. No change in either cholinergic marker was evident for the POA/HYP and HPC. The evolution of 14CO2 from [6-14C]glucose was generally, though not significantly, higher for synaptosomes from euthermic animals compared to hibernating animals. The results are discussed with reference to the involvement of cholinergic mechanisms in the control of hibernation.     

Biochemical alterations in denervated skeletal muscle and transected sciatic nerve in the hibernating ground squirrel

Biochemical intermediates associated with energy metabolism were measured in innervated and denervated anterior tibialis muscles of nonhibernating ground squirrels to gain insight into mechanisms associated with changes that occur during hibernation and subsequent to denervation. Steady-state concentrations of glucose, glucose 6-phosphate, and lactate in the innervated muscles were decreased 42, 75, and 78%, respectively, during hibernation. Upon awakening, the concentration of glucose was increased approximately two-fold and that of lactate was elevated about 10-fold and glucose-6-phosphate more than 15-fold. These findings were interpreted as indicating that glycolysis was reduced during hibernation and elevated suddenly and markedly immediately after hibernation. A similar effect was noted in denervated muscles of hibernating animals. The reduction in concentrations of glycolytic intermediates of innervated and denervated muscles during hibernation was accompanied by a decrease in the total reserves of high-energy phosphate; however, ATP : ADP ratios were not changed significantly. Denervation produced significant decreases in concentrations of glucose 6-phosphate and significant increases in glutamate in muscles of hibernating and nonhibernating animals. Decreases in glucose 6-phosphate were accompanied by significant decreases in fructose 1,6-diphosphate in denervated muscles of hibernating animals. Subsequent to denervation, phosphocreatine increased by 55% in muscles from hibernating animals but did not change in muscles from nonhibernating animals. The changes in phosphocreatine in denervated muscles from hibernating animals are accompanied by an increase in the total energy reserves of these muscles. Metabolic changes in muscles from hibernating animals appear to occur in the absence of any degeneration of transected sciatic nerves. High energy phosphate as well as choline acetylase and acetylcholinesterase are maintained at normal activities in nerves from hibernating squirrels for at least 3 weeks subsequent to separation of axons from nerve cell bodies.     

Distribution of dopamine immunoreactivity in the forebrain and midbrain of the snake Python regius: a study with antibodies against dopamine

The distribution of dopamine (DA) immunoreactivity in the forebrain and midbrain of the ball python, Python regius, was studied by using recently developed antibodies against DA. In order to determine general and species-specific features of the DA system in reptiles, we have selected the ball python as a representative of a reptilian radiation that hitherto has not been the subject of (immuno)histochemical studies. Dopamine-containing cell bodies were found around the glomeruli and in the external plexiform layer of both the main and accessory olfactory bulb, but not in the telencephalon proper. In the diencephalon, DA cells were observed in several parts of the periventricular hypothalamic nucleus, in the periventricular organ, the ependymal wall of the infundibular recess, the lateral hypothalamic area, the magnocellular ventrolateral thalamic nucleus, and the pretectal posterodorsal nucleus. In the midbrain, DA cells were found in the ventral tegmental area, the substantia nigra, and the presumed reptilian homologue of the mammalian A8 cell group. Dopaminergic fibers and varicosities were observed throughout the whole brain, particularly in the telencephalon and diencephalon. The nucleus accumbens, striatum, olfactory tubercle, and nucleus of the accessory olfactory tract appear to have the most dense innervation, but the lateral septal nucleus, the dorsal ventricular ridge, and the nucleus sphericus also show numerous DA-containing fibers and varicosities. Except for the lateral cortex, cortical areas are not densely innervated by DA fibers. The DA system of the snake Python regius shares many features with that of lizards and turtles as determined with the same antibodies. The taxonomically close relationship between lizards and snakes, which together constitute the Squamata, is reflected in a similar distribution of DA fibers and varicosities to the dorsal ventricular ridge and the lateral cortex, and in the limited number of CSF-contacting DA neurons in the hypothalamus.     

Slow axoplasmic transport in the hibernating and nonhibernating ground squirrel

Our objectives were to examine the effects of temperature upon slow axoplasmic transport in crushed and intact sciatic nerves of hibernating and nonhibernating ground squirrels (Spermophilus tridecemlineatus). Tritiated leucine was injected into the spinal cord near the ventral horn cells of the sciatic nerve and, after protein incorporation, allowed to transport for 5, 10, 15, or 20 days. Slow transport profiles were obtained from crushed and intact sciatic nerves of hibernating and nonhibernating animals. No significant differences (P < 0.05) were demonstrated in the transport velocities between crushed and noncrushed nerves in either hibernating or nonhibernating ground squirrels. Furthermore, no significant differences were found in transport velocities between hibernating and nonhibernating groups of animals. Slow axoplasmic transport velocities are not only unaffected by crush injury in this system, but also unaffected by normally lethal temperatures. This suggests that the slow component in the nerves of hibernating animals may be responsible for maintaining neurotrophic control throughout hibernation. The data also suggest that different mechanisms may be involved in slow versus fast axoplasmic transport.