Depletion of catecholaminergic neurons of the rostral ventrolateral medulla in multiple systems atrophy with autonomic failure

The ventrolateral portion of the intermediate reticular formation of the medulla (ventrolateral medulla, VLM), including the C1/A1 groups of catecholaminergic neurons, is thought to be involved in control of sympathetic cardiovascular outflow, cardiorespiratory interactions, and reflex control of vasopressin release. As all these functions are affected in patients with multiple systems atrophy (MSA) with autonomic failure, we sought to test the hypothesis that catecholaminergic (tyrosine hydroxylase [TH]-positive) neurons of the VLM are depleted in these patients. Medullas were obtained at autopsy from 4 patients with MSA with prominent autonomic failure and 5 patients with no neurological disease. Patients with MSA had laboratory evidence of severe adrenergic sudomotor and cardiovagal failure. Tissue was immersion fixed in 2% paraformaldehyde at 4°C for 24 hours and cut into 1-cm blocks in the coronal plane from throughout the medulla. Serial 50-μm sections were collected and one section every 300 μm was stained for TH. There was a pronounced depletion of TH neurons in the rostral VLM in all cases of MSA. There was also significant reduction of TH neurons in the caudal VLM in 3 MSA patients compared with 3 control subjects. In 2 MSA cases and in 2 control subjects, the thoracic spinal cord was available for study. There was also depletion of TH fibers and sympathetic preganglionic neurons (SPNs) in the 2 MSA cases examined. Thus, depletion of catecholaminergic neurons in the VLM may provide a substrate for some of the autonomic and endocrine manifestations of MSA.     

Delayed injury of hippocampal interneurons after neonatal hypoxia‐ischemia and therapeutic hypothermia in a murine model

Delayed hippocampal injury and memory impairments follow neonatal hypoxia‐ischemia (HI) despite the use of therapeutic hypothermia (TH). Death of hippocampal pyramidal cells occurs acutely after HI, but characterization of delayed cell death and injury of interneurons (INs) is unknown. We hypothesize that injury of INs after HI is: (i) asynchronous to that of pyramidal cells, (ii) independent of injury severity, and (iii) unresponsive to TH. HI was induced in C57BL6 mice at p10 with unilateral right carotid ligation and 45 min of hypoxia (FiO₂ = 0.08). Mice were randomized to normothermia (36 °C, NT) or TH (31 °C) for 4 hr after HI and anesthesia‐exposed shams were use as controls. Brains were studied at 24 hr (p11) or 8 days (p18) after HI. Vglut1, GAD65/67, PSD95, parvalbumin (PV) and calbindin‐1 (Calb1) were measured. Cell death was assessed using cresyl violet staining and TUNEL assay. Hippocampal atrophy and astroglyosis at p18 were used to assess injury severity and to correlate with number of PV + INs. VGlut1 level decreased by 30% at 24 hr after HI, while GAD65/67 level decreased by ～50% in forebrain 8 days after HI, a decrease localized in CA1 and CA3. PSD95 levels decreased in forebrain by 65% at 24 hr after HI and remained low 8 days after HI. PV + INs increased in numbers (per mm²) and branching between p11 and p18 in sham mice but not in NT and TH mice, resulting in 21–52% fewer PV + INs in injured mice at p18. Calb1 protein and mRNA were also reduced in HI injured mice at p18. At p18, somatodendritic attrition of INs was evident in all injured mice without evidence of cell death. Neither hippocampal atrophy nor astroglyosis correlated with the number of PV + INs at p18. Thus, HI exposure has long lasting effects in the hippocampus impairing the development of the GABAergic system with only partial protection by TH independent of the degree of hippocampal injury. © 2018 Wiley Periodicals, Inc.     

Mild Prolonged Hypothermia for Large Intracerebral Hemorrhage

Background

Perihemorrhagic edema (PHE) develops after intracerebral hemorrhage (ICH). It can worsen the clinical situation by its additional mass effect. Therapeutic hypothermia (TH) might be an effective method to control PHE, but has not been sufficiently studied in ICH patients.

Methods

We report data on n = 25 consecutive patients with large supratentorial ICH (volume > 25 ml) who were treated by mild TH of 35 °C for 8–10 days. Body temperature was controlled by endovascular cooling catheters. We followed the clinical course during hospital stay and measured volumes of ICH and PHE in regularly performed serial cranial computed tomography. Outcome was assessed after 3 and 12 months. These data were compared to a historical group of n = 25 patients with large ICH.

Results

While PHE continuously increased in the historical control group up to day 10, PHE volumes in the hypothermia group remained stable. There was a significant difference from day 3 after symptom onset. Shivering (36 %) and pneumonia (96 %) were the most frequent complications during TH. Mortality rate was 8.3 % in TH versus 16.7 % in the control group after 3 months and 28 versus 44 % after 1 year.

Conclusions

These data support the promising results of our first case series on TH in large ICH. TH prevents the development of PHE and its complications. Side effects of TH appeared often, but could be treated sufficiently. Therefore, TH might represent a new therapy for PHE after large ICH, but has to be further tested in randomized trials.     

Combination therapy of normobaric oxygen with hypothermia or ethanol modulates pyruvate dehydrogenase complex in thromboembolic cerebral ischemia

Pyruvate dehydrogenase complex (PDH) is a brain mitochondrial matrix enzyme. PDH impairment after stroke is particularly devastating given PDH's critical role in the link between anaerobic and aerobic metabolism. This study evaluates the restoration of oxidative metabolism and energy regulation with a therapeutic combination of normobaric oxygen (NBO) plus either therapeutic hypothermia (TH) or ethanol. Sprague‐Dawley rats were subjected to middle cerebral artery occlusion with an autologous embolus. One hour after occlusion, tissue‐type plasminogen activator (t‐PA) was administered alone or with NBO (60%), EtOH (1.0 g/kg), or TH (33°C), either singly or in combination. Neurological deficit score and infarct volume were assessed 24 hr after t‐PA‐induced reperfusion. PDH activity and reactive oxygen species (ROS) levels were measured 3 and 24 hr after t‐PA. Western blotting was used to detect PDH and pyruvate dehydrogenase kinase (PDK) protein expression. After t‐PA in ischemic rats, NBO combined with TH or EtOH most effectively decreased infarct volume and neurological deficit. The combined therapies produced greater increases in PDH activity and protein expression as well as greater decreases in PDK expression. Compared with the monotherapeutic approaches, the combined therapies provided the most significant declines in ROS generation. Reperfusion with t‐PA followed by 60% NBO improves the efficacy of EtOH or TH in neuroprotection by ameliorating oxidative injury and improving PDH regulation. Comparable neuroprotective effects were found when treating with either EtOH or TH, suggesting a similar mechanism of neuroprotection and the possibility of substituting EtOH for TH in clinical settings. © 2016 Wiley Periodicals, Inc.     

Hypothermic Treatment for Acute Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition that affects approximately 11,000 patients each year in the United States. Although a significant amount of research has been conducted to clarify the pathophysiology of SCI, there are limited therapeutic interventions that are currently available in the clinic. Moderate hypothermia has been used in a variety of experimental and clinical situations to target several neurological disorders, including traumatic brain and SCI. Recent studies using clinically relevant animal models of SCI have reported the efficacy of therapeutic hypothermia (TH) in terms of promoting long-term behavioral improvement and reducing histopathological damage. In addition, several clinical studies have demonstrated encouraging evidence for the use of TH in patients with a severe cervical spinal cord injury. Moderate hypothermia (33°C) introduced systemically by intravascular cooling strategies appears to be safe and provides some improvement of long-term recovery of function. TH remains an experimental clinical approach and randomized multicenter trials are needed to critically evaluate this potentially exciting therapeutic intervention targeting this patient population.     

A chimeric tyrosine/tryptophan hydroxylase

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t_1/2 of 84 min at 37°C, TPH was much less stable (t _1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at _1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.     

Nerve,muscle, and neuromuscular junction electrophysiology at high temperature

Although the effect of low temperature on the peripheral nervous system has been systematically studied, the effect of high temperature has not. We investigated the effect of elevating limb temperature from 32°C to 42°C by performing sequential motor studies, antidromic sensory studies, and 3-Hz repetitive stimulation in normal subjects. In addition, we recorded single motor units by using threshold stimulation. On average, motor amplitude and duration decreased by 27% and 19%, respectively, whereas sensory amplitude and duration decreased by 50% and 26%, respectively. Neuromuscular transmission remained normal at 42°C. Single motor unit recordings revealed a reduction in amplitude of 26%, similar to the overall reduction in compound motor amplitude. These findings demonstrate that significant reductions in sensory and motor amplitudes can occur in normal nerves at high temperature; we hypothesize that these changes are secondary to alterations in nerve and muscle ion channel function. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 431–436, 1997     

Effects of septal lesions and chronic estrogen treatment on dopamine,GABA and lordosis behavior in male rats

GORDON, J. H., D. M. NANCE, C. J. WALLIS AND R. A. GORSKI. Effects of septal lesions and chronic estrogentreatment on dopamine, GABA and lordosis behavior in male rats. BRAIN RES. BULL. 4(1) 85–89, 1979.—Septal lesions (SL) in female rats result in an increased sensitivity to the behavioral effects of acute estradiol benzoate (ACUTE-EB; 2 μg/day × 3) treatment as measured by the lordosis quotient (LQ; number of lordotic responses × 100/number of mounts). Male rats, intact or castrated, do not show this enhanced behavioral response to ACUTE-EB unless they are treated with EB (2 μg/day) for 2–4 weeks immediately following the production of SL. The present study was undertaken to examine possible neurochemical alterations which could account for the enhanced behavioral sensitivity to ACUTE-EB seen in the SL male rat treated chronically with EB during the postlesion period (SL-EB). Three groups, normal males, SL-EB and SL males chronically treated with oil (SL-oil), were subdivided and treated with ACUTE-EB or oil and decapitated. The brains were removed, frozen and stored at ?50°C prior to dissection and assay. Tyrosine hydroxylase (TH) activity was assayed in the dopamine (DA) rich areas of the forebrain (striatum, STR; nucleus accumbens septi, ACB; and olfactory tubercle). The TH activity was significantly suppressed in both the STR and ACB of the SL-EB males treated with ACUTE-EB. The glutamic acid decarboxylase (GAD) activity in both the substantia nigra and ventral tegmentum was significantly increased in the SL-EB males given ACUTE-EB relative to that of all other groups. In summary, SL-EB males given ACUTE-EB show (1) an enhanced LQ, (2) decreased TH activity in the region of DA terminals, and (3) increased GAD activity in the region of DA cell bodies. The increase in GAD activity is suggested to be a result of an altered neuronal feedback because of plastic changes that occur during chronic EB treatment following production of SL. This probable increase in inhibitory tone in the region of the DA cell bodies may explain the observation that the SL-EB male exhibits decreased DA turnover following ACUTE-EB treatment. Moreover, since DA may be inhibitory to the display of lordosis behavior, the SL-EB males may show an enhanced LQ, at least partially, because of this reduction in DA activity.     

Cooling of the epileptic focus suppresses seizures with minimal influence on neurologic functions

Purpose: Focal brain cooling is effective for suppression of epileptic seizures, but it is unclear if seizures can be suppressed without a substantial influence on normal neurologic function. To address the issue, a thermoelectrically driven cooling system was developed and applied in free‐moving rat models of focal seizure and epilepsy. Methods: Focal seizures limited to the unilateral forelimb were induced by local application of a penicillin G solution or cobalt powder to the unilateral sensorimotor cortex. A proportional integration and differentiation (PID)–controlled, thermoelectrically driven cooling device (weight of 11 g) and bipolar electrodes were chronically implanted on the eloquent area (on the epileptic focus) and the effects of cooling (20, 15, and 10°C) on electrocorticography, seizure frequency, and neurologic changes were investigated. Key Findings: Cooling was associated with a distinct reduction of the epileptic discharges. In both models, cooling of epileptic foci significantly improved both seizure frequency and neurologic functions from 20°C down to 15°C. Cooling to 10°C also suppressed seizures, but with no further improvement in neurologic function. Subsequent investigation of sensorimotor function revealed significant deterioration in foot‐fault tests and the receptive field size at 15°C. Significance: Despite the beneficial effects in ictal rats, sensorimotor functions deteriorated at 15°C, thereby suggesting a lower limit for the therapeutic temperature. These results provide important evidence of a therapeutic effect of temperatures from 20 to 15°C using an implantable, hypothermal device for focal epilepsy.     

Hypothermia blunts acetylcholine increase in CSF of traumatically brain injured rats

Activation of muscarinic acetylcholine (ACh) receptors contributes to the pathophysiological consequences of moderate experimental traumatic brain injury (TBI). Hypothermia (30°C) provides protection in experimental TBI. We measured ACh levels in CSF and plasma 5 min after moderate fluid percussion TBI under normothermic or hypothermic conditions, because ACh in the CSF has been correlated with the severity of behavioral deficits after TBI. Three groups were examined: TBI with hypothermic brain (30°C), TBI with normothermic brain (37°C), or sham TBI with normothermic brain (37°C). ACh concentrations in CSF were significantly higher in 37°C TBI rats, but not in 30°C TBI rats compared to shams. ACh concentratons in plasma did not differ between groups. These results suggest that a contributing factor to the neuroprotective effects of moderate hypothermia in TBI may be related to the reduction of excessive ACh levels in the central nervous system following injury.     

Heparin precipitable fraction (HPF) from dermatoligical patients. I. Characterization of the thrombin-clottable protein.

A heparin precipitable fraction (HPF) was obtained upon cooling (+4°C) of heparinized plasma from dermatological inpatients, mainly affected with psoriasis arthropathica. HPF, isolated by centrifugation (+4°C) and redissolved in 0.15 M NaCl at 37°C, consisted of about 60% thrombin-clottable protein. This portion could also be removed by heating at 56°C for 10 min. Polycrylamide gel electrophoresis demonstrated the larger part of HPF to be fibrinogen. In addition varying amounts of albumin and an unidentified protein (X-component) and traces of other plasma proteins were observed. SDS gel electrophoresis demonstrated the fibrinogen-related protein to have the same molecular weight as purified human fibrinogen. Upon reduction with beta-mercaptoethanol only intact Aα, Bβ and γ-chains were observed. Quantitative N-terminal amino acid analysis showed insignificant amounts of N-terminal glycine indicating the absence of soluble fibrin. Eighteen out of 21 patients with HPF had a negative ethanol gelation test in citrated plasma, and 17 out of 21 patients demonstrated no detectable serum FDP. Possibly, HPF exists in plasma as a complex between native fibrinogen, the X-component and heparin.     

Reversible changes in the activities and amounts of tyrosine hydroxylase in dopamine neurons of the substantia nigra in response to axonal injury as studied by immunochemical and immunocytochemical methods

The effects of unilateral electrolytic lesions of axons of nigrostriatal dopamine neurons of the rat brain on the activity and amount of the neurotransmitter synthesizing enzyme tyrosine hydroxylase (TH) in the axon terminals in the striatum and in the parent cell bodies in the substantia nigra (A9 group) were examined. Lesions in posterolateral hypothalamus damaging axons close to cell bodies resulted, by 72 h, in a permanent anterograde reduction of TH activity to 20% of control in the ipsilateral striatum. By immunochemical titration and immunocytochemical localization with a specific antibody to TH, the reduction was demonstrated as due to loss of enzyme protein. In the reactive A9 cell bodies TH activity was increased to 175% of control by 24 h followed by a gradual and permanent fall to 40% of control by day 14. The retrograde reduction of TH was due to a loss of enzyme protein in turn reflecting retrograde cell death. Striatal lesions of moderate size resulted in a fall in the activity and amount of TH in A9 substantia nigra cell bodies by day 7 to 60–65% of control, with full recovery by day 21. We conclude that the retrograde reaction in central dopamine neurons is dependent upon the proximity of the lesion to the parent cell body: proximal lesions result in retrograde cell death; distal lesions result in a reversible retrograde reduction in the amount and activity of TH. The findings confirm previous evidence that: (a) a reduced accumulation of neurotransmitter synthesizing enzymes is a biochemical concomitant of the retrograde reaction in central neurons; (b) that intrinsic neurons of the CNS may undergo a retrograde response which is reversible. The reduction of TH may indicate a reordering of protein biosynthesis favoring production of protein required for axonal regeneration at the expense of those involved in neurotransmission.     

Sequestration requirements for the degradation of 125I-labeled β nerve growth factor bound to embryonic sensory neurons

Nerve growth factor interacts with responsive cells by binding to cell surface membrane receptors. There are two different receptors on both embryonic sensory and sympathetic neurons, a high-affinity (type I) receptor and a lower-affinity (type II) receptor. Sequestration, which we have defined as bound nerve growth factor that becomes inaccessible to the external milieu with time, occurs through the type I receptor on both sensory and sympathetic neurons. We describe here a process subsequent to sequestration involving internalization and degradation of bound nerve growth factor and showing that bound nerve growth factor is not degraded under the following conditions: (1) low temperature, ie 4°C; (2) when a large excess of unlabeled nerve growth factor is added concomitantly with the labeled nerve growth factor and the temperature is raised from 4°C to 37°C; (3) when metabolic inhibitors sodium fluoride and dinitrophenol are added concomitantly with the labeled nerve growth factor and the temperature is raised from 4° to 37°C. On the other hand, conditions that allow bound nerve growth factor to be degraded are the following: (1) incubation of the sensory nerve cells at low temperature (ie, 4°C) only in the presence of labeled nerve growth factor, then raising the temperature to 37°C; (2) when sodium fluoride and dinitrophenol are added when the temperature is raised to 37°C; (3) when excess unlabeled nerve growth factor is added when the temperature is raised to 37°C. These studies are consistent with the idea that nerve growth factor has to bind to the cells in order to be degraded; however, binding is not sufficient for degradation to occur. Second, the bound nerve growth factor must be sequestered in order to be degraded. Third, the process of internalization of the bound nerve growth factor, unlike sequestration, is not an energy-dependent process. Thus, it seems reasonable to suggest the following steps for the interaction of nerve growth factor with responsive cells: binding to a cell surface membrane receptor, followed by sequestration of the bound nerve growth factor, and finally, internalization of the sequestered nerve growth factor.     

Simple intravenous fluid regimens to control fever in hospitalized stroke patients: A theoretical evaluation

Fever is an independent predictor of worse outcome in stroke patients. We hypothesized that a peripheral infusion of saline in chilled or ice slurry form can be a practical adjuvant therapy to maintain euthermia. We developed a theoretical model simulating systemic body cooling in response to 0 °C saline and 50% ice slurry. Temperature elevations up to 39 °C were studied with respect to the time needed to reach a core temperature of 37 °C. Mathematical modeling identified a cooling rate of 0.48 °C/hr and 0.24 °C/hr using a 450 mL/hr infusion of 50% ice slurry and chilled saline. A reduction of the infusion rate to 150 mL/hr decreased euthermia time by a factor of 3; however, the total amount of coolant remained constant. Thus, based on mathematical modeling, peripheral infusions of saline in chilled or ice slurry form can be used as an adjunct therapy to achieve euthermia and control fever. Using intravenous coolants in an on-demand, temperature-guided and supervised treatment setting seems most reasonable to avoid potentially unsafe use of extended fluid volumes and infusion times.     

Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission

Mice lacking expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme of the catecholamine biosynthetic pathway, in dopaminergic neuronal cell types were generated by a transgenic rescue approach to clarify the role of dopamine signaling during postnatal development. Introduction of the TH transgene directed by the dopamine β-hydroxylase gene promoter into TH knockout mice restored noradrenaline and adrenaline synthesis, preventing perinatal lethality and cardiac dysfunction in the knockout mice. Lack of TH expression in the cells that normally express the dopaminergic phenotype resulted in a marked reduction of dopamine accumulation in the tissues, which led to multiple behavioral abnormalities at the juvenile stage. These abnormalities were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine-induced hyperactivity, cataleptic behavior, and defects in active avoidance learning. In contrast, development of the pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained, despite defective dopamine synthesis. These results demonstrate that dopamine neurotransmission is essential for controlling spontaneous and voluntary movement and associative learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. J. Neurosci. Res. 54: 450–464, 1998. © 1998 Wiley-Liss, Inc.     

Dopa-responsive dystonia]

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic and sustained response to relatively low doses of levodopa. There are at least three causative genes for DRD: 1) the GCH1 gene on chromosome 14q22.1-q22.2, coding for the enzyme GTP cyclohydrolase I (GTPCH) that catalyzes the rate-limiting step in the tetrahydrobiopterin (BH4; the cofactor for tyrosine hydroxylase [TH]) biosynthesis, 2) the TH gene on 11p15.5, and 3) an as yet undefined gene on 14q13 (DYT14). In our series, 86% of families with DRD or dystonia with motor delay (an intermediate phenotype between GTPCH-deficient DRD [mild] and GTPCH-deficient hyperphenylalaninemia [severe]) had identifiable GCH1 or (rarely) TH mutations. Neurochemical data suggest that striatal dopamine reduction in GTPCH-deficient DRD (the major form of DRD) is caused not only by decreased TH activity resulting from a low cofactor level but also by actual loss of TH protein without nerve terminal loss. This TH protein reduction in the striatum (especially in the putamen) may be due to a diminished regulatory effect of BH4 on stability of TH molecules or to a dysfunction of TH protein transport from the substantia nigra to the striatum.     

Clinical study of paramyotonia congenita with and without myotonia in a warm environment

Fourteen patients with paramyotonia congenita were examined clinically. Patients of 3 farnilies had no myotonia in a warm environment while in a cold environment they developed paradoxical myotonia (myotonia aggravated by repeated muscle contraction). Patients of a 4th family had myotonia associated with after-activity in a warm environment which was not paradoxical. This myotonia was aggravated by cooling. In a warm environment the resting muscles of all patients showed no spontaneous electromyographic activity except for occasional myotonic runs. On cooling, spontaneous fibrillations developed. This was most intense at 32°C–28°C (muscle temperature). On deeper cooling it ceased. In contrast, 5 patients with myotonia congenita did not show such activity during cooling. In all paramyotonic patients cooling (30°C–25°C) produced muscle paralysis, which outlasted rewarming by several hours. At 32°C–30°C muscle relaxation was slowed. Recording of electromyographic activity and isometric contractions of the long finger flexors during cooling revealed that the slowing of muscle relaxation in paramyotonia is not as closely linked to after–activity as is the slowing of muscle relaxation in myotonia congenita.     

Sodium Pentobarbitone and the Suppression of Luteinizing Hormone Pulses in the Female Rat: the Role of Hypothermia

The role of hypothermia in the suppression of pulsatile luteinizing hormone (LH) release by the barbiturate sodium pentobarbitone has been investigated in ovariectomized rats. Each animal was fitted with an intraperitoneal miniature radio transmitter to monitor core temperature and with an indwelling intravenous and intraperitoneal catheter. During the 6-h sampling period the animal's core temperature was recorded automatically every 5 min and a 25 μl blood sample was obtained concurrently using an automated system. After the initial 3 h of sampling either the drug or the vehicle was administered via the intraperitoneal cannula from outside the cage, thus ensuring minimal disturbance to the animal. Administration of sodium pentobarbitone (40 mg/kg) at an ambient temperature of 21°C resulted in a significant hypothermia throughout the 3-h post-injection period. During this period there was a significant reduction in mean LH concentration, and in the frequency and amplitude of the LH pulses. When the drug was administered at an ambient temperature of 35°C there was no reduction in core temperature and no significant change in the LH pulse parameters. Vehicle treatment was without significant effect on core temperature or on the LH pulse parameters when administered at an ambient temperature of either 21°C or 35°C. These results indicate that the effects of this barbiturate on the pulsatile release of LH are secondary to the induced hypothermia and suggest that hypothermia per se may be able to disrupt LH pulses. It is therefore imperative to reassess the significance of previous studies that have implicated particular neurotransmitter systems in the control of LH pulses; unrecognized hypothermic effects of the treatments may have been the primary cause of the pulse suppression, rather than a direct involvement of the neurotransmitter in question in the regulation of LH releasing hormone. The neurophysiological process by which the hypothermic state may inhibit LH pulses remains to be determined.