A massively parallel strategy for STR marker development, capture, and genotyping.

Short tandem repeat (STR) variants are highly polymorphic markers that facilitate powerful population genetic analyses. STRs are especially valuable in conservation and ecological genetic research, yielding detailed information on population structure and short-term demographic fluctuations. Massively parallel sequencing has not previously been leveraged for scalable, efficient STR recovery. Here, we present a pipeline for developing STR markers directly from high-throughput shotgun sequencing data without a reference genome, and an approach for highly parallel target STR recovery. We employed our approach to capture a panel of 5000 STRs from a test group of diademed sifakas (Propithecus diadema, n = 3), endangered Malagasy rainforest lemurs, and we report extremely efficient recovery of targeted loci-97.3-99.6% of STRs characterized with ≥10x non-redundant sequence coverage. We then tested our STR capture strategy on P. diadema fecal DNA, and report robust initial results and suggestions for future implementations. In addition to STR targets, this approach also generates large, genome-wide single nucleotide polymorphism (SNP) panels from flanking regions. Our method provides a cost-effective and scalable solution for rapid recovery of large STR and SNP datasets in any species without needing a reference genome, and can be used even with suboptimal DNA more easily acquired in conservation and ecological studies.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

Isolated adult turtle brainstems exhibit central hypoxic chemosensitivity.

During hypoxia, red-eared slider turtles increase ventilation and decrease episodic breathing, but whether these responses are due to central mechanisms is not known. To test this question, isolated adult turtle brainstems were exposed to 240 min of hypoxic solution (bath PO2 = 32.6 ±â€¯1.2 mmHg) and spontaneous respiratory-related motor bursts (respiratory event) were recorded on hypoglossal nerve roots. During hypoxia, burst frequency increased during the first 15 min, and then decreased during the remaining 35-240 min of hypoxia. Burst amplitude was maintained for 120 min, but then decreased during the last 120 min. The number of bursts/respiratory event decreased within 30 min and remained decreased. Pretreatment with either prazosin (α1-adrenergic antagonist) or MDL7222 (5-HT3 antagonist) blocked the hypoxia-induced short-term increase and the longer duration decrease in burst frequency. MDL7222, but not prazosin, blocked the hypoxia-induced decrease in bursts/respiratory event. Thus, during bath hypoxia, isolated turtle brainstems continued to produce respiratory motor output, but the frequency and pattern were altered in a manner that required endogenous α1-adrenergic and serotonin 5-HT3 receptor activation. This is the first example of isolated reptile brainstems exhibiting central hypoxic chemosensitivity similar to other vertebrate species.

Endomorphin-2 (Endo2) and substance P (SubP) co-application attenuates SubP-induced excitation and alters frequency plasticity in neonatal rat in vitro preparations.

Substance P (SubP) and endomorphin-2 (Endo2) are co-localized presynaptically in vesicles of neurons adjacent to inspiratory rhythm-generating pre-Botzinger Complex (preBotC) neurons but the effects of co-released SubP and Endo2 on respiratory motor control are not known. To address this question, SubP alone or a combination of SubP and Endo2 (SubP/Endo2) were bath-applied in a sustained (15-min) or intermittent (5-min application, 5-min washout, x3) pattern at 10-100 nM to neonatal rat brainstem-spinal cord preparations. During neuropeptide application, SubP/Endo2 co-applications generally attenuated SubP-induced increases in burst frequency and decreases in burst amplitude. With respect to frequency plasticity (long-lasting increase in burst frequency 60 min post-neuropeptide application), SubP-induced frequency plasticity was increased with sustained SubP/Endo2 co-applications at 20 and 100 nM. Intermittent SubP/Endo2 co-applications tended to decrease the level of frequency plasticity induced by intermittent SubP alone applications. SubP/Endo2 co-applications revealed potentially new functions for neurokinin-1 (NK1R) and mu-opioid (MOR) receptors on respiratory rhythm-generating medullary neurons.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations.

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

Antinociceptive efficacy of buprenorphine and hydromorphone in red-eared slider turtles (Trachemys scripta elegans).

Despite the frequent clinical use of buprenorphine in reptiles, its antinociceptive efficacy is not known. In a randomized, complete cross-over study, the antinociceptive efficacy of buprenorphine (0.2 mg/kg s.c.) was compared with hydromorphone (0.5 mg/kg s.c.), and saline (0.9% s.c. equivalent volume) in 11 healthy red-eared slider turtles (Trachemys scripta elegans). Additionally, buprenorphine at 0.1 and 1 mg/kg was compared with saline in six turtles. Hindlimb withdrawal latencies were measured after exposure to a focal, thermal noxious stimulus before and between 3 hr and up to 96 hr after drug administration. Buprenorphine did not significantly increase hindlimb withdrawal latencies at any time point compared with saline. In contrast, hydromorphone administration at 0.5 mg/kg significantly increased hindlimb withdrawal latencies for up to 24 hr. These results show that hydromorphone, but not buprenorphine, provides thermal antinociception in red-eared slider turtles.

Mu-opioid receptor-dependent transformation of respiratory motor pattern in neonates in vitro.

Endogenous opioid peptides activating mu-opioid receptors (MORs) are part of an intricate neuromodulatory system that coordinates and optimizes respiratory motor output to maintain blood-gas homeostasis. MOR activation is typically associated with respiratory depression but also has excitatory effects on breathing and respiratory neurons. We hypothesized that low level MOR activation induces excitatory effects on the respiratory motor pattern. Thus, low concentrations of an MOR agonist drug (DAMGO, 10-200 nM) were bath-applied to neonatal rat brainstem-spinal cord preparations while recording inspiratory-related motor output on cervical spinal roots (C4-C5). Bath-applied DAMGO (50-200 nM) increased inspiratory motor burst amplitude by 40-60% during (and shortly following) drug application with decreased burst frequency and minute activity. Reciprocal changes in inspiratory burst amplitude and frequency were balanced such that 20 min after DAMGO (50-200 nM) application, minute activity was unaltered compared to pre-DAMGO levels. The DAMGO-induced inspiratory burst amplitude increase did not require crossed cervical spinal pathways, was expressed on thoracic ventral spinal roots (T4-T8) and remained unaltered by riluzole pretreatment (blocks persistent sodium currents associated with gasping). Split-bath experiments showed that the inspiratory burst amplitude increase was induced only when DAMGO was bath-applied to the brainstem and not the spinal cord. Thus, MOR activation in neonates induces a respiratory burst amplitude increase via brainstem-specific mechanisms. The burst amplitude increase counteracts the expected MOR-dependent frequency depression and may represent a new mechanism by which MOR activation influences respiratory motor output.

Protective role of hemoglobin and fetal hemoglobin in early kidney disease for children with sickle cell anemia.

Patients with sickle cell anemia are at risk for organ damage including kidney disease. Microalbuminuria may be an early marker of disease progression. This retrospective review analyzed laboratory and clinical findings in children with sickle cell anemia according to the presence or absence of MA during well clinic sickle cell visits. Results were analyzed in sum as well as by therapeutic intervention (not on therapy,hydroxyurea therapy, or chronic transfusion therapy). Thirty two of 144(22%) children had MA, including 20 of 82 (24%) children not on a therapeutic intervention (chronic transfusion or hydroxyurea). In children not on therapy, low hemoglobin, low fetal hemoglobin and high lactate dehydrogenase were associated with MA. Frequency of positive screens for MA for the different treatment groups were: Hydroxyurea 13%; chronic transfusion 26% and children on no treatment 24%. However,the difference between the hydroxyurea group and the chronic transfusion or no treatment groups did not reach statistical significance.Increased hemoglobin and fetal hemoglobin may provide protection against kidney disease in sickle cell anemia and should be evaluated in a randomized, prospective clinical trial.

Evaluation of the analgesic effects of oral and subcutaneous tramadol administration in red-eared slider turtles.

OBJECTIVE: To determine the dose- and time-dependent changes in analgesia and respiration caused by tramadol administration in red-eared slider turtles (Trachemys scripta). DESIGN: Crossover study. ANIMALS: 30 adult male and female red-eared slider turtles. PROCEDURES: 11 turtles received tramadol at various doses (1, 5, 10, or 25 mg/kg [0.45, 2.27, 4.54, or 11.36 mg/lb], PO; 10 or 25 mg/kg, SC) or a control treatment administered similarly. Degree of analgesia was assessed through measurement of hind limb thermal withdrawal latencies (TWDLs) at 0, 3, 6, 12, 24, 48, 72, and 96 hours after tramadol administration. Nineteen other freely swimming turtles received tramadol PO (5, 10, or 25 mg/kg), and ventilation (V(E)), breath frequency, tidal volume (V(T)), and expiratory breath duration were measured. RESULTS: The highest tramadol doses (10 and 25 mg/kg, PO) yielded greater mean TWDLs 6 to 96 hours after administration than the control treatment did, whereas tramadol administered at 5 mg/kg, PO, yielded greater mean TWDLs at 12 and 24 hours. The lowest tramadol dose (1 mg/kg, PO) failed to result in analgesia. Tramadol administered SC resulted in lower TWDLs, slower onset, and shorter duration of action, compared with PO administration. Tramadol at 10 and 25 mg/kg, PO, reduced the V(E) at 12 hours by 51% and 67%, respectively, and at 24 through 72 hours by 55% to 62% and 61 % to 70%, respectively. However, tramadol at 5 mg/kg, PO, had no effect on the V(E). CONCLUSIONS AND CLINICAL RELEVANCE: Tramadol administered PO at 5 to 10 mg/kg provided thermal analgesia with less respiratory depression than that reported for morphine in red-eared slider turtles.

Comparison of Thermal and Mechanical Noxious Stimuli for Testing Analgesics in White's Tree Frogs (Litoria caerulea) and Northern Leopard Frogs (Lithobates pipiens).

Determining the clinical efficacy of analgesic drugs in amphibians can be particularly challenging. The current study investigated whether a thermal nociceptive stimulus is useful for the evaluation of analgesic drugs in 2 amphibian species. The objectives of this study were 2-fold: 1) compare 2 models of nociception (thermal and mechanical) using 2 frog species; White's Tree Frogs (Litoria caerulea; WTF) and Northern Leopard Frogs (Lithobates pipiens; NLF) after administration of saline or morphine sulfate; and 2) evaluate antinociceptive efficacy of morphine sulfate at 2 doses in a common amphibian research species, the NLF, using a mechanical stimulus. Neither WTF nor NLF displayed consistent drug-dependent changes in withdrawal responses to a noxious thermal stimulus applied using the Hargreaves apparatus, but NLF exposed to the noxious mechanical stimulus demonstrated a significant dose-dependent antinociceptive response to morphine sulfate. These results indicate that morphine is not antinociceptive in WTF, supporting previously reported results, and demonstrate the importance of using an appropriate experimental antinociceptive test in amphibians. Our data suggest that nociception in amphibian species may be best evaluated by using mechanical nociceptive models, although species differences must also be considered.

Respiratory and antinociceptive effects of dexmedetomidine and doxapram in ball pythons (Python regius).

OBJECTIVE: To determine the effects of dexmedetomidine, doxapram, and dexmedetomidine plus doxapram on ventilation ([Formula: see text]e), breath frequency, and tidal volume (Vt) in ball pythons (Python regius) and of doxapram on the thermal antinociceptive efficacy of dexmedetomidine. ANIMALS: 14 ball pythons. PROCEDURES: Respiratory effects of dexmedetomidine and doxapram were assessed with whole-body, closed-chamber plethysmography, which allowed for estimates of [Formula: see text]e and Vt. In the first experiment of this study with a complete crossover design, snakes were injected, SC, with saline (0.9% NaCl) solution, dexmedetomidine (0.1 mg/kg), doxapram (10 mg/kg), or dexmedetomidine and doxapram, and breath frequency, [Formula: see text]e, and Vt were measured before and every 30 minutes thereafter, through 240 minutes. In the second experiment, antinociceptive efficacy of saline solution, dexmedetomidine, and dexmedetomidine plus doxapram was assessed by measuring thermal withdrawal latencies before and 60 minutes after SC injection. RESULTS: Dexmedetomidine significantly decreased breath frequency and increased Vt but did not affect [Formula: see text]e at all time points, compared with baseline. Doxapram significantly increased [Formula: see text]e, breath frequency, and Vt at 60 minutes after injection, compared with saline solution. The combination of dexmedetomidine and doxapram, compared with dexmedetomidine alone, significantly increased [Formula: see text]e at 30 and 60 minutes after injection and did not affect breath frequency and Vt at all time points. Thermal withdrawal latencies significantly increased when snakes received dexmedetomidine or dexmedetomidine plus doxapram, versus saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Concurrent administration of doxapram may mitigate the dexmedetomidine-induced reduction of breathing frequency without disrupting thermal antinociceptive efficacy in ball pythons.

Analgesic Efficacy of Tramadol and Morphine in White's Tree Frogs (Litoria caerulea).

Published data are sparse regarding the recognition of clinically relevant pain and appropriate analgesia in amphibians. The amphibian analgesia literature has primarily focused on nociceptive pathways in a single species, the northern leopard frog (Rana pipiens). The objective of the current study was to assess the analgesic efficacy and safety of oral tramadol and subcutaneous morphine in a commonly maintained zoo and pet species, White's tree frog (Litoria caerulea). We hypothesized that tramadol and morphine would provide dose-dependent antinociception, as measured by significant increases in hindlimb withdrawal latency after exposure to a noxious thermal stimulus. Two randomized, placebo-controlled, complete crossover studies were performed, with tramadol (n = 12) administered at 15, 25, and 40 mg/kg PO and morphine (n = 12) administered at 5 and 10 mg/kg SC. Hindlimb withdrawal latency was measured for a maximum of 72 h. No adverse side effects or signs of sedation were observed with any dose or drug evaluated. No significant difference in withdrawal latency was detected between the control and either tramadol or morphine. These negative results were surprising, suggesting that the thermal nociceptive model may not be biologically relevant in amphibian species.

Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats.

Neuroplasticity is a fundamental property of the respiratory control system, enabling critical adaptations in breathing to meet the challenges, but little is known whether neonates express neuroplasticity similar to adults. We tested the hypothesis that, similar to adults, tyrosine receptor kinase B (TrkB) or adenosine A2a receptor activation in neonates are independently sufficient to elicit respiratory motor facilitation, and that co-induction of TrkB and A2a receptor-dependent plasticity undermines respiratory motor facilitation. TrkB receptor activation with 7,8-dihydroxyflavone (DHF) in neonatal brainstem-spinal cord preparations induced a long-lasting increase in respiratory motor output in 55 % of preparations, whereas adenosine A2a receptor activation with CGS21680 only sporadically induced respiratory motor plasticity. CGS21680 and DHF co-application prevented DHF-dependent respiratory motor facilitation, whereas co-application of MSX-3 (adenosine A2a receptor antagonist) and DHF more rapidly induced respiratory motor plasticity. Collectively, these data suggest that mechanisms underlying respiratory neuroplasticity may be only partially operational in early neonatal life, and that adenosine A2a receptor activation undermines TrkB-induced respiratory plasticity.

Time and dose-dependent impairment of neonatal respiratory motor activity after systemic inflammation.

Neonatal respiratory impairment during infection is common, yet its effects on respiratory neural circuitry are not fully understood. We hypothesized that the timing and severity of systemic inflammation is positively correlated with impairment in neonatal respiratory activity. To test this, we evaluated time- and dose-dependent impairment of in vitro fictive respiratory activity. Systemic inflammation (induced by lipopolysaccharide, LPS, 5 mg/kg, i.p.) impaired burst amplitude during the early (1 h) inflammatory response. The greatest impairment in respiratory activity (decreased amplitude, frequency, and increased rhythm disturbances) occurred during the peak (3 h) inflammatory response in brainstem-spinal cord preparations. Surprisingly, isolated medullary respiratory circuitry within rhythmic slices showed decreased baseline frequency and delayed onset of rhythm only after higher systemic inflammation (LPS 10 mg/kg) early in the inflammatory response (1 h), with no impairments at the peak inflammatory response (3 h). Thus, different components of neonatal respiratory circuitry have differential temporal and dose sensitivities to systemic inflammation, creating multiple windows of vulnerability for neonates after systemic inflammation.

Hibernation induces pentobarbital insensitivity in medulla but not cortex.

The 13-lined ground squirrel (Ictidomys tridecemlineatus), a hibernating species, is a natural model of physiological adoption to an extreme environment. During torpor, body temperature drops to 0-4 degrees C, and the cortex is electrically silent, yet the brain stem continues to regulate cardiorespiratory function. The mechanisms underlying selective inhibition in the brain during torpor are not known. To test whether altered GABAergic function is involved in regional and seasonal differences in neuronal activity, cortical and medullary slices from summer-active (SA) and interbout aroused (IBA) squirrels were placed in a standard in vitro recording chamber. Silicon multichannel electrodes were placed in cortex, ventral respiratory column (VRC), and nucleus tractus solitarius (NTS) to record spontaneous neuronal activity. In slices from IBA squirrels, bath-applied pentobarbital sodium (300 microM) nearly abolished cortical neuronal activity, but VRC and NTS neuronal activity was unaltered. In contrast, pentobarbital sodium (300 microM) nearly abolished all spontaneous cortical, VRC, and NTS neuronal activity in slices from SA squirrels. Muscimol (20 microM; GABA(A) receptor agonist) abolished all neuronal activity in cortical and medullary slices from both IBA and SA squirrels, thereby demonstrating the presence of functional GABA(A) receptors. Pretreatment of cortical slices from IBA squirrels with bicuculline (100 microM; GABA(A) receptor antagonist) blocked pentobarbital-dependent inhibition of spontaneous neuronal activity. We hypothesize that GABA(A) receptors undergo a seasonal modification in subunit composition, such that cardiorespiratory neurons are uniquely unaffected by surges of an endogenous positive allosteric modulator.

Effects of opioid receptor activation on thermal antinociception in red-eared slider turtles (Trachemys scripta).

OBJECTIVE: To determine the effects of mu-, delta-, and kappa-opioid receptor (MOR, DOR, and KOR, respectively) activation on thermal antinociception in red-eared slider turtles Trachemys scripta. ANIMALS: 51 adult turtles. PROCEDURES: Infrared heat stimuli were applied to the plantar surface of turtle hind limbs. Thermal hind limb withdrawal latencies (HLWLs) were measured before (baseline) and at intervals after SC administration of various doses of saline (0.9% NaCl) solution (SS), MOR, DOR, or KOR agonists (3 to 13 turtles/treatment). Treatment with a DOR antagonist SC prior to DOR agonist administration was also evaluated. RESULTS: Treatment with an MOR agonist ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate salt [DAMGO; 1.3 or 6.6 mg/kg]) increased HLWLs (from baseline) at 2 to 8 hours after injection; at the higher dose, the maximum mean increase was 5.6 seconds at 4 hours. Treatment with a DOR agonist ([D-Ala(2), D-Leu(5)]-enkephalin acetate salt [DADLE; 25 mg/kg]) increased mean HLWL by 11.3 seconds at 4 hours; however, treatment with DADLE (5.8 mg/kg) or with another DOR agonist ([D-Pen(2),(5)]-enkephalin hydrate [DPDPE; 1.2 or 6.3 mg/kg]) did not alter HLWL, compared with SS effects. Administration of a DOR antagonist (naltrindole hydrochloride; 10 mg/kg) prior to DADLE administration (25 mg/kg) increased mean HLWL by 2.7 seconds at 4 hours. One KOR agonist, U50488 ([-]-trans-[1S,2S]-U50488 hydrochloride hydrate; 6.7 mg/kg) decreased HLWL steadily from 2 to 24 hours (less than baseline value); another KOR agonist, U69593 ([+]-[5alpha,7alpha,8beta]-N-Methyl-N-[7-{1-pyrrolidinyl}-1-oxaspiro{4.5}dec-8-yl]-benzene-acet-amide; 6.7 or 26 mg/kg) did not alter HLWLs, compared with SS effects. CONCLUSIONS AND CLINICAL RELEVANCE: Opioid-dependent thermal antinociception in turtles appeared to be attributable mainly to MOR activation with a relatively minor contribution of DOR activation.

Respiratory frequency plasticity during development.

Respiratory frequency plasticity is a long-lasting increase in breathing frequency due to a perturbation. Mechanisms underlying respiratory frequency are poorly understood, and there is little evidence of frequency plasticity in neonates. This hybrid review/research article discusses available literature regarding frequency plasticity and highlights potential research opportunities. Also, we include data demonstrating a model of frequency plasticity using isolated neonatal rat brainstem-spinal cord preparations. Specifically, substance P (SubP) application induced a long-lasting (>60 min) increase in spontaneous respiratory motor burst frequency, particularly in brainstem-spinal cords with the pons attached; there were no male/female differences. SubP-induced frequency plasticity is dependent on the application pattern, such that intermittent (rather than sustained) SubP applications induce more frequency plasticity. SubP-induced frequency plasticity was blocked by a neurokinin-1 receptor antagonist. Thus, the newborn rat respiratory control system has the capacity to express frequency plasticity. Identifying mechanisms that induce frequency plasticity may lead to novel methods to safely treat breathing disorders in premature and newborn infants.

One bout of neonatal inflammation impairs adult respiratory motor plasticity in male and female rats.

Neonatal inflammation is common and has lasting consequences for adult health. We investigated the lasting effects of a single bout of neonatal inflammation on adult respiratory control in the form of respiratory motor plasticity induced by acute intermittent hypoxia, which likely compensates and stabilizes breathing during injury or disease and has significant therapeutic potential. Lipopolysaccharide-induced inflammation at postnatal day four induced lasting impairments in two distinct pathways to adult respiratory plasticity in male and female rats. Despite a lack of adult pro-inflammatory gene expression or alterations in glial morphology, one mechanistic pathway to plasticity was restored by acute, adult anti-inflammatory treatment, suggesting ongoing inflammatory signaling after neonatal inflammation. An alternative pathway to plasticity was not restored by anti-inflammatory treatment, but was evoked by exogenous adenosine receptor agonism, suggesting upstream impairment, likely astrocytic-dependent. Thus, the respiratory control network is vulnerable to early-life inflammation, limiting respiratory compensation to adult disease or injury.

Respiratory pattern in midline-lesioned brainstems and hemibrainstems from adult turtles.

Discrete midline lesions uncouple left and right respiratory motor output in mammals, but not in frogs and lampreys. To address this question in reptiles, isolated adult turtle brainstems were cut along the midline while recording respiratory motor output (bursts of action potentials) on left and right hypoglossal (XII) nerves. XII motor bursts were synchronized as long as a small portion of the midline was still intact. When turtle brainstems were completely cut along the midline and separated into hemibrainstems, XII motor bursts were produced that could be abolished by mu-opioid receptor (MOR) activation or exposure to high pH (7.80) solution. Also, 13/57 hemibrainstems expressed episodic discharge (>1.75bursts/episode). To test whether crossed connections were necessary to express a long-lasting increase in burst frequency (i.e., frequency plasticity), phenylbiguanide (PBG, 5-HT(3) receptor agonist, 20microM) was bath-applied to hemibrainstems. Although PBG significantly increased burst frequency by 0.43+/-0.10bursts/min after 60min, no frequency plasticity was observed because burst frequency returned to near baseline levels after a 2-h washout. Thus, crossed connections in turtle brainstems synchronize respiratory motor output and are not required for normal respiratory pattern formation, but are required for PBG-dependent frequency plasticity.

Analgesic efficacy of butorphanol and morphine in bearded dragons and corn snakes.

OBJECTIVE: To test the hypothesis that administration of butorphanol or morphine induces antinociception in bearded dragons and corn snakes. DESIGN: Prospective crossover study. ANIMALS: 12 juvenile and adult bearded dragons and 13 corn snakes. PROCEDURES: Infrared heat stimuli were applied to the plantar surface of bearded dragon hind limbs or the ventral surface of corn snake tails. Thermal withdrawal latencies (TWDLs) were measured before (baseline) and after SC administration of physiologic saline (0.9% NaCl) solution (equivalent volume to opioid volumes), butorphanol tartrate (2 or 20 mg/kg [0.91 or 9.1 mg/lb]), or morphine sulfate (1, 5, 10, 20, or 40 mg/kg [0.45, 2.27, 4.5, 9.1, or 18.2 mg/lb]). RESULTS: For bearded dragons, butorphanol (2 or 20 mg/kg) did not alter hind limb TWDLs at 2 to 24 hours after administration. However, at 8 hours after administration, morphine (10 and 20 mg/kg) significantly increased hind limb TWDLs from baseline values (mean +/- SEM maximum increase, 2.7+/-0.4 seconds and 2.8+/-0.9 seconds, respectively). For corn snakes, butorphanol (20 mg/kg) significantly increased tail TWDLs at 8 hours after administration (maximum increase from baseline value, 3.0+/-0.8 seconds); the low dose had no effect. Morphine injections did not increase tail TWDLs at 2 to 24 hours after administration. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with doses used in most mammalian species, high doses of morphine (but not butorphanol) induced analgesia in bearded dragons, whereas high doses of butorphanol (but not morphine) induced analgesia in corn snakes.