Control of breathing in newborn mice lacking the beta‐2 nAChR subunit

Aim: To study the ventilatory and arousal/defence responses to hypoxia in newborn mutant mice lacking the β2 subunit of the nicotinic acetylcholine receptors. Methods: Breathing variables were measured non‐invasively in mutant (n =31) and wild‐type age‐matched mice (n = 57) at 2 and 8 days of age using flow barometric whole‐body plethysmography. The arousal/defence response to hypoxia was determined using behavioural criteria. Results: On day 2, mutant pups had significantly greater baseline ventilation (16%) than wild‐type pups (P < 0.02). Mutant pups had a decreased hypoxic ventilatory declines. Arousal latency was significantly shorter in mutant than in wild‐type pups (133 ± 40 vs. 146 ± 20 s, respectively, P < 0.026). However, the duration of movement elicited by hypoxia was shorter in mutant than in wild‐type pups (14.7 ± 5.9 vs. 23.0 ± 10.7 s, respectively, P < 0.0005). Most differences disappeared on P8, suggesting a high degree of functional plasticity. Conclusion: The blunted hypoxic ventilatory decline and the shorter arousal latency on day 2 suggested that disruption of the β2 nicotinic acetylcholine receptors impaired inhibitory processes affecting both the ventilatory and the arousal response to hypoxia during postnatal development.     

Control of breathing in the newborn

Anatomical differences between infants and adults (e.g., fetal hemoglobin, central nervous system maturation) imply that with regard to control of breathing, the newborn infant cannot be considered simply a small adult. Resting breathing of the neonate may be regular or quiet variable. Although breathing patterns are different in rapid eye movement and quiet sleep states, it is not clear whether ventilation is different. Both resting ventilation and CO₂ sensitivity increase with postnatal age. The ventilatory response to hypoxia in the neonate is unusual in that there is a transient increase in ventilation at the onset of hypoxia, but this response is not sustained in the steady state. The reasons for this difference from the adult response to hypoxia are uncertain. A possible association between abnormal control of respiration which might predispose to respiratory failure and the occurrence of apneas or periodic breathing has been sought in many studies, but results are inconclusive. It is speculated that respiratory responsiveness to transient changes in chemical stimuli might be important for the long-term well-being of the infant. Furthermore, it is suggested that modelling of respiratory behavior of the neonate may be quite useful because of ethical limits on the experimental protocols that are applicable to the human infant.     

Fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the epsilon2 (NR2B) subunit

The N-methyl-D-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the epsilon2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although epsilon2(-/-) mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the epsilon2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from epsilon2(-/-) neurons expressed an NMDA receptor-mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg(2+) or AP5. Whole cell currents from epsilon2(-/-) neurons were also more sensitive to block by low concentrations of Zn(2+) and much less sensitive to the epsilon2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile from epsilon2(-/-) neurons are consistent with the expression of zeta1/epsilon1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the epsilon2 subunit. Thus epsilon1 can substitute for the epsilon2 subunit at synapses and epsilon2 is not required for targeting of NMDA receptors to the postsynaptic membrane.     

Unaltered pain-related behavior in mice lacking NMDA receptor GluRepsilon 1 subunit

Noxious afferent input following tissue damage and inflammation triggers a state of neuronal hyperexcitability-a phenomenon of central sensitization-which manifests behaviorally as allodynia and hyperalgesia. At the molecular level, maintenance of central sensitization is largely dependent on the N-methyl-D-aspartate receptor (NMDAR) activation. NMDARs are composed of GluRzeta1 (NR1) and one of four GluRepsilon (NR2) subunits, which determine the functional properties of native NMDARs. Although there is accumulating evidence to implicate GluRepsilon 2-containing NMDARs in pain mechanisms, the functional significance of GluRepsilon 1-containing NMDARs in this setting has not been examined in detail. Here, we used hind paw injection of formalin, complete Freund's adjuvant and a nerve injury model to investigate the effects of GluRepsilon 1 subunit gene deletion on pain-related behavior in mice. In all of the models tested, GluRepsilon 1-deficient mice exhibited responses similar to wild-type controls. These results suggest that GluRepsilon 1 disruption does not result in altered nociceptive behavior in mice. Although the contribution of other nociceptive pathways cannot be ruled out, we speculate that the preserved function of GluRepsilon 2-containing NMDARs could explain unaltered nociceptive behavior in mutant mice.     

Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem-spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90 min) of incubation with 0, 25, or 250 U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.     

Anxiety-like behaviors in mice lacking GIT2

G protein-coupled receptor kinase-interactor 2 (GIT2) is a signaling scaffold protein that also functions as GTPase-activating protein (GAPs) for ADP-ribosylation factor (Arf) small GTP-binding proteins. GIT2 has been implicated in the regulation of G protein-coupled receptor trafficking and cell adhesion and migration. To evaluate possible neurobehavioral functions of GIT2 in vivo, we evaluated GIT2-knockout (KO) mice for abnormalities in emotionality and mood. Male and female GIT2-KO mice presented with anxiety-like behaviors in the zero-maze and light–dark emergence tests. Immobility times in tail suspension were reduced in GIT2-KO males, but were normal in GIT2-KO females. Hence, GIT2-KO mice display anxiety-like behavior in an absence of depressive-like responses.     

Neurological phenotype and synaptic function in mice lacking the CaV1.3 alpha subunit of neuronal L-type voltage-dependent Ca2+ channels

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.     

Aberrant development of thymocytes in mice lacking laminin-2

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4- CD8-) and SP (CD4+ CD8-, CD4- CD8+) populations and a significant decrease in the DP (CD4+ CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice.     

Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of P/Q-type Ca2+ channels

The role of voltage-gated Ca(2+) (Ca(V)) channels in pain mechanisms has been the object of intense investigation using pharmacological approaches and, more recently, using mutant mouse models lacking the Ca(V)alpha(l) pore-forming subunit of N-, R- and T-type channels. The role of P/Q-type channels in nociception and pain transmission has been investigated by pharmacological approaches but remains to be fully elucidated. To address this issue, we have analyzed pain-related behavioral responses of null mutant mice for the Ca(V)2.1alpha(1) subunit of P/Q-type channels. Homozygous null mutant Ca(V)2.1alpha(1)-/- mice developed dystonia at 10-12 days after birth and did not survive past weaning. Tested at ages where motor deficit was either absent or very mild, Ca(V)2.1alpha(1)-/- mice showed reduced tail withdrawal latencies in the tail-flick test and reduced abdominal writhes in the acetic acid writhing test. Adult heterozygous Ca(V)2.1alpha(1)+/- mice did not show motor deficits in the rotarod and activity cage tests and did not show alterations in pain responses in the tail-flick test and the acetic acid writhing test. Strikingly, they showed a reduced licking response during the second phase of formalin-induced inflammatory pain and a reduced mechanical allodynia in the chronic constriction injury model of neuropathic pain. Our findings show that P/Q-type channels play an antinociceptive role in sensitivity to non-injurious noxious thermal stimuli and a pronociceptive role in inflammatory and neuropathic pain states, pointing to an important role of Ca(V)2.1 channels in central sensitization.     

Hyperexcitability and reduced low threshold potassium currents in auditory neurons of mice lacking the channel subunit Kv1.1

A low voltage-activated potassium current, I _KL, is found in auditory neuron types that have low excitability and precisely preserve the temporal pattern of activity present in their presynaptic inputs. The gene Kcna1 codes for Kv1.1 potassium channel subunits, which combine in expression systems to produce channel tetramers with properties similar to those of I _KL, including sensitivity to dendrotoxin (DTX). Kv1.1 is strongly expressed in neurons with I _KL, including auditory neurons of the medial nucleus of the trapezoid body (MNTB). We therefore decided to investigate how the absence of Kv1.1 affected channel properties and function in MNTB neurons from mice lacking Kcna1 . We used the whole cell version of the patch clamp technique to record from MNTB neurons in brainstem slices from Kcna1 -null (−/−) mice and their wild-type (+/+) and heterozygous (+/−) littermates. There was an I _KL in voltage-clamped −/− MNTB neurons, but it was about half the amplitude of the I _KL in +/+ neurons, with otherwise similar properties. Consistent with this, −/− MNTB neurons were more excitable than their +/+ counterparts; they fired more than twice as many action potentials (APs) during current steps, and the threshold current amplitude required to generate an AP was roughly halved. +/− MNTB neurons had excitability and I _KL amplitudes identical to the +/+ neurons. The I _KL remaining in −/− neurons was blocked by DTX, suggesting the underlying channels contained subunits Kv1.2 and/or Kv1.6 (also DTX-sensitive). DTX increased excitability further in the already hyperexcitable −/− MNTB neurons, suggesting that −/− I _KL limited excitability despite its reduced amplitude in the absence of Kv1.1 subunits.     

Ganglioside expression in tissues of mice lacking beta2-microglobulin

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other (brain, liver, lungs and muscle) tissues of C57BL/6 mice lacking the gene for beta2-microglobulin (beta2M), a constitutive component of the MHC class I molecule. Ganglioside fractions in the tissues of mice homozygous (beta2M-/-) and heterozygous (beta2M-/+) for the gene deletion were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific polyclonal antibodies. Ubiquitous gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control beta2M-/+ mice, whereas the homozygous knockout mice had substantially decreased expression of these structures. The lungs of the beta2M-/- mice also had reduced expression of T-lymphocyte-specific GM1b-type gangliosides (GM1b and GalNAc-GM1b). beta2M-deficient mice also had more GM1a and GD1a gangliosides in the liver, and several neolacto-series gangliosides were increased in the brain and lungs. This study provides in vivo evidence that the beta2M molecule can influence the acquisition of a distinct ganglioside assembly in different mouse organs, implicating its non-immunological functions.     

Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABA(A) receptor alpha1 subunit

GABA(A) receptors (GABA(A)-Rs) are pentameric structures consisting of two alpha, two beta, and one gamma subunit. The alpha subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the alpha1 subunit to native GABA(A)-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and alpha1 subunit knock-out (alpha1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from alpha1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the alpha1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from alpha1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the alpha1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the alpha1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.     

Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels

CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.     

Enhancement of neurogenesis by running wheel exercises is suppressed in mice lacking NMDA receptor epsilon 1 subunit

Neurogenesis continues throughout adulthood in the dentate gyrus in mice, and is regulated by environmental, endocrine, and pharmacological stimuli. Although running wheel exercises have been reported to enhance neurogenesis, details of molecule mechanisms of the enhancement are not well understood. We report here that the hippocampal neurogenesis is enhanced when wild-type mice are raised in cages with running wheels for 3 weeks, but the wheel exercise does not enhance the neurogenesis in mice lacking the NMDA receptor epsilon1 subunit. Brain-derived neurotrophic factor (BDNF) has been reported to affect neuronal cell proliferation and survival. We examined the BDNF levels in the hippocampi of wild-type and epsilon1 knockout mice, and found that the BDNF level was increased through wheel exercises in the wild-type but not in the knockout mice. The enhancement of neurogenesis by the wheel exercise was also found to be reversible: when the exercise-stimulated wild-type mice were returned to the environment without running wheels for 3 weeks, the neurogenesis was the same as that in the mice which had never experienced the exercise. These results suggest that the wheel exercise may activate NMDA receptors in the hippocampus, which in turn may enhance BDNF production and neurogenesis.