Martha M. Bosma

The Type 1 Inositol 1,4,5-Trisphosphate Receptor Gene Is Altered in the opisthotonos Mouse

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the optmutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group I mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones

Spontaneous [Ca2+]i transients were measured in the mouse neocortex from embryonic day 16 (E16) to postnatal day 6 (P6). On the day of birth (P0), cortical neurones generated widespread, highly synchronous [Ca2+]i transients over large areas. On average, 52% of neurones participated in these transients, and in 20% of slices, an average of 80% participated. These transients were blocked by TTX and nifedipine, indicating that they resulted from Ca2+ influx during electrical activity, and occurred at a mean frequency of 0.91 min−1. The occurrence of this activity was highly centred at P0: at E16 and P2 an average of only 15% and 24% of neurones, respectively, participated in synchronous transients, and they occurred at much lower frequencies at both E16 and P2 than at P0. The overall frequency of [Ca2+]i transients in individual cells did not change between E16 and P2, just the degree of their synchronicity. The onset of this spontaneous, synchronous activity correlated with a large increase in Na+ current density that occurred just before P0, and its cessation with a large decrease in resting resistance that occurred just after P2. This widespread, synchronous activity may serve a variety of functions in the neonatal nervous system.

Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons

The mechanism of periodic gonadotropin-releasing hormone (GnRH) secretion from hypothalamic neurons is difficult to elucidate due to the diffuse distribution of GnRH neurons and the complex interaction of neuronal inputs onto them. Recent use of transgenic techniques allowed construction of an immortalized GnRH neuronal cell line (GT1), which has neuronal markers and secretes GnRH in a periodic fashion. Using the patch-clamp recording technique in the whole-cell and nystatin perforated-patch configuration, the present experiments show that this cell line expressed a tetrodotoxin-sensitive Na channel, two types of Ca channels, three types of outward K channels and a K inward rectifier. The latter current was suppressed in some cells by GnRH or somatostatin. In addition, a gamma-aminobutyric acid (GABA) response, presumably through GABAA receptors, is recorded. In long-term current-clamp recordings, spontaneous depolarizing activity was found to increase, and then decrease, between 20–35 min after removal of the cells from serum- and steroid-containing medium. In some cases, more than one cycle of activity was seen. Under voltage clamp, an inward current was recorded at similar times, with reversal at about −15 mV. Thus, two mechanisms of cell interaction, GABAA responses and feedback through GnRH responses, and one mechanism of endogenous periodic electrical activity were observed in these cells, which could synchronize periodic GnRH release.

Development of Synchronized Activity of Cranial Motor Neurons in the Segmented Embryonic Mouse Hindbrain

Spontaneous electrical activity synchronized among groups of related neurons is a widespread and important feature of central nervous system development. Among the many places from which spontaneous rhythmic activity has been recorded early in development are the cranial motor nerve roots that exit the hindbrain, the motor neuron pool that, at birth, will control the rhythmic motor patterns of swallow, feeding and the oral components of respiratory behaviour. Understanding the mechanism and significance of this hindbrain activity, however, has been hampered by the difficulty of identifying and recording from individual hindbrain motor neurons in living tissue. We have used retrograde labelling to identify living cranial branchiomeric motor neurons in the hindbrain, and [Ca2+]i imaging of such labelled cells to measure spontaneous activity simultaneously in groups of motor neuron somata. We find that branchiomeric motor neurons of the trigeminal and facial nerves generate spontaneous [Ca2+]i transients throughout the developmental period E9.5 to E11.5. During this two‐day period the activity changes from low‐frequency, long‐duration events that are tetrodotoxin insensitive and poorly coordinated among cells, to high‐frequency short‐duration events that are tetrodotoxin sensitive and tightly coordinated thoughout the motor neuron population. This early synchronization may be crucial for correct neuron‐target development.

Na(V)1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms.

NaV1.1 is the primary voltage-gated Na+ channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that NaV1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a+/−), which encodes the pore-forming α-subunit of the NaV1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a+/− mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a+/− mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a+/− mice, and this communication is barely detectable in the homozygous gene KO (Scn1a−/−). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of NaV1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na+ channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of NaV1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.

Signaling Mechanisms during the Response of Pituitary Gonadotropes to GnRH

Publisher Summary The pituitary gland is a versatile hormonal interface between the nervous system and the rest of the body, a warehouse of peptides waiting for nervous requisitions. In mammals, the anterior pituitary can secrete at least six major peptide hormones from at least five cell types. Each cell is under the control of specific releasing hormones and neurotransmitters secreted into the pituitary portal circulation by hypothalamic neurons. This chapter describes a study of G protein-mediated modulation of ion channels, which involved working with muscarinic activation of a K+ channel in cardiac trial cells and GnRH and muscarinic inhibition of a K+ channel in frog sympathetic ganglia, where GnRH was a neurotransmitter. The work with GnRH has explained the activities of gondaotrope, where a chain of events is triggered by GnRH. The primary secretory response to GnRH is unambiguously attributable to a steady rise of IP3that leads to an oscillatory release of Ca2+ from intracellular stores. Each elevation brings [Ca2+]i well above the 300 nM level that suffices to initiate exocytosis of numerous secretory granules. The secretory stimulus for gonadotropes does not initiate a major entry of extracellular Ca2+ as a primary signal for exocytosis. The electrophysiological responses of stimulated gonadotropes include a hyperpolarization during periods of [Ca2+]i rise instead of a depolarization.

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

SummaryCell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/μm2, a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na+, Ca2+ and K+, but not Cl−. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca2+ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.

Midline serotonergic neurones contribute to widespread synchronized activity in embryonic mouse hindbrain

Spontaneous, synchronous activity occurs in motor neurones of the embryonic mouse hindbrain at the stage when rhombomeric segmentation disappears (embryonic day 11.5). The mechanisms generating and synchronizing the activity, however, and the extent to which it is widespread in the hindbrain, are unknown. We show here that spontaneous activity is initiated in the midline of the hindbrain, and propagates laterally to encompass virtually the entire hindbrain synchronously and bilaterally. Separation of the midline region from lateral regions abolishes or slows activity laterally, but not medially. The early differentiating neurones of the midline raphe system are present in the rostral midline and express serotonin at E11.5. Their axons ramify extensively in the marginal zone, cross the midline, and extend at the midline both rostrally into the midbrain and caudally towards the caudal hindbrain. Blockers of serotonin receptors, specifically the 5‐HT2A receptor, abolish synchronous activity in the hindbrain, while blockers of other neurotransmitter systems, including GABA and glutamate, do not. In addition, the 5‐HT2A receptor is expressed in the marginal regions in the entire medial‐to‐lateral extent of the hindbrain and in the midline commissural region. Thus, the serotonergic neurones of the developing midline raphe system may play a role in initiating and propagating spontaneous synchronous activity throughout the hindbrain.

Identification of the delayed rectifier potassium channel, Kv1.6, in Cultured astrocytes

Astrocytes are an abundant glial cell type of the central nervous system that appear to play a role in regulating extracellular potassium concentrations in brain, thereby contributing to the maintenance of normal neuronal activity. Voltage‐gated potassium conductances, shown to be present in astrocytes, may be involved in this and other astrocytic functions. Toward defining the role of voltage‐gated potassium channels in astrocytes, total RNA prepared from cultured mouse cortical astrocytes was screened, using a reverse transcriptase‐polymerase chain reaction (RT‐PCR) approach, for the expression of several members of the Shaker‐like potassium channel subfamily (Kv1.1–Kv1.6). A relatively high level of Kv1.6 transcript was identified by RT‐PCR and then confirmed and quantitated by ribonuclease protection assays using a Kv1.6‐specific riboprobe. Immunocytochemical staining showed double‐labeling of glial fibrillary acidic protein‐positive cells with antibody specific for the Kv1.6 channel. The Kv1.6 protein expression was variable among the individual astrocytes. Outward voltage‐gated currents were studied in astrocytes in primary culture using the Nystatin‐perforated patch voltage clamp technique. Outward potassium currents were observed in all cells studied, and this current was partially blocked by perfusion with 100 nM dendrotoxin (DTX) in 14 of 16 cells tested. This DTX‐sensitive current appeared to be a sustained outward potassium current, consistent with the suggestion that the Shaker‐like potassium channel Kv1.6 underlies a portion of the delayed rectifier potassium current in cultured mouse cortical astrocytes. GLIA 20:127–134, 1997.

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

SummaryWhole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca2+ currents found in this cell. The peak of the low threshold current (channel I) occurred at −20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca2+ and Mg2+ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca2+ or Mg2+ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×106m−1 sec−1 for Mg2+ and 4.5×108m−1 sec−1 for Ca2+. The Ca2+ block was somewhat relieved at negative potentials, whereas the Mg2+ block was not, suggesting that Ca2+, but not Mg2+, can exit from the binding site toward the cell interior.