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Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

The tumor suppressor patched1 (PTC1), a product of the mammalian homologue of the Drosophila segment polarity gene patched, is a receptor for hedgehog (HH) and is crucial for embryonic development. Although little is known about the signal transduction pathways leading to the activation of ptc1, increased ptc1 transcription has always been associated with elevated HH activity and decreased activity of cAMP-dependent protein kinase A. Here, we demonstrate that in the mammalian pineal gland, ptc1 expression exhibits a dramatic diurnal rhythm with peak expression at midnight. ptc1 mRNA expression in the pineal is regulated by a clock mechanism mediated by the superior cervical ganglion. Most importantly, ptc1 transcription can be induced by agents activating the cAMP signal transduction pathway both in vivo and in vitro and appears to be independent of HH signaling.

1. The membrane properties, morphology and physiological responses of peripherally located sensory neurones that innervate body wall muscle of the leech Hirudo medicinalis have been investigated using intracellular recording and dye injection techniques. 2. The peripheral neurones and their dendrites were visualized directly in whole mounts of the body wall by intracellular injection of horseradish peroxidase or Lucifer Yellow. They lie along the course of segmental nerves between the layers of longitudinal and oblique body wall muscle and within the sheath of the nerve. They have a distinctive morphology with two expanded, fan‐shaped dendrites arranged in series separated by the cell body and a 300 micron long cylindrical process. Both dendrites are associated with longitudinal muscle of the ventral body wall but with separate bands of muscle fibres. The axons project into the ventral nerve cord and arborize within the ipsilateral half of the segmental ganglion. No processes extend across the mid‐line of the ganglion or enter the connectives to neighbouring ganglia. 3. 'Resting' membrane potentials recorded from the peripheral cell body or from the axon as it entered the segmental ganglion ranged from ‐30 to ‐70 mV. The transmembrane potential recorded depended on the amount by which the body wall was stretched: the most hyperpolarized values were recorded from the most stretched preparations. Although the peripheral cell body can generate overshooting action potentials these are not actively propagated to the CNS. Rather, imposed voltage changes spread decrementally along the axon. Input resistances measured in the cell body ranged from 14 to 26 M omega. The space constant, estimated from the spread of hyperpolarizing current injected into the cell body, was 2.4 mm. 4. The response of the neurones to change in length of the longitudinal muscle recorded from the axon near its terminal arborization within the ventral nerve cord is a graded DC signal: the neurones thus relay information to CNS synapses in analogue form. Spiking activity recorded extracellularly in the anterior segmental nerve root in response to stretch of the body wall is due to activation of touch mechanosensory cells that innervate the skin. 5. Unlike stretch receptors innervating skeletal muscle in vertebrates or arthropods, the leech neurones respond to stretch of the body wall muscle with maintained hyperpolarizing potentials and to release of stretch with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Electrical connexions between mesoderm cells have been examined during the formation of somites in Xenopus laevis, Bombina orientalis and Ambystoma mexicanum. 2. In Xenopus the resting potentials of presumptive myotome cells (‐65 + 2 mV, S.E. of mean) and somite muscle cells (‐65 +/‐ 0‐6 mV S.E. of mean) were 40 mV, greater than dermatome cells (‐25 +/‐ 0‐6 mV, S.E. of mean). Similar differences were found in Bombina and Ambystoma. 3. In all three species cells of the dermatome layer of the mesoderm were electrically coupled to each other. Cells of the presumptive myotome layer in the unsegmented region of the mesoderm were also electrically coupled. 4. In Xenopus dermatome and myotome layers of the mesoderm were not electrically coupled to each other either before or after somite formation. In the other two species dermatome and myotome layers were uncoupled once the somites had formed. 5. In all three species the position of the intersomite border in the unsegmented mesoderm region is marked by the breaking of electrical contracts between those cells destined to form the next somite and the rest of the unsegmented mesoderm. 6. In the axolotl each somite remains electrically insulated from its neighbour. In Xenopus and Bombina electrical connexions are re‐established between somite muscle cells once the morphogenetic movements underlying somite formation are complete. 7. Presumptive myotome cells in Xenopus and Ambystoma acquire the membrane property of inward‐going rectification before incorporation into a somite. 8. Once Xenopus and Bombina embryos show spontaneous movements large end‐plate potentials are recorded from the head somites. Excitation spreads from somite to somite along the low resistance intercellular pathway allowing simultaneous contraction of several somites before extensive somite innervation. 9. The structure of developing somite muscle of Xenopus has been studied with the electron microscope. 10. Close membrane contacts of the gap junction type have been seen between undifferentiated presumptive myotome cells, muscle cells in the same somite and between muscle cells in adjacent somites. 11. Myofilament organization first begins in mesoderm cells when they are forming a new somite. Complete sarcomeres appear in the head somites when the embryo begins spontaneous flexion movements.

1. The mean resting membrane potential of cells in the neural plate of axolotl embryos increases from ‐25 to ‐45 mV (maximum values from ‐35 to ‐60 mV) when the embryos move from early to mid‐neural plate stages of development. 2. Increasing the extracellular [K] to 20 mM shortly before the spontaneous increase in resting potential occurs causes neural plate cells to hyperpolarize. 3. A moderate increase in [K]omicron does not hyperpolarize the membrane of neural plate cells at earlier stages or at late neural fold stages. 4. Both the hyperpolarization produced by K and the normal increase in resting potential are blocked by cardiac glycosides. 5. Cells of the ventral ectoderm do not display an increase in resting potential during the neural fold stages of development and do not hyperpolarize in response to a moderate increase in [K]omicron. 6. The results suggest that activation of the Na pump may be an early consequence of neural induction. They are discussed in the light of this suggestion.

1. The receptor terminals of individual mechanosensory neurones responding to light touch (T cells) have been visualized directly in the skin of the leech by injecting horseradish peroxidase (HRP) into their cell bodies in the central nervous system. The axons of injected cells could be followed from their origin in the neuropile of the ganglion to their terminals in the skin. 2. The axons of T cells run through ipsilateral nerve roots in the body wall to the base of the layer of epithelial cells in the skin. Here axons branch extensively and turn between the epithelial cells to end a few microns from the skin surface. These terminals are situated in intercellular spaces immediately below the junctional complex joining the outer ends of the epithelial cells. 3. The T cell terminals are free nerve endings with a beaded appearance; they contain large mitochondria and clusters of vesicles. 4. An individual T cell makes about 100 endings within a square millimetre of skin in the centre of its territory, and is estimated to make a total of several hundred endings. 5. The distribution of T cell endings observed directly agrees with physiological studies of receptive field organization and emphasizes the high degree of specificity of connexions of these neurones with their peripheral targets.

The physiological responses, receptive fields and morphology of individual nociceptor (N) neurones have been studied in the leech. In each of the midbody ganglia there are four N cells (two on either side). Each N cell has a distinctive territory that it supplies in the periphery, on the surface or internally. 1. Both N cells respond selectively to noxious mechanical stimuli applied to the skin but not to touch, light, pressure or stretch. The receptive field of each cell is well defined and covers roughly the same area, extending from the dorsal midline to the ventral midline, with considerable overlap. 2. One of the N cells, situated more medially in the ganglion, also fires at high frequencies in response to mechanical stimulation, such as pinching or squeezing, of the connective tissue lining the viscera. In contrast, the other N cell (situated laterally in the ganglion) is activated by pressure or pinches applied to the opening of the excretory duct but not the gut. 3. Following injection of horseradish peroxidase into the soma, axons of N cells appear as unspecialized fine processes about 1 micrometer in diameter, in the dermis of the leech, deep to the layer of epidermal cells. In addition, at specific sites in the skin, the N cell situated laterally in the ganglion makes distinctive coiled terminals in association with the expanded dendrites of large neurones in the periphery, the functions of which are unknown. This finding raises the possibility that lateral N cells may perform some additional role as yet not understood.

1. Individual sensory neurones responding to touch (T) and to noxious (N) stimuli applied to the skin of the leech were killed by injecting pronase into their cell bodies, situated within the C.N.S. This procedure destroys one neurone in its entirety without damaging the cells. 2. When three out of four N cells within a ganglion have been killed, the receptive field of the remaining N sensory cell expands to cover the denervated area of skin. Similarly the field of the touch cell that innervates dorsal skin spreads across the mid line to innervate contralateral skin after the three touch cells on that side have been deleted. 3. The spread is graded and develops with time. The earliest effects appear within 4 weeks and the full spread develops by 3 months. 4. No detectable spread of receptive fields occurs if only two N cells, one on each side, are killed. 5. Following deletion of N cells, the receptive fields of T and pressure sensory cells are unaffected. Similarly, if T cells have been killed, the fields of N cells or pressure cells do not become enlarged. 6. These results represent a modality‐specific mechanism by which one sensory cell can be influenced to extend the territory it supplies in the periphery in response to a minimal lesion without its own terminals having been damaged.

Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurin inhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins.

We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.

New growth in cutaneous mechanosensory neurones elicited by axotomy or axon crush was studied using intracellular injection of horseradish peroxidase at different times after the lesion, ranging from a few days to over a year. Cutting or crushing major, large-calibre axon branches of mechanosensory neurones elicits sprouting of new processes, either centrally within the ganglion neuropile or at the site of the lesion in the peripheral nerve. In contrast, cutting or crushing fine-calibre axon branches supplying accessory parts of the receptive field does not elicit sprouting of the main arbor or main axon branches. Different modalities of mechanosensory neurone respond differently to lesions of their axons. Cutting the axons of high-threshold units responding to noxious stimulation of the skin elicits sprouting of additional processes from the axon hillock region within the central nervous system (CNS), whereas cutting or crushing the axons of low-threshold cells responding to light touch of the skin elicits sprouting at the site of the lesion only, and not within the CNS. In addition to the new growth directed into the peripheral nerve, damaged nociceptive neurones also form new processes that wrap the somata of particular cells within the ganglion. Sprouted processes of axotomized neurones are retained for long periods after the lesion (up to 425 days). The electrical properties of touch and nociceptive cells were studied between 1 and 60 days after axotomy, by intracellular recording from the centrally located cell bodies. The amplitude, width and maximum dV/dt of the action potential and after-hyperpolarization, as well as the resting potential and input resistance, did not change significantly after axotomy, despite the considerable process sprouting known to occur during this time.