Edited GluR2, a gatekeeper for motor neurone survival?

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder of motor neurones. Although the genetic basis of familial forms of ALS has been well explored, the molecular basis of sporadic ALS is less well understood. Recent evidence has linked sporadic ALS with the failure to edit key residues in ionotropic glutamate receptors, resulting in excessive influx of calcium ions into motor neurones which in turn triggers cell death. Here we suggest that edited AMPA glutamate (GluR2) receptor subunits serve as gatekeepers for motor neurone survival.

Three-dimensional culture of leech and snail ganglia for studies of neural repair.

Three-dimensional (3D) collagen gels provide a stable matrix in which isolated regenerating ganglia from leech and snail can be maintained for studies of the molecular and cellular mechanisms underlying the regenerative process. Segmental ganglia from leech, or supraoesophageal, suboesophageal or buccal ganglia from snail were maintained for up to 3 weeks in 3D matrices of mammalian Type I collagen. The collagen matrix supports the regenerative outgrowth of axon tracts as well as the migration of microglial cells, important elements in the repair process. Proteins or soluble factors or target tissue may be added to the basic collagen matrix to manipulate the environment of the regenerating tissue. We describe techniques for immunostaining of regenerating axons and microglial cells within the gel matrix in combination with staining of cell nuclei, and the use of intracellular labelling to distinguish axons of identified neurons within the regenerative outgrowth.

Hirudo medicinalis: a platform for investigating genes in neural repair.

We have used the nervous system of the medicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlike mammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity. We have constructed subtractive cDNA probes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell. This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP synthase are upregulated at 24 h postinjury in leech nerve cord. To investigate the functional role of regulated genes in neuron regrowth we are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1. Our approach thus identifies genes regulated at different times after injury that may underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.

Identifying genes for neuron survival and axon outgrowth in Hirudo medicinalis.

We have studied the molecular basis of nervous system repair in invertebrate (Hirudo medicinalis) nerve cells. Unlike in mammals, neurons in invertebrates survive injury and regrow processes to restore the connections that they held before the damage occurred. To identify genes whose expression is regulated after injury, we have used subtractive probes, constructed from regenerating and non-regenerating ganglia from the leech Hirudo medicinalis, to screen cDNA libraries made from whole leech CNS or from identified microdissected neurons. We have identified genes of known or predicted function as well as novel genes. Known genes up-regulated within hours of injury and that are widely expressed in invertebrate and mammalian cells include thioredoxin and tubulin. Other known genes, e.g. Cysteine Rich Intestinal Protein (CRIP), have previously been identified in mammalian cells though not in regenerating adult neurons. Two regulated genes identified, myohemerythrin and the novel protein ReN3 are exclusively expressed in invertebrates. Thus our approach has enabled us to identify genes, present in a neuron of known function, that are up- and down-regulated within hours of axotomy, and that may underpin the intrinsic ability of invertebrate neurons to survive damage and initiate regrowth programmes.

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech.

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.

HmCRIP, a cysteine-rich intestinal protein, is expressed by an identified regenerating nerve cell.

A Hirudo medicinalis cDNA isolated from regenerating CNS tissue at 24 h post-axotomy was identified as a leech homologue of the mammalian cysteine-rich intestinal proteins (CRIPs) and named HmCRIP. HmCRIP is up-regulated within 6 h of axotomy, peaking at 24 h. This is the first demonstration of a CRIP homologue in regenerating CNS and in a serotonergic neurone. In rodents CRIP is an important factor in the regulation of the inflammatory immune response through control of Th1/Th2 differentiation. The role of HmCRIP in the regeneration competent environment of the annelid central nervous system is discussed.

Developmentally regulated markers in the postnatal cervical spinal cord of the opossum Monodelphis domestica.

The aim of this study was to identify developmentally regulated immunocytochemical markers to assess development in the cervical spinal cord of Monodelphis domestica. We demonstrate that two commercially available antibodies exhibit altered patterns of distribution during early postnatal development. Although neurofilament staining was present at birth, only the phosphorylated form, recognised by monoclonal antibodies 2F11 or SMI31 could be detected. Non-phosphorylated neurofilament, recognised by monoclonal antibody SMI32, only became detectable around postnatal day 4 (P4) but was restricted to cells in the ventral horn until 5 weeks postnatum. By 7.5 weeks, SMI32 immunoreactivity (IR) was found throughout the grey matter in a pattern similar to that in the adult for both SMI32 and microtubule-associated protein 2 (MAP2). The intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM), were detectable at birth in radially oriented, fibrous cells, but GFAP-IR was restricted to the ventral half of the cord. This ventral to dorsal gradient of GFAP-IR diminished during the first week of postnatal life, disappearing by P8. Many astrocyte-like, GFAP-positive cells were clearly present by 38 days and, in the adult, were abundant in the white matter. A few VIM-IR cells remained in the adult cord, also within the white matter. We suggest that SMI32 and GFAP are useful, developmentally regulated markers for studies of opossum spinal cord development. We are currently using these markers to investigate the pronounced rostral to caudal gradient in the postnatal spinal cord and to assess development in the cultured spinal cord.

Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels.

We describe the application of three-dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type I collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site: and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.

A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration.

We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

cDNA libraries from identified neurons.

One approach to studying the changes in gene expression which underlie differentiation is to construct cDNA libraries from different tissues or at different stages of development. However, generating representative cDNA libraries from heterogeneous tissues such as the nervous system is often a real problem. Here, we describe a reproducible method for the construction of large and complex cDNA libraries from a few leech Retzius or P neurons (equivalent to about 50 pg of mRNA) using polymerase chain reaction-based technology. The libraries contain about 10(6) independent recombinants and are remarkably free from contaminating rRNA or polymerase chain reaction artefacts. Sequence analysis of randomly picked clones shows that the libraries contain a high proportion (more than 90%) of cDNAs larger than 500 b.p. As expected, many of the clones are novel, but two (alpha-tubulin and cyclophilin-A) have been extensively characterized in other species. To our knowledge, this is the first report of a cDNA library from identified neurons.

Advantages of using microfabricated extracellular electrodes for in vitro neuronal recording.

We describe fabrication methods and the characterisation and use of extracellular microelectrode arrays for the detection of action potentials from neurons in culture. The 100 microns2 platinised gold microelectrodes in the 64 electrode array detect the external current which flows during an action potential with S:N ratios of up to 500:1, giving a maximum recorded signal of several millivolts. The performance of these electrodes is enhanced if good sealing of the cells over the electrodes is obtained and further enhanced if the electrodes and the cells lie in a deep groove in the substratum. The electrodes can be used for both recording and stimulation of activity in cultured neurons and for recording from multiple sites on a single cell. The use of such electrodes to obtain recordings from invertebrate neurons is described. The particular advantages of these electrodes, their long term stability, non-invasive nature, high packing density, and utility in stimulation, are demonstrated.

Simultaneous multisite recordings and stimulation of single isolated leech neurons using planar extracellular electrode arrays.

Planar extracellular electrode arrays provide a non-toxic, non-invasive method of making long-term, multisite recordings with moderately high spatial frequency (recording sites per unit area). This paper reports advances in the use of this approach to record from and stimulate single identified leech neurons in vitro. A modified enzyme treatment allowed identified neurons to be extracted with very long processes. Multisite extracellular recordings from the processes of such isolated neurons revealed both the velocity and direction of action potential propagation. Propagation in two cell types examined was from the broken stump towards the cell body (antidromic). This was true for spontaneous action potentials, action potentials produced by injecting current into the cell body and extracellular stimulation of the extracted process via a planar extracellular electrode. These results extend previous findings which have shown that the tip of the broken stump of extracted neurons has a high density of voltage-activated sodium channels. Moreover they demonstrate the applicability of extracellular electrode arrays for recording the electrical excitability of single cells.

Stretch receptors and body wall muscle in leeches.

1. A new kind of muscle receptor has been described in the medicinal leech, the first stretch receptor in a soft-bodied invertebrate to be identified and characterized. 2. The sensory responses to stretch in these receptors are different from those of other more well known muscle receptors such as vertebrate spindles or crustacean stretch receptors. 3. The leech receptors provide the sensory innervation for the tubular muscle layers of the body wall, and they respond to stretch of the muscle with hyperpolarizing potentials that are conducted passively to the CNS. 4. This review summarizes their unusual morphology, membrane properties and sensory responses to stretch; their association with particular muscle fibre types in leech body wall; their synaptic connections; and their role in a well characterized rhythmic behaviour in the leech-swimming.

New growth elicited in adult leech mechanosensory neurones by peripheral axon damage.

1. 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. 2. 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. 3. 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. 4. 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. 5. Sprouted processes of axotomized neurones are retained for long periods after the lesion (up to 425 days). 6. 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.

Hyperpolarizing responses to stretch in sensory neurones innervating leech body wall muscle.

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)

Physiological responses, receptive fields and terminal arborizations of nociceptive cells in the leech.

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.

Expanded receptive fields of cutaneous mechanoreceptor cells after single neurone deletion in leech central nervous system.

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.

Morphology and distribution of touch cell terminals in the skin of the leech.

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.