Laura Lossi

BDNF as a pain modulator

At least some neurotrophins may be powerful modulators of synapses, thereby influencing short- and long-term synaptic efficiency. BDNF acts at central synapses in pain pathways both at spinal and supraspinal levels. Neuronal synthesis, subcellular storage/co-storage and release of BDNF at these synapses have been characterized on anatomical and physiological grounds, in parallel with trkB (the high affinity BDNF receptor) distribution. Histological and functional evidence has been provided, mainly from studies on acute slices and intact animals, that BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotrasmission in spinal cord. Recent studies have unraveled some of the neuronal circuitries and mechanisms involved, highlighting the key role of synaptic glomeruli in lamina II as the main sites for such a modulation.

In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS

Apoptosis has been recognized to be an essential process during neural development. It is generally assumed that about half of the neurons produced during neurogenesis die before completion of the central nervous system (CNS) maturation, and this process affects nearly all classes of neurons. In this review, we discuss the experimental data in vivo on naturally occurring neuronal death in normal, transgenic and mutant animals, with special attention to the cerebellum as a study model. The emerging picture is that of a dual wave of apoptotic cell death affecting central neurons at different stages of their life. The first wave consists of an early neuronal death of proliferating precursors and young postmitotic neuroblasts, and appears to be closely linked to cell cycle regulation. The second wave affects postmitotic neurons at later stages, and is much better understood in functional terms, mainly on the basis of the neurotrophic concept in its broader definition. The molecular machinery of late apoptotic death of postmitotic neurons more commonly follows the mitochondrial pathway of intracellular signal transduction, but the death receptor pathway may also be involved.Undoubtedly, analysis of naturally occurring neuronal death (NOND) in vivo will offer a basis for parallel and future studies aiming to elucidate the mechanisms of pathologic neuronal loss occurring as the result of conditions such as neurodegenerative disorders, trauma or ischemia.

Ghrelin in Central Neurons

Ghrelin, an orexigenic peptide synthesized by endocrine cells of the gastric mucosa, is released in the bloodstream in response to a negative energetic status. Since discovery, the hypothalamus was identified as the main source of ghrelin in the CNS, and effects of the peptide have been mainly observed in this area of the brain. In recent years, an increasing number of studies have reported ghrelin synthesis and effects in specific populations of neurons also outside the hypothalamus. Thus, ghrelin activity has been described in midbrain, hindbrain, hippocampus, and spinal cord. The spectrum of functions and biological effects produced by the peptide on central neurons is remarkably wide and complex. It ranges from modulation of membrane excitability, to control of neurotransmitter release, neuronal gene expression, and neuronal survival and proliferation. There is not at present a general consensus concerning the source of ghrelin acting on central neurons. Whereas it is widely accepted that the hypothalamus represents the most important endogenous source of the hormone in CNS, the existence of extra-hypothalamic ghrelin-synthesizing neurons is still controversial. In addition, circulating ghrelin can theoretically be another natural ligand for central ghrelin receptors. This paper gives an overview on the distribution of ghrelin and its receptor across the CNS and critically analyses the data available so far as regarding the effects of ghrelin on central neurotransmission.

Neuropeptides as synaptic transmitters

Neuropeptides are small protein molecules (composed of 3–100 amino-acid residues) that have been localized to discrete cell populations of central and peripheral neurons. In most instances, they coexist with low-molecular-weight neurotransmitters within the same neurons. At the subcellular level, neuropeptides are selectively stored, singularly or more frequently in combinations, within large granular vesicles. Release occurs through mechanisms different from classical calcium-dependent exocytosis at the synaptic cleft, and thus they account for slow synaptic and/or non-synaptic communication in neurons. Neuropeptide co-storage and coexistence can be observed throughout the central nervous system and are responsible for a series of functional interactions that occur at both pre- and post-synaptic levels. Thus, the subcellular site(s) of storage and sorting mechanisms into different neuronal compartments are crucial to the mode of release and the function of neuropeptides as neuronal messengers.

Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS

Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro. We will discuss here the main advantages/disadvantages linked to the use of CNS slices for histological analysis of neuronal proliferation and death, as well as the main findings obtained in the most popular types of preparations, i.e. the cortical, hippocampal, cerebellar and retinal slices.

Neurotrophins in spinal cord nociceptive pathways.

Neurotrophins are a well-known family of growth factors for the central and peripheral nervous systems. In the course of the last years, several lines of evidence converged to indicate that some members of the family, particularly NGF and BDNF, also participate in structural and functional plasticity of nociceptive pathways within the dorsal root ganglia and spinal cord. A subpopulation of small-sized dorsal root ganglion neurons is sensitive to NGF and responds to peripheral NGF stimulation with upregulation of BDNF synthesis and increased anterograde transport to the dorsal horn. In the latter, release of BDNF appears to modulate or even mediate nociceptive sensory inputs and pain hypersensitivity. We summarize here the status of the art on the role of neurotrophins in nociceptive pathways, with special emphasis on short-term synaptic and intracellular events that are mediated by this novel class of neuromessengers in the dorsal horn. Under this perspective we review the findings obtained through an array of techniques in naïve and transgenic animals that provide insight into the modulatory mechanisms of BDNF at central synapses. We also report on the results obtained after immunocytochemistry, in situ hybridization, and monitoring intracellular calcium levels by confocal microscopy, that led to hypothesize that also NGF might have a direct central effect in pain modulation. Although it is unclear whether or not NGF may be released at dorsal horn endings of certain nociceptors in vivo, we believe that these findings offer a clue for further studies aiming to elucidate the putative central effects of NGF and other neurotrophins in nociceptive pathways.

Ultrastructural evidence for a pre- and postsynaptic localization of full-length trkB receptors in substantia gelatinosa (lamina II) of rat and mouse spinal cord

Brain‐derived neurotrophic factor (BDNF) exerts its trophic effects by acting on the high‐affinity specific receptor trkB. BDNF also modulates synaptic transmission in several areas of the CNS, including the spinal cord dorsal horn, where it acts as a pain modulator by yet incompletely understood mechanisms. Spinal neurons are the main source of trkB in lamina II (substantia gelatinosa). Expression of this receptor in dorsal root ganglion (DRG) cells has been a matter of debate, whereas a subpopulation of DRG neurons bears trkA receptors and contains BDNF. By the use of two different trkB antibodies we observed that 7.7% and 10.8% of DRG neurons co‐expressed BDNF + trkB but not trkA, respectively, in rat and mouse. Ultrastructurally, full‐length trkB (fl‐trkB) receptors were present at somato‐dendritic membranes of lamina II neurons (rat: 66.8%; mouse: 73.8%) and at axon terminals (rat: 33.2%; mouse: 26.2%). In both species, about 90% of these terminals were identified as primary afferent fibres (PAFs) considering their morphology and/or neuropeptide content. All fl‐trkB‐immunopositive C boutons in type Ib glomeruli were immunoreactive for BDNF and, at individual glomeruli and axo‐dendritic synapses, fl‐trkB receptors were located in a mutually exclusive fashion at pre‐ or postsynaptic membranes. Thus, only a small fraction of fl‐trkB‐immunoreactive dendrites were postsynaptic to BDNF‐immunopositive PAFs. This is the first ultrastructural description of fl‐trkB localization at synapses between first‐ and second‐order sensory neurons in lamina II, and suggests that BDNF may be released by fl‐trkB‐immunopositive PAFs to modulate nociceptive input in this lamina of dorsal horn.

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord.

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full‐length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch‐clamp recordings, calcium imaging and immunocytochemistry on slices from 8–12 days post‐natal rats. In this preparation, BDNF (100–500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN‐PAFs). Effect was blocked by trk antagonist K252a or anti‐trkB antibody clone 47. A pre‐synaptic mechanism was demonstrated after (i) patch‐clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA‐mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK1 receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN‐PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN‐PAFs. This is the first demonstration that trkB receptors expressed by PN‐PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.

The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn

Ghrelin is mainly described for its effects on feeding behavior and metabolism. However, central nervous system distribution of its receptor [type 1a GH secretagogue receptor (GHSR)] and modulation of neurotransmission in the hypothalamus suggest broader effects than originally predicted. Systemically administrated ghrelin inhibits inflammatory pain after behavioral observations. Therefore, we investigated the expression and function of type 1a GHSR in mouse spinal cord by molecular biology, biochemistry, histology, and electrophysiology. The mRNA and protein were detected in tissue extracts by RT-PCR and Western blotting. In situ, receptor mRNA and immunoreactivity were localized to cell bodies within the medial aspect of the deep dorsal horn. Patch clamp recordings on laminae IV-VI demonstrated that bath-applied ghrelin (100 nm) induced a strong increase of spontaneous gamma-aminobutyric acid/glycine-mediated current frequency (463 +/- 93% of the control) and amplitude (150 +/- 16% of the control) in about 60% of recorded neurons. Specificity of type 1a GHSR activation was confirmed by the lack of effect of the deacylated form of ghrelin (des-acyl-ghrelin) and after preincubation with the specific receptor antagonist [d-Lys(3)]GHRP-6. In the presence of tetrodotoxin, the effect of the peptide was strongly reduced, mainly indicating an action potential-dependent mechanism. The functional link between ghrelin and pain was confirmed by inhibition in vitro of the c-fos response to capsaicin activation of nociceptive fibers, after quantification of Fos-immunoreactive nuclei in laminae IV-VI. Our results are the first demonstration of the presence of functional type 1a GHSRs in the spinal cord and indicate that ghrelin may exert antinociceptive effects by directly increasing inhibitory neurotransmission in a subset of deep dorsal horn neurons.

Molecular morphology of neuronal apoptosis: analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex.

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.