Preplaque ('preclinical') Aß-induced inflammation and nerve growth factor deregulation in transgenic models of Alzheimer's disease-like amyloid pathology.

BACKGROUND: Alzheimer's disease (AD) neuropathology likely begins decades before clinical symptoms are manifested. Investigations on the early stages of the amyloid pathology are crucial for the discovery of diagnostic biomarkers or new therapeutic targets. Our transgenic (tg) animal models are most suitable to study early AD pathological events, as the pathology evolves in a well-staged manner, starting with intracellular Aß accumulation and ending with plaque deposition. OBJECTIVE: To determine the occurrence of key inflammatory markers and to look for signs of nerve growth factor (NGF) dysmetabolism at preplaque and postplaque stages in tg models of AD-like amyloid pathology and in human AD brains. METHODS: We used our own tg lines (mice and rat), high-quality human brain material and applied neurochemical and immunohistochemical experimental approaches. RESULTS: In both tg models, we observed an intracellular accumulation of oligomeric Aß in cortical and hippocampal pyramidal neurons. This coincided with an upregulation of key inflammatory markers (iNOS, MHCII, COX-2) and a recruitment of microglia towards Aß-burdened neurons. Using human AD brains, we showed alterations in the NGF metabolic pathway, which were mirrored in our tg rat model at early and late stages of amyloid plaque generation. CONCLUSION: A proinflammatory process and, consequently, the deregulation of the NGF metabolic pathway could be amongst the earliest pathological events in the progression of AD.

Liver X Receptor Activation with an Intranasal Polymer Therapeutic Prevents Cognitive Decline without Altering Lipid Levels.

The progressive accumulation of amyloid-beta (Aß) in specific areas of the brain is a common prelude to late-onset of Alzheimer's disease (AD). Although activation of liver X receptors (LXR) with agonists decreases Aß levels and ameliorates contextual memory deficit, concomitant hypercholesterolemia/hypertriglyceridemia limits their clinical application. DMHCA (N,N-dimethyl-3ß-hydroxycholenamide) is an LXR partial agonist that, despite inducing the expression of apolipoprotein E (main responsible of Aß drainage from the brain) without increasing cholesterol/triglyceride levels, shows nil activity in vivo because of a low solubility and inability to cross the blood brain barrier. Herein, we describe a polymer therapeutic for the delivery of DMHCA. The covalent incorporation of DMHCA into a PEG-dendritic scaffold via carboxylate esters produces an amphiphilic copolymer that efficiently self-assembles into nanometric micelles that exert a biological effect in primary cultures of the central nervous system (CNS) and experimental animals using the intranasal route. After CNS biodistribution and effective doses of DMHCA micelles were determined in nontransgenic mice, a transgenic AD-like mouse model of cerebral amyloidosis was treated with the micelles for 21 days. The benefits of the treatment included prevention of memory deterioration and a significant reduction of hippocampal Aß oligomers without affecting plasma lipid levels. These results represent a proof of principle for further clinical developments of DMHCA delivery systems.

Early-stage inflammation and experimental therapy in transgenic models of the Alzheimer-like amyloid pathology.

BACKGROUND: Intracellular accumulation of beta-amyloid (Abeta) is one of the early features in the neuropathology of Alzheimer's disease (AD) and Down's syndrome. This can be reproduced in cell and transgenic animal models of the AD-like amyloid pathology. In a transgenic rat model, our lab has previously shown that the intracellular accumulation of Abeta is sufficient to provoke cognitive impairments and biochemical alterations in the cerebral cortex and hippocampus in the absence of amyloid plaques. OBJECTIVE: To investigate an early, pre-plaque inflammatory process in AD-like transgenic models and establish whether the neurotoxic effects of Abeta oligomers and proinflammatory responses can be arrested with minocycline. METHODS: For these studies, we used naïve mice and transgenic animal models of the AD-like amyloid pathology and applied neurochemical, immunohistochemical and behavioral experimental approaches. RESULTS: In the early stages of the AD-like amyloid pathology, intracellular Abeta oligomers accumulate within neurons of the cerebral cortex and hippocampus. Coincidental with this, behavioral impairments occur prior to the appearance of amyloid plaques, together with an upregulation of MHC-II, i-NOS and COX-2, well-known proinflammatory markers. Treatment with minocycline corrected behavioral impairments, lowered inflammatory markers and levels of Abeta trimers. CONCLUSION: A pharmacological approach targeting the early neuroinflammatory effects of Abeta might be a promising strategy to prevent or delay the onset of AD.

Variations in excitatory and inhibitory postsynaptic protein content in rat cerebral cortex with respect to aging and cognitive status.

Age-related cognitive impairments are associated with structural and functional changes in the cerebral cortex. We have previously demonstrated in the rat that excitatory and inhibitory pre- and postsynaptic changes occur with respect to age and cognitive status; however, in aged cognitively impaired animals, we have shown a significant imbalance in postsynaptic markers of excitatory versus inhibitory synapses, using markers of excitatory versus inhibitory neurotransmitter-related scaffolding proteins [postsynaptic density-95 (PSD95)/synapse associated protein-90 (SAP90) and gephyrin, respectively]. The present study focuses on whether the expression of various excitatory and inhibitory postsynaptic proteins is affected by ageing and cognitive status. Thus, aged animals were segregated into aged cognitively impaired (AI) and aged cognitively unimpaired (AU) groups using the Morris water maze. We applied Western immunoblotting to reveal the expression patterns of a number of relevant excitatory and inhibitory receptors in the prefrontal and parietal cortices of young (Y), AU and AI animals, and performed semi-quantitative analyses to statistically tabulate changes among the three animal groups. A significant increase in the inhibitory postsynaptic scaffold protein, gephyrin, was observed in the parietal cortex of AI animals. Similarly, an increase in GABA(A) receptor subunit alpha1 was observed in the parietal cortex of AI animals. An increase in the excitatory N-methyl-d-aspartate receptor subunit N-methyl-d-aspartate receptor 1 expression was observed in the parietal cortex of AI animals, whereas a significant decrease in AMPA receptor subunit glutamate receptor 2 expression was found in the prefrontal cortex of AI animals. Finally, the excitatory, postsynaptic neuronal cell-adhesion receptor, neuroligin-1, was found to be significantly increased in both the prefrontal and parietal cortical areas of AI animals.

Substance P and cholecystokinin-like peptides in Helix neurons and cholecystokinin and serotonin in a giant neuron.

Immunohistochemical procedures revealed that the serotonin-containing cell situated in each metacerebral ganglion of the snail Helix also contains a cholecystokinin-like peptide, but is devoid of substance P. In radioimmunoassays the cholecystokinin-like material showed similarities to the carboxy terminal regions of mammalian cholecystokinin and gastrin. Since much is known about the morphology and synaptic connections of this serotonin neuron, the role of the cholecystokinin-like peptides can now be investigated by neurophysiological methods, thus opening the way to discovering whether a neuron can use more than one transmitter.

Revisiting the Cholinergic Hypothesis in Alzheimer's Disease: Emerging Evidence from Translational and Clinical Research.

Scientific evidence collected over the past 4 decades suggests that a loss of cholinergic innervation in the cerebral cortex of patients with Alzheimer's disease is an early pathogenic event correlated with cognitive impairment. This evidence led to the formulation of the "Cholinergic Hypothesis of AD" and the development of cholinesterase inhibitor therapies. Although approved only as symptomatic therapies, recent studies suggest that long-term use of these drugs may also have disease-modifying benefits. A Cholinergic System Workgroup reassessed the role of the cholinergic system on AD pathogenesis in light of recent data, including neuroimaging data charting the progression of neurodegeneration in the cholinergic system and suggesting that cholinergic therapy may slow brain atrophy. Other pathways that contribute to cholinergic synaptic loss and their effect on cognitive impairment in AD were also reviewed. These studies indicate that the cholinergic system as one of several interacting systems failures that contribute to AD pathogenesis.

NGF-cholinergic dependency in brain aging, MCI and Alzheimer's disease.

Forebrain cholinergic neurons are highly dependent on nerve growth factor (NGF) for phenotype maintenance. We have established that in addition to "target-derived" NGF neurotrophic stimulation, cholinergic neurons also respond dose-dependently, to intra-parenchymal NGF administration in the somato-dendritic region of the nucleus Basalis, thus illustrating the potential of alternative reparative therapies which would by-pass the undesirable effects of diffuse neurotrophin application. Moreover, our lab has also observed that the steady-state number of cortical cholinergic synapses is dependent on continuous NGF supply, as anti-NGF monoclonal antibodies and TrkA receptor antagonists deplete pre-existing cholinergic bouton numbers. Furthermore, the application of either NGF or TrkA NGF-mimetic agonists successfully rescues the age-dependent loss of cortical cholinergic boutons in aged-impaired rats. The vulnerability of the cortical cholinergic system has also been demonstrated in transgenic animal models of the Alzheimer's disease (AD) amyloid pathology. It is of interest to note however, that an up-regulation of cholinergic presynaptic boutons has been observed in certain transgenic mouse models prior to plaque formation. This observation is similar to the visibly increased immunoreactivity of cortical and hippocampal choline acetyltransferase (ChAT) fibers in patients with Mild Cognitive Impairment (MCI). A series of ex-vivo experiments conducted by our group have demonstrated that contrary to popular belief, proNGF, as opposed to mature NGF, is released from the cerebral cortex in an activity-dependent manner. In addition, proNGF appears to be released with a series of pro-enzymes and enzymes, which are involved in its subsequent maturation to NGF and degradation in the extracellular space. Given that proNGF is known to be upregulated in AD patients a dysregulation in the maturation or degradation of mature NGF might explain the preferential vulnerability of the cholinergic system in the AD pathology.

BACE1 inhibition by microdose lithium formulation NP03 rescues memory loss and early stage amyloid neuropathology.

Lithium is first-line therapy for bipolar affective disorder and has recently been shown to have protective effects in populations at risk for Alzheimer's disease (AD). However, the mechanism underlying this protection is poorly understood and consequently limits its possible therapeutic application in AD. Moreover, conventional lithium formulations have a narrow therapeutic window and are associated with a severe side effect profile. Here we evaluated a novel microdose formulation of lithium, coded NP03, in a well-characterized rat model of progressive AD-like amyloid pathology. This formulation allows microdose lithium delivery to the brain in the absence of negative side effects. We found that NP03 rescued key initiating components of AD pathology, including inactivating GSK-3ß, reducing BACE1 expression and activity, and reducing amyloid levels. Notably, NP03 rescued memory loss, impaired CRTC1 promoter binding of synaptic plasticity genes and hippocampal neurogenesis. These results raise the possibility that NP03 be of therapeutic value in the early or preclinical stages of AD.

Loss of presynaptic and postsynaptic structures is accompanied by compensatory increase in action potential-dependent synaptic input to layer V neocortical pyramidal neurons in aged rats.

Reduction in both presynaptic and postsynaptic structures in the aging neocortex may significantly affect functional synaptic properties in this area. To directly address this issue, we combined whole-cell patch-clamp recording of spontaneously occurring postsynaptic currents (PSCs) with morphological analysis of layer V pyramidal neurons in the parietal cortex of young adult (1- to 2-month-old) and aged (28- to 37-month-old) BN x F344 F(1) hybrid rats. Analysis of spontaneous PSCs was used to contrast functional properties of basal synaptic input with structural alterations in the dendritic tree of pyramidal neurons and density of terminals in contact with these cells. We observed significant changes in a number of morphological parameters of pyramidal neurons in aged rats. These include smaller cell body size and fewer basal dendritic branches (but not of oblique dendrites and dendritic tufts) and spines. Ultrastructural analysis also revealed a lower density of presynaptic terminals per unit length of postsynaptic membrane of labeled pyramidal neurons in the aged brain. This reduction in both presynaptic and postsynaptic elements was paralleled by a significant decrease in frequency of tetrodotoxin-insensitive miniature (action potential-independent) PSCs (mPSCs). The frequency of excitatory and inhibitory mPSCs was reduced to the same extent. In contrast, no significant change was observed in the frequency of spontaneous PSCs recorded in absence of tetrodotoxin (sPSCs), indicating an increase in action potential-dependent (frequency(sPSCs) - frequency(mPSCs)) input to pyramidal neurons in the aged group. This functional compensation may explain the lack of drastic loss of spontaneous neuronal activity in normal aging.

Endogenous beta-amyloid peptide synthesis modulates cAMP response element-regulated gene expression in PC12 cells.

Extracellular-regulated kinases play a fundamental role in several neuroplasticity processes. In order to test whether endogenous beta-amyloid peptides play a role in the activation of extracellular-regulated kinase, we investigated the Rap1-extracellular-regulated kinase pathway in PC12 cells expressing human beta-amyloid precursor protein containing familial Alzheimer's disease mutations. In PC12 cells transfected with mutant human beta-amyloid precursor proteins that lead to higher levels of endogenous beta-amyloid, we observed an up-regulation of phospho-extracellular-regulated kinase and higher levels of activity-induced cAMP response element-directed gene expression. These results suggest that moderate levels of endogenous beta-amyloid peptides stimulate cAMP response element-directed gene expression. This stimulation was via a Rap1/MEK/extracellular-regulated kinase signaling pathway, as it was blocked by inhibition of Rap1 and MEK activities, and it requires beta-amyloid precursor protein cleavage at the gamma-site as it was abolished by a gamma-secretase inhibitor. Interestingly, in agreement with the previous observations, micromolar levels of extracellular fibrillar beta-amyloid blocked the cAMP response element-regulated gene expression stimulated by potassium and forskolin. This indicates that beta-amyloid can provoke different responses on cAMP response element-directed gene expression, such that low beta-amyloid levels may play a physiological role favoring synaptic plasticity under normal conditions while it would inhibit this mechanism under pathological conditions.

The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity.

The rat monoclonal antibody YC5/45 has been shown to react specifically with serotonin containing neurones, and does not detect dopamine containing sites. On the other hand the monoclonal antibody recognizes dopamine, since it is capable of inhibiting the binding of 3H-YC5/45 to sheep red blood cells coated with serotonin albumin conjugate, or the direct agglutination of the same cells by the monoclonal antibody. Tryptamine and 5-methoxytryptamine are even better inhibitors, while derivatives with a modified-CH2-CH2-NH2 group are inactive. Treatment of the active compounds with paraformaldehyde increases their efficiency dramatically. The binding of antibody was also tested by cross linking with paraformaldehyde: dopamine, serotonin, tryptamine, 5-methoxytryptamine etc., to serum albumin. Only the serotonin derivative was active. The result shows that the discrimination specificity observed in immunohistochemistry is due to the paraformaldehyde fixation. It is concluded that for histochemical detection of serotonin by YC5/45, two reactive sites are necessary. The -CH2-CH2-NH2 group is probably modified by formaldehyde to produce a cyclic derivative to form the antibody recognition site. A different formaldehyde reactive site is required for cross linking to the tissue and this is provided by the presence of the 5-OH of serotonin which is absent in the others.

A progressive deposition of paired helical filaments (PHF) in the brain characterizes the evolution of dementia in Alzheimer's disease. An immunocytochemical study with a monoclonal antibody against the PHF core.

Using the monoclonal antibody (mAb) 6.423 which recognizes epitopes of the pronase-resistant core of paired helical filaments (PHF), we studied postmortem frontal cortex from Alzheimer's disease (AD) patients with short (Group II) and long (Group III) histories of clinical dementia. Four cases with clinically unconfirmed dementia and a postmortem diagnosis of AD (Group I) were also studied. In Group I, the 6,423 mAb was negative whereas in Group II, the antibody recognized primarily neurofibrillary tangles (NFT). In contrast, brains in Group III contained a dense network of 6,423-immunoreactive (IR) thread-like structures ("ghost" neurites) and plaque-like structures with granular appearance, in addition to NFT. The number of 6,423-IR structures appeared to be related to the duration of clinical dementia and the age of onset. Furthermore, "ghost" neurites were more abundant in young AD cases. The possible significance of the 6,423-IR pattern in the pathogenesis of AD is discussed.

Towards trophic factor pharmacology?

Recent developments in neurobiology have lent support to the idea that neural degeneration can be prevented, if not reverted, by the judicious application of substances which specifically affect the trophism of neurones in the adult. A great many of these substances have been proposed and some fully identified chemically. In addition to well characterized neurotrophic factors, other molecules can bring about equally dramatic effects in experimental animals. These investigations are leading us to a new field in neuropharmacology, that of "trophic factor pharmacology." Clinical expectations are high and the anticipation felt in the laboratory equally so.

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain.

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM1. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity 3H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K+ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.

Ultrastructural and neurochemical analysis of synaptic input to trigemino-thalamic projection neurones in lamina I of the rat: a combined immunocytochemical and retrograde labelling study.

The synaptology of lamina I thalamic projection neurones in the spinal trigeminal nucleus of the rat was investigated by combining electron microscopic immunocytochemistry with the retrograde transport of horseradish peroxidase. Fifteen retrogradely labelled neurones were serially sectioned and their dendrites were traced for up to 160 microns in order to characterise the synaptic input to their cell bodies and proximal dendrites. Projection neurones receive synapses from dome-shaped substance P and enkephalin immunoreactive terminals, which make simple axosomatic or axodendritic synapses. In addition, the cells receive synapses from numerous nonimmunoreactive terminals including a wide range of different dome-shaped terminals and various scalloped or glomerular terminals. Dome-shaped terminals synapse with small stubby spines in addition to cell bodies or dendritic shafts and they are probably derived from lamina II interneurones and from descending bulbospinal pathways. Glomerular terminals occur in two main classes: small type A terminals with dark axoplasm and larger type B terminals. Type B terminals participate in synaptic triads in which a peripheral terminal synapses both axoaxonically with the glomerular terminal and axodendritically with the projection neurone. Type A and type B terminals closely resemble the central terminals of spinal cord lamina II glomeruli and are probably derived from C and A delta I degrees afferent fibers. The results indicate that lamina I projection neurones are under pre- and postsynaptic control from diverse sources. Their complex synaptic organisation highlights the key role that such cells play in the rostrad transmission of somatosensory information.

The distribution of substance P immunoreactive fibers in the rat central nervous system.

A detailed account of the distribution of immunoreactive substance P-containing structures in the rat central nervous system is presented, from results obtained by applying an indirect immunofluorescent technique. High densities of substance P-containing nerve terminals were present in sensory nuclei and other non-sensory structures such as thalamus, hypothalamus and extrapyramidal system. Substance P-reactive neuron cell bodies were present in spinal root ganglia, nucleus habenulae medialis, nucleus interpeduncularis, caudoputamen and globus pallidus. Most of the neocortex and the cerebellar cortices had no substance P-positive elements. The results support the hypothesis that substance P may be a widespread neurotransmitter in the central nervous system.

Choline acetyltransferase-immunoreactive profiles are presynaptic to primary sensory fibers in the rat superficial dorsal horn.

The specific aim of this study was to search for morphological counterparts to the known antinociceptive effects of cholinomimetic drugs at the spinal cord level. For this, the light microscopic and ultrastructural distribution of choline acetyltransferase immunoreactivity was studied in laminae I-III of the rat cervical spinal cord. Immunoreactivity was present in cell bodies in lamina III, and in dendrites and axons of all three laminae. Immunoreactive axonal varicosities were often presynaptic to the central varicosities of type II synaptic glomeruli in lamina II and lamina III, less often presynaptic to the central elements of type I glomeruli in lamina II, and often presynaptic to dendrites in both type I and type II glomeruli. In addition, immunoreactive dendrites were often postsynaptic to the central varicosities of glomeruli of all morphological types. These results indicate that 1) primary sensory fibers excite cholinergic interneurons; 2) the acetylcholine released by the axon terminals of these interneurons modulates both nociceptive and non-nociceptive sensory information at the spinal cord level through both pre- and postsynaptic mechanisms. Furthermore, our results reinforce current ideas on reciprocal sensory interaction between thick and fine afferent fibers.

Immunocytochemical localization of substance P in the spinal trigeminal nucleus of the rat: a light and electron microscopic study.

The neuropeptide substance P is a transmitter candidate for certain primary afferent fibers which terminate in the substantia gelatinosa. In this study the light and electron microscopic localization of substance P in the substantia gelatinosa of the spinal trigeminal nucleus of the rat has been studied using immunocytochemical procedures. Substance P immunoreactive fibers were observed mainly in lamina I and outer lamina II. Ultrastructural analysis revealed immunoreactivity in unmyelinated fibers and in axon terminals which contained agranular spherical vesicles and large dense-cored vesicles and which made predominantly simple asymmetric axodendritic synaptic contacts. Immunoreactive terminals only rarely formed the central terminal of synaptic glomeruli and in only one example was a stained terminal possibly postsynaptic to an unstained terminal. The majority of synapses were onto small dendrites in outer lamina II and in some cases these dendrites were themselves presynaptic to other dendrites. Immunoreactive terminals also synapsed with the soma and proximal dendrites of large neurons on the border of laminae I and II. The results show that there are at least two distinct targets for substance P immunoreactive terminals in the substantia gelatinosa, namely the large lamina I neurons and lamina II probable interneurons. Some of the former may be projection neurons while some of the latter may correspond to the inhibitory islet cells described by Gobel and colleagues in the cat. In addition the results indicate that few substance P immunoreactive terminals receive axoaxonic synapses and emphasize instead the role of postsynaptic interactions. In particular the results suggest several sites at which substance P might interact postsynaptically with the neuropeptide enkephalin.

Ultrastructure and distribution of substance P-immunoreactive sensory collaterals in the guinea pig prevertebral sympathetic ganglia.

A light and electron microscopic study has been made of the substance P-immunoreactive networks formed by sensory nerve fibres in the prevertebral sympathetic ganglia of the guinea pig to seek confirmation that these networks arise from collateral branches of sensory fibres passing through the ganglia and to explore the synaptic and other specialized relationships established by these networks. Slices from coeliac-superior mesenteric and inferior mesenteric ganglia of young adult males, perfusion-fixed by paraformaldehyde, were immunostained with a monoclonal antibody to substance P, and the immunolabelling was visualized by a peroxidase reaction. Immunolabelled fibres passing through the ganglia were seen by light microscopy to give off varicose collaterals that ramified in the ganglionic neuropil. Electron microscopy showed that the parent fibres were almost exclusively unmyelinated. Many collaterals ran directly beneath the basal lamina bordering the intraganglionic tissue spaces, and the varicosities either remained superficially exposed under the basal lamina or sank deeper into the supporting Schwann cells, becoming apposed to dendrites of the ganglionic neurones, upon which they formed synapses, or to other nerve terminals. The incidence of these specific associations was quantified, singly and in combination. Synapses could be situated at the same level as unlabelled synapses on the same dendrite, and exposed varicosities could lie within 0.5 micron of exposed, postsynaptic dendrites. These observations confirm a collateral, synaptic nature for the networks and suggest additional nonsynaptic modes of release and sites of transmitter action. They are consistent with the hypothesis that the system serves a nocifensor function of axon reflex type.

Electron microscopic and immunohistochemical evidence that unmyelinated ventral root axons make u-turns or enter the spinal pia mater.

The occurrence of unmyelinated and small myelinated axons and of nerve fibres with a substance P-like immunoreactivity was studied in juxtamedullary L7 ventral root fascicles and surrounding pia mater of kittens and cats. Electron microscopic analysis of thin transverse serial sections from this region in adult cats showed that the number of unmyelinated axon profiles decreases rapidly as the CNS is approached, reaching zero near or at the CNS/PNS border. No unmyelinated axons were found to enter the CNS through this root. At least to some extent, the disappearance of unmyelinated ventral root axons from the juxtamedullary root fascicles was due to presence of intrafascicular axonal hairpin loops and to a shift of axons from ventral root fascicles to the pia mater. It was also found that, in the L7 ventral root, the content of unmyelinated axons in the pia mater is lower in kittens than in cats. Fluorescence microscopic examination of specimens incubated with substance P antiserum showed that some looping axons and ventral root-pia mater axons were substance P immunoreactive. These observations suggest the hypothesis that sensory unmyelinated axons might grow through the L7 ventral root and enter the pia mater during postnatal development. Moreover they show that the occurrence of sensory unmyelinated axons in ventral roots does not necessarily contradict the law of Magendie .