Chronic embryonic MK-801 exposure disrupts the somatotopic organization of cutaneous nerve projections in the chick spinal cord

DEVELOPMENTAL BRAIN RESEARCH ELSEVIER

Developmental Brain Research 82 (1994) 152-166

Research report

Chronic embryonic MK-801 exposure disrupts the somatotopic organization of cutaneous nerve projections in the chick spinal cord Bruce Mendelson * Department of Anatomy (Slot 510), University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205-7199, USA Accepted 24 May 1994

Abstract

The effect of altering neural activity on the development of the central projections of cutaneous and muscle sensory neurons was studied in the embryonic chick spinal cord. Animals were treated chronically with MK-801, a non-competitive N-methyl-Daspartate receptor antagonist, during the period when both cutaneous and muscle sensory afferents form connections in the spinal cord. Daily applications of MK-801 began on embryonic day 5, 1 day before sensory collaterals penetrate the spinal cord gray matter, and continued until the animals were analyzed (at embryonic day 14). The patterns of cutaneous and muscle sensory nerve projections were determined by applying fluorescent tracers to individual, identified peripheral nerves. MK-801 treatment did not overtly alter the pattern of muscle afferent projections. However, in the MK-801-treated embryos; the somatotopic organization of cutaneous afferent projections was dramatically altered. Normally, the projections formed by the lateral femoral cutaneous and the medial femoral cutaneous nerves are located immediately adjacent to one another in the lumbar dorsal horn, with little overlap. In the MK-801-treated embryos, the projections from these two cutaneous nerves both expanded significantly within dorsal horn laminae to become almost completely superimposed. These data suggest that MK-801 disrupts the development of the somatotopic organization of cutaneous afferent projections in the spinal cord.

Keywords: Sensory neuron; Dorsal horn; Development; Axonal growth; Dorsal root ganglion

1. Introduction

In some regions of the developing nervous system, patterns of neural electrical activity have been shown to influence the development of synaptic connectivity. The importance of activity has been demonstrated elegantly in the developing visual system where changes in either the amount or pattern of neural activity during development can induce dramatic changes in synaptic connectivity [29,64,67]. Altering patterns of neural activity also disrupts the formation of appropriate connections in auditory pathways [37] and in portions of the somatosensory system [65]. In these systems, neural activity is involved in the sharpening of topographic maps of sensory information. In the spinal cord, some neural circuits can form appropriately in the absence of normal patterns of activity, while other neuronal

* Fax: (1) (501) 686-6382. E-mail: [email protected]. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

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properties appear to develop in an activity-dependent manner. Activity blockade during development does not alter the formation of the connections necessary for coordinated swimming behavior in amphibians [4,24,25] nor does it alter the pattern of monosynaptic connections between muscle sensory and motor neurons that form the stretch reflex circuit [15,52]. However, disrupting stretch reflex activity during development does alter the amplitude of monosynaptic sensory input to motor neurons [52]. Studies using an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons have also shown that electrical activity can regulate synaptic efficacy [13,55], and altering neural activity modulates the expression of a specific proteoglycan by motoneurons during development [33]. Several studies have indicated that excitatory amino acid neurotransmitters (EAAs) participate in chemical synaptic transmission between somatic sensory afferents and spinal cord neurons [31,40,73]. Glutamate has been found in the somata and central processes of both

B. Mendelson / Developmental Brain Research 82 (1994) 152-166

large [47] and small [3,46] dorsal root ganglion neurons. The postsynaptic sites of EAA action include both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. In the dorsal horn, most monosynaptic primary afferent evoked responses appear to be mediated by non-NMDA receptors while the majority of polysynaptic activity requires NMDA receptor function [63,73]. In particular, NMDA receptors appear necessary for the sensitization of spinal cord neurons following either repetitive stimulation of nociceptive afferents [10,72] or tissue damaging stimuli [8,61]. Receptor binding studies have shown that NMDA receptors are found primarily in the dorsal horn in the adult mammalian spinal cord [32,54], but are found throughout the spinal cord gray matter at early developmental stages [20,34]. The ventral horn binding correlates with studies showing that primary afferent-evoked monosynaptic potentials recorded in embryonic rat motoneutons are mediated by both NMDA and non-NMDA receptors [57,74]. Therefore, it is likely that at early developmental times, NMDA-mediated synaptic activity occurs throughout most spinal cord laminae. In an isolated chick spinal cord preparation, 90-min exposure to 50/zM NMDA induced cytopathological changes in both the dorsal and ventral horns, and these alterations were blocked by concurrent MK-801 application [66]. These data indicate that NMDA receptors are present in the developing chick spinal cord. The anticonvulsant MK-801 is known to be a noncompetitive NMDA receptor antagonist [69]. Although MK-801 can act as an antagonist at nicotinic acetylcholine receptors [16,60], many of its actions on the nervous system appear to be due to its ability to block the function of the NMDA receptor. In spinal cord neurons, MK-801 directly antagonizes NMDA-induced excitation [7,11]. MK-801 is also a potent neuroprotective agent. MK-801 application prior to ischemic [18] or contusive insult [19] to the nervous system reduces the extent of neuronal cell death normally induced by the procedures. Interestingly, in adult rats, chronic MK-801 application for 7-30 days increases the density of primary afferent fibers that are immunoreactive for calcitonin gene-related peptide (CGRP) both in the spinal cord and in the periphery [17,23]. This treatment did not induce any change in number of CGRP-containing primary afferent somata [49,50] implying that the MK801 either induced sprouting of CGRP-immunoreactive fibers or caused an increase in synthesis or transport of CGRP. MK-801 treatment has also been found to promote neurite elongation in cultured dentate granule cells [1]. Therefore, MK-801 is not only a neuroprotective agent, it can also alter normal patterns of neurite outgrowth. In the present study, the effects of chronic MK-801 treatment on the development of the central projections of primary afferent neurons were examined. Chick

153

embryos were exposed to MK-801 during the period when primary sensory afferents are forming synaptic contacts with spinal cord neurons. Subsequently, identified populations of sensory afferents were labeled with lipophilic tracers in order to examine the patterns of sensory afferent arborizations in the spinal cord. The distribution of muscle afferent collaterals was not overtly altered following the MK-801 treatment. However, the somatotopic organization of cutaneous afferents in the dorsal horn was significantly disrupted. The projections formed by the lateral femoral cutaneous (LFC) and medial femoral cutaneous (MFC) nerves which are normally located immediately adjacent to one another with no significant overlap, were largely superimposed after the MK-801 treatment. These data suggest that the effects of MK-801 treatment in the spinal cord are cell type specific, inducing alterations in cutaneous afferent morphology with little effect on muscle afferents. Some of these results have been presented in the form of an abstract [51].

2. Materials and methods 2.1. Animals Fertile chick eggs (Gallus g. domesticus) were obtained from a local supplier. The embryos were maintained at 37°C in a forced draft incubator and staged according to Hamburger and Hamilton [22]. In some cases, animals sacrificed at embryonic day 14 (El4) were determined to be midway between two anatomically defined stages. These animals were staged with an accuracy of 'half stages'. That is, those animals midway between stages 39 and 40 were designated stage 39.5 and those midway between 40 and 41 were designated 40.5.

2.2. Drug administration To determine if chronic MK-801 treatment affects the development of primary sensory afferents, chick embryos were exposed daily to MK-801 during the time when primary sensory afferents are forming synaptic contacts in the spinal cord. To administer the solutions, a window was made in each egg at E3, stage 19-20 (St 19-20) and sealed with a coverslip and melted paraffin. The MK-801, dissolved in sterile chick Tyrode's solution (the constituents of Tyrode's solution are given in [52]) was administered through the window directly onto the well-vascularized chorioallantoic membrane. The window was subsequently resealed with paraffin. Groups of embryos were treated daily with specific doses of MK-801: 0.1, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg in sterile Tyrode's. These doses were determined with respect to the average weight of the contents of the eggs, excluding the weight of the shell. Control groups were treated with equivalent volumes of sterile Tyrode's. These doses were chosen because they bracket doses previously found to induce increases in CGRP immunostaining of primary afferents in the spinal cord [17,48,49] and to inhibit the expression of a specific glycoprotein by motoneurons during development [33]. Applications of MKo801 or sterile Tyrode's began on E5, 1 day before sensory afferent collaterals penetrate the spinal cord gray matter [12,53] and continued until El4 when the neuroanatomical analyses were performed.

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B. Mendelson / Developmental Brain Researck 82 (1904) 157 / ~0

2.3. Neuroanatomical analyses To examine the detailed anatomy of sensory or muscle afferent arbors individual, identified cutaneous or muscle nerves were labeled with lipophilic tracers [28]. At El4, animals were perfused first with 20 ml of Tyrode's solution followed by 20 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH, 7.3). The spinal cords with lumbosacral dorsal root ganglia and peripheral nerves intact, were dissected from the rest of the embryo. Subsequently, specific lipophilic tracers were applied to particular, identified peripheral nerves. To study the somatotopic organization of cutaneous projections in the dorsal horn, the LFC and M F C nerves were labeled with either Dil (l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, Molecular Probes, Inc.) or D i A (4-(4-dihexadecylaminostyryl)-Nmethylpyridinium iodide, Molecular Probes, Inc.). In most animals, Dil was applied to the LFC nerves on both sides of the embryo and DiA was applied to the M F C nerve on one side only. In other animals, DiI was applied to the sartorius (sart) muscle nerve in order to analyze the effects of MK-801 treatment on muscle afferent collaterals. After the dye application, the spinal cords were stored in 4% paraformaldehyde in the dark at 37°C for 1 - 6 weeks to allow for diffusion of the label. The whole m o u n t preparations were viewed periodically with a compound microscope equipped with epifluores-

cent illumination to monitor diffusion ol lhc m,c,_~. I h c (rob animals analyzed in this study were those in which the tracers ~'erc confined to the originally labeled nerves. After diffusion ol the dyes, transverse 5 0 / * m Vibratome sections were cut and mounted in serial order in phosphate buffer. All of the analyses were performed on sections taken from between the rostral margin of the dorsal roo! (DR) of lumbosacral segment 1 (LS1) and the rostral margin of the D R of L82, a region that normally contains dense LI::('~ M F C and sart projections [53,71]. The somatotopic organization of LFC and MFC nerve projections in the dorsal horn was analyzed in 2 ways. In the firs1 method, the sections were viewed and photographed using epifluorescent mi. croscopy and high contrast film. Two modifications were made to the standard filter cubes used R)r fluorescent microsc~py to aid in discriminating the fluorescence due to Dil labeling from thai due to DiA labeling. To observe DiI and not DiA, an O M - 5 4 6 / 1 0 exciter filter (Olympus) was added to a standard rhodamine filter cube, and to view DiA and not Dil. an O M - 5 3 0 / 2 0 barrier filter (Olympus) was added to a standard fluorescein filter cube. Negatives and prints of control and MK-80l-treated sections were then compared. A second method was used to analyze the consistency of any MK-801-induced effect among different sections and to quantify any d o s e - r e s p o n s e relationship. In this procedure, the LFC collaterals that are located

Fig. 1. Observation of whole-mount preparations indicated that DiI and D i A applied to identified cutaneous or muscle nerves labeled neurons at the segmental levels reported previously by investigators using other labeling techniques [26,27,38,70]. A: a photomicrograph of a whole mount preparation, shown dorsal side up, where the LFC nerve had been labeled with DiI. The D R G of T7 and L S I - 3 are indicated, and the filled arrowheads point to the dorsal root entry zones of LS1 and LS2. Rostral is toward the top of the page and lateral is toward the left. The DiI-labeled L F C sensory neurons are located primarily in the D R G of LS1 and LS2 with a few labeled somata in the D R G of T7 and LS3. B: a photomicrograph of a whole m o u n t preparation, shown ventral side up, where the sart nerves on both sides had been exposed to DiI. T h e ventral roots (VR's) of L S 1 - 3 are labeled, M indicates the midline and rostral is toward the top of the page. The pattern of labeled sart motoncurons is bilaterally symmetrical, the somata are located laterally in the spinal cord, primarily in segments LSI and LS2, with a few neurons adjacent to the rostral edge of the V R of LS3. Both micrographs are shown at the same magnification and the scale bar = 500 /zm.

B. Mendelson /Developmental Brain Research 82 (1994) 152-166

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in lamina 2 in LS segments 1 and 2 were analyzed using image analysis techniques. The percentage of lamina 2 occupied by LFC collaterals was determined in all of the sections taken between the rostral margin of the LS1 DR and the LS2 DR where the dorsal horns were complete (i.e. not torn or wrinkled during sectioning and mounting). In this manner, 7-15 sections on each side of the spinal cord were analyzed in each embryo. Transverse sections were viewed using the epifluorescent techniques outlined above and digitized using a Pulnix 745 CCD camera, CCU-84 camera controller (Motion Analysis, Inc.), and a Macintosh Ilci computer running IMAGE software (Rasband, NIH). On the digitized image of each section, the boundaries of lamina 2, and the boundaries of the DiI-labeled LFC fibers within lamina 2 were traced sequentially with a CalComp digitizing pad. The IMAGE software then calculated the cross-sectional area of lamina 2 and the cross-sectional area of lamina 2 that was occupied by labeled collaterals. Subsequently, the percentage of the cross-sectional area of lamina 2 occupied by LFC collaterals was determined.

3. Results

The adequacy and specificity of the DiI and DiA labeling were confirmed using a number of criteria. In the whole-mount preparations, the dyes were observed to be confined to the particular nerves labeled. Embryos where any dye was observed in inappropriate tissue or where dye had leaked onto an adjacent nerve were not analyzed. The positions of individual sensory and motor pools were also mapped in all animals. The whole mount preparation from every embryo was analyzed. In these preparations, it is possible to observe individual sensory and motor somata (Fig. 1). Therefore, it was possible to determine the complete rostrocaudal extent of each labeled sensory and motor pool. In all of the animals used in this study, the sensory and motor pools were found to occupy the segmental levels

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Table 1 The effects of chronic MK-801 treatment on developmental stage and weight measured at El4 Dose of MK-801

n

Stage

Weight (g)

Control (Tyrode's-treated) 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg 2.0 mg/kg 3.0 mg/kg 4.0 mg/kg

19 5 13 8 14 9 8

39.9 + 0.2 40.0_+0.0 40.0_+0.1 39.9+0.4 40.0+0.2 39.3-+0.9** 39.8 + 0.4

9.9 + 1.7 9.0+0.7 11.25:1.6 * 10.2_+ 1.0 10.8 -+ 1.3 8.0+1.5 * 10.1 + 1.6

All of the embryos were analyzed at E14. n, number of animals analyzed. Control embryos were treated with sterile Tyrode's. Animals were staged according to Hamburger and Hamilton [22]. The data are means+ 1 S.D. The numbers of animals are greater than those presented in Fig. 3 because some animals were processed for immunohistochemical analyses (data not shown). The 0.5 mg/kg group was found to be significantly heavier than the Tyrode's-treated controls (* P = 0.05, unpaired t-test). The animals treated with 3.0 mg/kg were morphologically about 1/2 stage behind the control group and were significantly lighter (** P = 0.01, , p = 0.01). However, there were no significant differences observed between the 4.0 mg/kg embryos and the controls.

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Fig. 2. Exposure to MK-801 does not alter the distribution of cells in spinal cord laminae. Both micrographs are transverse 504zm sections from El4, St 40 embryos taken at the level of LS1. The density of neurons in the control embryo (A), is similar to that in the embryo treated with 4.0 mg/kg of MK-801 (B). In the chicken, spinal cord laminae 1 and 2 are located lateral to laminae 3 and 4 [44]. Black arrowheads mark the medial border of lamina 2 and white arrowheads indicate the ventral and inedial boundaries of lamina 3 in both A and B. Laminae 2 and 3 in the dorsal horn and lamina 9 in the ventral horn (black arrows) are easily defined and appear similar in both the control (A) and MK-801-treated (B) embryos. Both photomicrographs are shown at the same magnification and the scale bar = 200/J.m.

156

B. Mendelson / Dct'elopmemal Brain Research ,~¢2(I994) 152 lr~6

reported previously using other labeling techniques [26,27,38,70]. For example, sensory neurons labeled by applying DiI to the LFC nerve were located primarily in the dorsal root ganglia ( D R G ) of segments LS1 and LS2 with a few neurons in the D R G of T7 and LS3 (Fig. 1A). Motoneurons labeled by applying DiI to the sart nerve were consistently found in LS segments 1 and 2 with a few labeled motoneurons extending into the rostral portion of LS3 (Fig. 1B). In all of the animals analyzed, the fluorescent labeling was of uniform brightness throughout the rostrocaudal extent of the labeled pool, and individual sensory afferent collaterals ended in brightly labeled structures that appeared to be synaptic terminals. In control animals, the pattern of sensory afferent collateral labeling of both muscle and cutaneous nerves resembled that previously published using horseradish peroxidase labeling techniques [12,40,71]. It is therefore likely that the DiI and DiA applications labeled the complete extent of motor and sensory pools as well as the complete central sensory afferent projections. However, to minimize errors that could result from incomplete labeling, the quantitative analyses were restricted to sections taken from between the rostral margin of the D R of LS1 and the rostral margin of the D R of LS2, a region that contains dense LFC and MFC projections [53,71]. In animals where both DiI and DiA were used, DiI diffused faster than DiA. In most of the animals used in this study, the LFC was labeled with DiI and the MFC with DiA. In the animals where both nerves were labeled, there were many cases where the LFC was completely labeled at a time when the MFC was not. Due to this difference in diffusion rate, the projections of the MFC nerve were completely labeled in only 2 - 3 animals per dose of MK-801. Therefore, statistical analyses were performed only on the LFC projections where 4 or more animals per dose were completely labeled according to the above criteria.

with higher doses gave variable results. Those treated with 3.0 m g / k g MK-801 weighed Icss and wcre about one half of a stage behind the control group, but embryos exposed to 4.0 m g / k g were not significantly different from the controls. The MK-80I treatments did not adversely influence the survival rate ,~ff tile embryos. The survival rate of the Tyrode's-treated control animals was 79% (n = 47) as compared to 91% for all of the MK-801-treated groups (n - 145). This relationship held true for the animals treated with the highest doses of MK-801 since the survival rate for embryos treated with 3.0 m g / k g was 79% (n = 29) and the survival rate for the animals treated with 4.0 m g / k g was 91% (n = 18). The number of animals reported in the survival rate calculations are higher than those in Table 1 because some treated embryos were sacrificed both before and after El4 for studies not discussed in the current report. The survival rate was determined by calculating the ratio of the number of animals alive at the time of sacrifice, to the total number of animals exposed to a given dose of MK-801 or to Tyrode's solution. These data suggest that none of the treatments produced strong toxic effects. The basic morphology of the spinal cords of MK801-treated animals also appeared normal. Fig. 2 shows transverse sections through the lumbosacral spinal cord taken from a normal chick (Fig. 2A) and an animal treated with 4.0 m g / k g of MK-801 (Fig. 2B). The

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In the present study, embryos were treated chronically with various doses of MK-801 or with equivalent volumes of sterile saline solution. The systemic MK-801 treatment did not produce overtly harmful effects on the embryos treated with low-medium doses of MK-801 (2.0 m g / k g or less) since the developmental stages at the time of fixation were not significantly different from saline-treated controls and the average body weights of the treated animals were similar to the controls (Table 1). The average body weights of the group treated with 0.5 m g / k g were slightly larger than the controls ( P = 0.05, students t-test). But this was probably due to the fact some of these animals were perfused late on embryonic day 14 and the embryos gain weight rapidly during these stages. Animals treated

2o 0

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Dose of MK-801 Fig. 3. The effect of chronic MK-80I exposure on the cross-sectional area of lamina 2. All of the analyses shown in this figure were performed on animals sacrificed at El4. The data represent measurements made from all of the embryos where the projectionsof LFC collaterals into lamina 2 were analyzed quantitatively. The number of animals in each treatment group are shown inside each bar. The mean values of the cross-sectional area Of lamina 2 measured in the groups of animals treated with 2.0 mg/kg and lower doses of MK-801 were not significantly different from the value obtained from control animals. The value from the animals treated with 3.0 mg/kg was significantly smaller than all of the other values (P < 0.001 for the 3.0 mg/kg group compared to each of the other groups, Scheffe's multiple comparison test). The bars represent mean -t-1 S.E.

B. Mendelson / Developmental Brain Research 82 (1994) 152-166

density of neuronal somata in both embryos appears similar as does the general laminar arrangement of the neurons. Note that the arrangement of laminae in the chick dorsal horn differs from that observed in mammals. In the chick, lamina 3 is located medial, rather than ventral to lamina 2 [2,44]. The intermediate laminae are rather difficult to distinguish, but laminae 2 and 3 in the dorsal horn and lamina 9 in the ventral

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horn can be recognized easily and are similar in morphology in both the control and MK-801-treated animals. Since the major anatomical changes induced by MK801 treatment were observed in lamina 2, the cross-sectional area of lamina 2 was measured in all of the embryos where the LFC projections were analyzed quantitatively (Fig. 3). The mean values from the groups

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Fig. 4. Exposure to MK-801 does not alter the distribution of sart muscle afferent collaterals in the spinal cord. Transverse sections from El4, St 40 chicks taken from a control embryo (A,B), an animal treated with 4.0 m g / k g of MK-801 (C) and an animal treated with 3.0 m g / k g MK-801 (D) in which the sart nerves were labeled with DiI. In all of the micrographs medial is to the left and a white asterisk indicates lamina 2. One population of afferent collaterals penetrates the gray matter dorsolaterally to project just ventral to lamina 2 to terminate in the region of laminae 4 and 5 (open arrows in all micrographs). Another population of sart collaterals terminates in intermediate and ventral laminae and presumably represents the projections of stretch sensitive muscle afferents (muscle spindle afferents). At spinal cord levels just rostral to the sart motor pool (A,C) most of the sart spindle-afferent collaterals penetrate the gray matter at a dorsomedial position (black arrows) and none penetrate dorsally (white arrowheads). At the level of the sart motor pool (B,D) most spindle-afferent collaterals penetrate the gray matter dorsally (black arrowheads) and project ventrally close to the medial border of lamina 2. There are no apparent major differences in the patterns of sart afferent collaterals in the control (A,B) versus the MK-801-treated (C,D) embryos. In B and D the LS1 ventral roots were cut so no sart motoneurons were labeled. All micrographs are shown at the same magnification and the scale bar = 100 ~m.

158

B. Mendelson / Det~elopmentalBrain Research 82 (1994) 152 -16~>

o f a n i m a l s t r e a t e d w i t h low a n d m o d e r a t e d o s e s o f MK-801 were not significantly different from those obtained from the Tyrode's-treated controls. However,

3.2. Effect o f MK-801 treatment on muscle a~]erent collaterals

the average value determined from embryos treated w i t h 3.0 m g / k g w a s s i g n i f i c a n t l y s m a l l e r t h a n t h e valu e s f r o m e a c h o f t h e o t h e r g r o u p s ( P < 0.001, S c h e f f e ' s multiple comparison test). For example, the average size o f l a m i n a 2 in T y r o d e ' s - t r e a t e d a n i m a l s w a s 5 8 , 6 5 2 + 658 /xm 2 as c o m p a r e d t o 5 1 , 5 6 0 + 6 1 8 # m 2 f o r t h e

In the spinal cord, MK-801 treatment induced specific c h a n g e s in t h e a r b o r i z a t i o n p a t t e r n s o f c u t a n e o u s a f f e r e n t s , b u t p r o d u c e d n o o b v i o u s c h a n g e s in t h e pattern of muscle afferent collateral arborization. Previous studies have shown that the morphology of muscle a f f e r e n t c o l l a t e r a l s in t h e l u m b o s a c r a l s p i n a l c o r d is w e l l d e v e l o p e d b y E l 4 [12,40,53]. I n t h e p r e s e n t s t u d y . sart motoneurons and muscle afferents were examined in fourteen El4 embryos treated with MK-8(/I: a were t r e a t e d w i t h 0.1 m g / k g , 1 w i t h 0.5 m g / k g , 2 w i t h 1.0 m g / k g , 4 w i t h 2.0 m g / k g , 1 w i t h 3.0 m g / k g a n d 2 w i t h 4.0 m g / k g . I n all o f t h e s e e m b r y o s , t h e s o m a t a o f t h e

3.0 m g / k g g r o u p ( m e a n + 1S.E., P < 0.001). T h e s e d a t a suggest that exposure to low and moderate doses of MK-801 does not induce any major alterations in dorsal h o r n m o r p h o l o g y , b u t t h e r e m a y b e s o m e d e c r e a s e in c e l l u l a r v i a b i l i t y in e m b r y o s t r e a t e d w i t h 3.0 m g / k g and higher doses.

Fig. 5. Effect of MK-801 treatment on the central projections of the LFC and MFC nerves in the dorsal horn. Transverse sections of the lumbar spinal cord taken from El4, St 40 embryos. In all 4 micrograpbs, filled arrowheads point toward the medial borders of lamina 2 on both sides, on one side, open arrows mark the ventrolateral border of lamina 2. M indicates the midline and d the first lumbosacral dorsal root (observed on one side of each micrograph). A: a saline-treated control embryo where the left MFC nerve was exposed to the fluorescent tracer DiA The labeled MFC central processes occupy only the dorsomedial part of lamina 2 (indicated by *). C: a control embryo where the LFC nerves on both sides were exposed to the fluorescent tracer DiI. The LFC fibers are restricted to the ventrolateral portions of lamina 2 (indicated by *). A and C are photomicrographs of the same section. In 'A' a modified fluorescein filter cube was used to view only DiA fluorescence and in 'C" a modified rhodamine filter cube was used to view only the Dil. By lining up the two m~crographs using the left dorsal root, one can appreciate that in normal embryos, the populations of MFC and LFC fibers occupy adjacent, but non-overlapping regions of the dorsal horn. B and D are micrographs from an embryo treated with 3.0 mg/kg MK-801. B: the MFC nerve was labeled on the right side. and the modified ftuorescein filter cube was used to show that the DiA-labeled MFC fibers fill most of lamina 2 (*). D: the LFC nerves on both sides were exposed to Dil and the modified rhodamine filter cube was used to view the DiI-labeled LFC fibers that also occupy most of lamina 2 (indicated by * ). B and D are micrograpbs of the same section that were photographed using different filter cubes. By lining up these two micrographs, one can see that in the MK-801-treated embryos, the central projections of the LFC and MFC nerves overlap appreciably. All photomicrographs are shown at the same magnification and the scale bar = 200 /xm.

B. Mendelson / Developmental Brain Research 82 (1994) 152-166

sart motoneurons were located in the appropriate lateral position in the lateral motor column and at the appropriate segmental levels (data not shown). The sart muscle afferent collaterals also exhibited the normal pattern of arborization. Two types of sart muscle afferent collaterals can be distinguished based upon where they terminate in the gray matter of the spinal cord. One group of collaterals penetrates the gray matter at a dorsolateral position and projects medially just ventral to lamina 2 (open arrows in Fig. 4). Most of these collaterals terminate in laminae 4 and 5. The other type of sart collateral appears to represent the projections from afferents that supply muscle spindles since they terminate in intermediate and ventral laminae, in close proximity to motoneuronal den-

159

drites and somata. However, these spindle afferents penetrate the gray matter at specific locations at particular rostrocaudal levels. Rostral to the sart motoneuronal somata, the sart spindle afferent collaterals penetrate the gray matter at a dorsomedial position (black arrows in Fig. 4A,C) and project ventrolaterally to arborize in intermediate and ventral laminae. Few, if any collaterals penetrate at the medial border of lamina 2 (white arrowheads in Fig. 4A,C). At the level of sart motoneuronal somata, most spindle afferent fibers penetrate the gray matter dorsally, close to the medial border of lamina 2 (black arrowheads in Fig. 4B,D) and project ventrally to arborize in intermediate and ventral laminae. Although no quantitative analyses were performed, no differences were observed between

Fig. 6. Transverse sections of the dorsal horns from a control embryo (A,C) and an embryo treated with 2.0 m g / k g MK-801 (B,D). Both embryos were sacrificed at El4, and the sections from both the control and MK-801-treated embryos were taken from the same longitudinal spinal cord level. The midline is at the right edge of each micrograph, arrowheads mark the medial border of lamina 2 both dorsally and ventrally, d, the first lumbosacral dorsal root; If, lateral funiculus. A: a control embryo where the MFC nerve was labeled with DiA. The MFC collaterals are densely packed into the dorsomedial region of lamina 2 (*) and are located in the ventromedial region of lamina 3 (m). C: a control animal where the LFC nerve was labeled with DiI showing that the LFC collaterals are densely packed into the ventrolateral part of lamina 2 (* in C) and are present in the dorsolateral region of lamina 3 (1). A and C are photomicrographs of the same section photographed using different filter cubes. B: a section from an embryo treated with 2.0 m g / k g MK-801 where the MFC nerve was labeled with DiA. The labeled MFC collaterals occupy most of lamina 2 (*). The area occupied by MFC collaterals in lamina 3 (m) also appears larger in cross-section than the lamina 3 projection observed in the control animal. D: a section from an animal treated with 2.0 m g / k g MK-801 where the LFC nerve was labeled with Dil. The labeled LFC collaterals also occupy most of lamina 2 (*). The region occupied by the LFC collaterals in lamina 3 (i) appears larger in this MK-801-treated embryo than that observed in the Tyrode's-treated control. B and D are photomicrographs of the same section photographed using different filter cubes. The density of fibers and boutons in the MK-801-treated embryos appears less than that observed in controls. All micrographs are shown at the same magnification, the scale bar = 200/~m.

160

B, Mendelson / Det elopmental Brain Research 82 (1994) 152~ 10~

t h e p a t t e r n o f s a r t a f f e r e n t c o l l a t e r a l s in c o n t r o l (Fig. 4 A , B) v e r s u s M K - 8 0 1 - t r e a t e d e m b r y o s (Fig. 4 C , D ) . T h e m u s c l e a f f e r e n t c o l l a t e r a l s p e n e t r a t e d t h e gray m a t t e r o f t h e spinal c o r d at s i m i l a r l o c a t i o n s in b o t h M K - 8 0 1 treated and control embryos, and none of the muscle a f f e r e n t c o l l a t e r a l s , in any o f t h e e m b r y o s e x a m i n e d , a r b o r i z e d e x t e n s i v e l y in l a m i n a e 1 - 3 . T h e r e f o r e , t h e r e w e r e n o a p p a r e n t t r e a t m e n t - i n d u c e d d i f f e r e n c e s in t h e

general distributions of either muscle afferent collaterals.

of t h e

two t y p e s

of

3.3. Effect o f MK-801 treatment on cutaneous afferent collaterals I n n o r m a l a n i m a l s , t h e c e n t r a l p r o j e c t i o n s of t h e L F C a n d M F C n e r v e s a r e a d j a c e n t to o n e a n o t h e r in

Fig. 7. DiI labeling shows that the MK-801 treatments disrupt LFC projections in lamina 2 in a dose-dependent manner. Transverse sections of the dorsal horns of animals treated with various doses of MK-801 where the LFC nerves were labeled with DiI. All of the sections were taken from approximately the same longitudinal level; between the rostral margin of the DR of LS1 and the rostral margin of the DR of LS2. In all of the micrographs: M, medial; the black arrowhead marks the dorsomedial boundary of lamina 2; the white arrowhead indicates the ventral border of lamina 2; and * denotes the LFC collaterals in lamina 2. A: in control animals, LFC collaterals are densely packed into the ventrolateral portion of lamina 2. In the 0.5 mg/kg group, some animals displayed only a slight enlargement in the LFC projection into lamina 2 (B) while m others, the projection expanded dramatically (C). At higher doses (D,E) the LFC collaterals were observed to fill almost the entire cross-sectional area of lamina 2 and the density of the fibers appeared less dense than in controls. All micrographs are shown at the same magnification and the scale bar = 100/zm.

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B. Mendelson / Developmental Brain Research 82 (1994) 152-166

the dorsal horn of the lumbosacral spinal cord and do not overlap appreciably [53,71]. The analyses of cutaneous projections in the present study were restricted to the longitudinal region between the rostral edges of the D R ' s of LS1 and LS2. This is a region where both the LFC and MFC have dense projections and where the collaterals from each nerve occupy significant amounts of the cross-sectional area of the dorsal horn (Figs. 5-7). At slightly more rostral locations the LFC collaterals occupy considerably more of the cross-sectional area of the dorsal horn than the MFC fibers and the converse situation exists caudal to LS2 [71]. Normally in the region analyzed, the LFC collaterals project exclusively to the ventrolateral portion of lamina 2 while the MFC collaterals are restricted to the dorsomedial part of lamina 2 (Figs. 5 and 6). The two populations of fibers do not appear to overlap appreciably in lamina 2, but rather there is a border between the two projection fields. In lamina 3, the projection patterns of the two nerves are also adjacent to one another, with the L F C collaterals located dorsolateral to the MFC fibers (Fig. 6). The pattern of cutaneous afferent projections in lamina 2 was dramatically altered by the MK-801 treatment. The density of collateral fibers within lamina 2 a p p e a r e d to be lower in the MK-801-treated embryos, and, in contrast to the normal situation, the LFC and M F C projection patterns overlapped extensively (Figs. 5 and 6). In control animals, the projections into lamina 2 are so dense that it is often difficult to resolve individual afferent collaterals at low magnification (Figs. 5-7). In the embryos treated with 0.5 m g / k g MK-801 some embryos displayed dense LFC projections in laminae 2 (Fig. 7B). However, in other embryos treated with 0.5 m g / k g MK-801, and all of the embryos treated with higher doses, the density of afferent fibers in the dorsal horn a p p e a r e d to be lower, individual collaterals and boutons could be identified easily (Figs. 5, 6 and 7 C - E ) . The cross-sectional area occupied by both the LFC and M F C collaterals in the dorsal horn increased following MK-801 treatment (Figs. 5-7). This was clearly observed in lamina 2 where there was no sharp border between the LFC and MFC projection fields in the MK-801-treated animals. Rather, the projections from both the MFC and LFC nerves filled most of the cross-sectional area of lamina 2. In Fig. 7 the effect of varying doses of MK-801 on the LFC projections is shown. In animals treated with 0.5 m g / k g some embryos displayed only a slight expansion of LFC projections (Fig. 7B) while in other embryos the L F C collaterals occupied almost the total cross-sectional area of lamina 2 (Fig. 7C). The extent of the expansion of the LFC projections reached a plateau in the animals treated with 1.0 m g / k g (Fig. 7D). In all of the embryos exposed to doses of 1.0 m g / k g or higher, the LFC

collaterals occupied almost the total cross-sectional area of lamina 2 (Fig. 7 D,E). Although the LFC projections in MK-801-treated animals appeared to expand in laminae 2, the projections did not expand ventrally into laminae 4-5. Preliminary observations suggest that the LFC and MFC projections into lamina 3 also expand following MK-801 treatment. In Fig. 6 the MFC projection into lamina 3 in the MK-801treated embryo ( ' m ' in Fig. 6B) appears larger than that observed in the control embryo ( ' m ' in Fig. 6A). This apparent expansion in lamina 3 can also be observed in the LFC projection by comparing the LFC fibers in the control embryo ('1' in Fig. 6C) to those in the embryo treated with 2.0 m g / k g MK-801 ('1' in Fig. 6D). However, the projections into lamina 3 were not analyzed quantitatively in the present study. To analyze the consistency of the MK-801-induced effect among different sections and to quantify the d o s e - r e s p o n s e relationship, the LFC projections into lamina 2 in LS segments 1 and 2 were analyzed using image analysis techniques. This region was chosen for analysis because the boundaries of lamina 2 can be distinguished easily in transverse sections (Fig. 2) and,

100-

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l

so1

E~ ~

e'~

60

50

Tyrode's

0.1 rng/kg

0.5 mg/kg

1.0 mg/kg

2.0 mg/kg

3.0 rng~kg

Dose of MK-801 Fig. 8. MK-801 disrupts LFC projections in lamina 2 in a dose-dependent manner. All of the analyses shown in this figure were performed at El4. The percentage of the area of lamina 2 occupied by LFC collaterals is plotted against the dose of MK-801. Each point represents data from one side of one animal (mean_+1 S.D.). The responses of individual animals in each treatment group were consistent except for those embryos exposed to the lowest doses of MK-801; 0.1 mg/kg and 0.5 mg/kg MK-801. In these embryos the magnitude of the MK-801-induced effect varied among individuals. Data from each treatment group were pooled in order to perform statistical comparisons between groups. When the mean values from each treatment group were compared, all of the MK-801-treated values were found to be significantly different from the Tyrode's-treated controls (P = 0.006 for the 0.1 mg/kg dose and P < 0.0001 for all of the higher doses, Scheffe's multiple comparison test). The effect appeared to plateau at 1.0 mg/kg since the value from the 1.0 mg/kg group was significantly larger than that from the 0.5 mg/kg group (91.0+0.5 compared to 79.2+0.8, P < 0.001) and there were no significant differences among the 1.0, 2.0 and 3.0 mg/kg groups. The number of animals analyzed in each group is shown in Fig, 3.

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B. Mendelson / Det,elopmental Brain Research 82 (1994) 152- i 6~

in normal animals, the LFC projection pattern occupies about 2 / 3 of the cross-sectional area of lamina 2, leaving about 1 / 3 of lamina 2 devoid of LFC-fibers. Fig. 8 shows the average area of lamina 2 occupied by LFC fibers in individual animals (each point represents the mean + 1 S.D. from the sections analyzed on one side of one animal). The responses of individual animals in each group were very consistent except for those treated with the lowest doses: 0.1 m g / k g and 0.5 mg/kg. As mentioned above, some animals treated with 0.5 m g / k g displayed only a slight increase in the LFC projection in the dorsal horn, while other individuals exhibited a large expansion of the LFC projection. The data from the individual animals in each treatment group were pooled in order to perform statistical comparisons between groups. The projections of LFC central-collaterals in saline-treated animals occupy 64.1% + 0.5 (mean + l S.E.M.) of the transverse area of lamina 2. In animals treated with 0.1 m g / k g of MK-801 the area of LFC projection into lamina 2 expanded significantly to occupy 68.4 + 0.9% of lamina 2 ( P = 0.006, Scheffe's multiple comparison test). All of the average values from animals treated with 0.5 m g / k g and higher doses were significantly larger than the values from the Tyrode's-treated controls ( P < 0.0001). The effect appeared to plateau following the 1.0 m g / k g treatment which induced LFC central-collaterals to occupy most of lamina 2 in the segments analyzed (91.0 + 0.5%). The further increases in average LFC projection in lamina 2 with the 2.0 m g / k g and 3.0 m g / k g doses of MK-801 were not significantly different from those observed after the 1.0 m g / k g treatment. The MFC projections into lamina 2 were also enlarged (Figs. 5 and 6). However, as mentioned above, the MFC projections were completely labeled in only 2-3 animals per dose of MK-801 so statistical analyses were performed only on the LFC projections, where 4 or more animals per dose were completely labeled (see Fig. 3 for the number of animals analyzed in each group).

4. Discussion

The present results indicate that embryonic MK-801 treatment disrupts the normal pattern of somatotopic organization of cutaneous nerve projections while not overtly affecting either muscle afferent projections or overall spinal cord morphology. Although MK-801 treatment can induce degenerative changes in certain types of neurons [56], many studies, using doses of MK-801 similar to those used in the present study, produced specific results that did not appear to involve overtly harmful neural effects. For example, in adult rats, chronic treatment with 1.0-2.0 m g / k g / d a y of MK-801 induced an increase in density of CGRP-im-

munoreactive primary afferent fibers [23,491 and substance P immunoreactivity in the spinal cord !117].Acute exposure to similar concentrations of MK-801 prior t{~ ischemia [18,45] or contusion [119] has been f~)und to be neuroprotective. In the present study, chronic MK-SI)I treatment induced the central projections of the LFC and MFC nerves to expand to occupy a much larger percentage of the cross-sectional area of the dorsal horn than in control embryos. Treatment wilh 0.1 m g / k g induced a significant increase in the LFC projection into lamina 2 and treatment with /t.5 m g / k g produced a further expansion that was significantly different from that measured after the 0.1 m g / k g treatment. At both of these low doses there was some variability in the magnitude of the responses measured among individual animals. This may have occurred because there is some slight variability in the rate of development among animals. Perhaps faster developing embryos were able to clear the MK-801 more efficiently than slower developing animals and were therefore exposed to slightly lower average MK-801 concentrations. The extent of the MK-801-induced effect reached a plateau at 1.0 m g / k g where LFC collaterals were induced to occupy more than 9 0 g of the cross-sectional area of lamina 2 in all treated animals. The further expansions measured in animals treated with 2.0 m g / k g and 3.1) m g / k g were not significantly different from those measured after the 1.0 m g / k g treatment. The average cross-sectional area of lamina 2 was measured in all of the embryos where the LFC projections were analyzed quantitatively. There were no significant differences in the cross-sectional areas between control animals and animals treated with doses as high as 2.0 mg/kg. However, there was a small, but significant decrease in the cross-sectional area of lamina 2 measured in embryos treated with 3.0 mg/kg. This may indicate that chronic treatment with high doses of MK-801 reduces cellular viability. Since the magnitude of the MK-801-induced effect on LFC fibers reached a maximum at 1.0 m g / k g , a dose where no spinal cord abnormalities were observed, it is unlikely that this effect on LFC collaterals was due to MK-801induced toxicity. The MK-801 treatments appeared to produce a cell-type specific effect. The organization of cutaneous afferents in lamina 2 was altered by doses of MK-801 as low as 0.5 m g / k g and the highest doses used (3.0-4,0 m g / k g ) also appeared to affect cutaneous afferents specifically. None of the MK-801-treated embryos exhibited any changes in the basic laminar organization of the spinal cord or any observable changes in muscle afferent organization. The MK-801 treatments also did not alter the positions of identified motoneurons. Within the dorsal horn, quantitative analyses were performed on the projections into lamina 2. However, preliminary qualitative analyses suggest that the projec-

B. Mendelson / Developmental Brain Research 82 (1994) 152-166

tions from both the LFC and MFC nerves also expanded in lamina 3. Previous anatomical and physiological analyses have shown that there are two separate somatotopic maps of sensory information in the dorsal horn of the chicken [70,71]. There is a lateral map within lamina 2 that appears to be primarily composed of processes from high threshold afferents, and a more medial map in lamina 3 that consists predominantly of collaterals from lower threshold sensory neurons. The present study shows that the high threshold sensory projections into lamina 2 are dramatically altered by MK-801 treatments, further quantitative studies will be necessary to determine if the projections from the lower threshold afferents respond similarly to the MK801 treatments. However, the overt morphology of the pattern of muscle afferent projections, and positions of the sart motoneurons did not appear to be altered by the MK-801 treatments. Therefore, the MK-801 treatments appeared to produce cell-type specific effects. The anticonvulsant MK-801 is a potent NMDA-receptor antagonist that readily passes through the blood-brain barrier [69]. MK-801 blocks NMDA-induced depolarizations in vitro [43,69] and systemic MK-801 treatment blocks NMDA-induced convulsions [68]. MK-801 has also been shown to antagonize nicotinic acetylcholine receptors [16,60]. Nicotinic acetylcholine receptors are present in the chick spinal cord during the stages when MK-801 was applied during this study [62] and the blockade of cholinergic transmission with d-tubocurarine alters the pattern of spontaneous motoneuronal activity [39]. Therefore the MK-801 applications may affect both EAA and cholinergic synaptic transmission. However, systemic application of nicotinic acetylcholine antagonists in the chick during these stages is known to block motoneuronal cell death [52,58,59]. This treatment leads to the presence of about 50% more motoneurons than are present in untreated embryos which drastically alters the morphology of the spinal cord. For example, in embryos treated with d-tubocurarine the cross-sectional area of the lateral motor column is significantly larger than in control embryos [52,59]. In the present study, no overt changes in the area or cell packing density of the lateral motor column were observed. So although MK801 may affect some aspects of cholinergic transmission, systemic treatment does not appear to produce the inhibition of motoneuronal cell death induced by classical nicotinic antagonists. Further studies using more specific neurotransmitter blockers will be necessary to determine unequivocally if the observed effect was due to blocking NMDA receptors, nicotinic receptors or a combination of receptor types. There are several possible cellular mechanisms that could account for the results observed. MK-801 may have induced afferent fibers to sprout into inappropriate regions of the dorsal horn. In adult rats, systemic

163

MK-801 treatment appears to induce sprouting of the central processes of primary afferent fibers that are immunoreactive for CGRP with no change in number of CGRP-containing primary afferent somata [23,50]. In vitro, MK-801 treatment has been shown to alter the pattern of neurite outgrowth of hippocampal neurons [1]. MK-801 treatment could also disrupt naturally occurring neuronal death. As mentioned above, other treatments that alter neural activity such as exposing embryos to nicotinic acetylcholine antagonists, inhibit motoneuronal cell death [59]. In the present study, MK-801 treatment was carried out during the period of sensory neuronal death [5]. It is therefore possible that the treatment spared some sensory neurons which would normally die, that project to inappropriate regions of lamina 2. MK-801 treatment has also been shown to modulate the expression of brain-derived neurotrophic factor in certain regions ,of the adult rat central nervous system [6,30]. Therefore, it is possible that trophic influences on cutaneous afferents were altered by the MK-801 treatment. But, perhaps the most likely mechanism involves the ability of MK-801 to block NMDA receptor-mediated neurotransmission in the dorsal horn. In most adult systems that have been studied, the monosynaptic transmission between primary afferent neurons and dorsal horn cells is mediated by nonNMDA EAA receptors, while most polysynaptic afferent-evoked responses require NMDA receptor activation [63,73]. However, in the rat, NMDA receptors are more widely distributed in the developing spinal cord as compared to the adult [34]. Perhaps at these earlier developmental stages there is monosynaptic NMDA receptor-mediated transmission between primary afferents and dorsal horn neurons. Blocking this transmission with MK-801 may inhibit the development of appropriate somatotopic projections in the dorsal horn just as the blockade of direct NMDA-induced responses during development of the visual system inhibits the refinement of the topographic map of visual information (reviewed in [9,21]). On the other hand, many polysynaptic afferent-evoked responses in the dorsal horn involve NMDA receptor activation. Perhaps MK-801 treatment disrupts the formation synaptic connections in local circuits of neurons in the dorsal horn. These circuits may be necessary for the formation of primary afferent somatotopy. For example, synaptic input to primary afferent collaterals in the dorsal horn may be altered by the MK-801. This, in turn, may induce abnormal collateral growth just as altering the inputs to identified neurons induces abnormal dendritic development [21,35]. Although the MK-801 treatments induced the central projections of the LFC and MFC nerves to expand to occupy larger than normal areas of laminae 2, the collaterals were not found in more ventral, inappropri-

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B. Mendelson / Det'elopmental Brain Research 82 (1994) 152 . 1C~O

ate laminae. This suggests that the growth of cutaneous afferent collaterals becomes restricted to appropriate laminae using recognition mechanisms that do not involve NMDA receptor activation. The refinement of the projection of individual nerves to particular regions of dorsal horn laminae is disrupted by the MK-801 treatment, perhaps by blocking NMDA receptor function. This series of events is similar to what is observed during development of the lower vertebrate visual system where a rough topographic map of retinal ganglion cell projections to the optic tectum can form in the absence of normal patterns of activity. But, the further refinement of this map is activity dependent (reviewed in [9,21]). Fitzgerald et al. [14] have shown that the receptive field sizes of single dorsal horn neurons appear to decrease in size as development proceeds. These developmental changes in receptive field size suggest a sharpening of the sensory projections. In the adult rat, the peripheral processes of muscle afferents can be redirected to supply skin by cutting and cross anastomosing individual muscle and cutaneous nerves. In this situation, the muscle afferents that have been redirected to supply skin peripherally, form novel functional connections with dorsal horn neurons appropriate for the new peripheral target. In this manner, an orderly somatotopic representation of skin is maintained [41,42]. It is likely that these changes in dorsal horn connectivity are also driven in part by mechanisms involving neural activity. The abnormal cutaneous nerve projections observed in the present study suggest that normal patterns of neural activity are necessary for the development of somatotopic organization in the dorsal horn. Neural activity has also been shown to be important in the development of synaptic connections in portions of the visual, auditory and somatosensory systems in the brain, regions where sensory information forms topographical projections [9,36,65,67]. These results point toward the generalization that normal patterns of activity are necessary for the appropriate development of the fine tuned maps of sensory information that form in the nervous system. In the future, using this relatively simple spinal cord system, it will be of interest to combine anatomical, electrophysiological and molecular techniques in order to further define the cellular mechanisms that coordinate activity-dependent neural development.

Acknowledgements It is a pleasure to thank Mitzi Bonner for her excellent technical assistance as well as Brian Davis and John Houle for helpful comments concerning this

manuscript. This work was supported by NIAAA 5 R29 AA09205.

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