Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse.

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons.

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca2+ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1-3 microM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca2+ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca2+ concentration in both neurites and cell bodies. We hypothesize that the ability of low concentrations of Ca2+ ionophores to raise intracellular Ca2+ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca2+ overload and consequent necrosis.

C-fiber depletion alters response properties of neurons in trigeminal nucleus principalis.

The effects of C-fiber depletion induced by neonatal capsaicin treatment on the functional properties of vibrissa-sensitive low-threshold mechanoreceptive (LTM) neurons in the rat trigeminal nucleus principalis were examined in adult rats. Neonatal rats were injected either with capsaicin or its vehicle within 48 h of birth. The depletion of unmyelinated afferents was confirmed by the significant decrease in plasma extravasation of Evan's blue dye induced in the hindlimb skin of capsaicin-treated rats by cutaneous application of mustard oil and by the significant decrease of unmyelinated fibers in both the sciatic and infraorbital nerves. The mechanoreceptive field (RF) and response properties of 31 vibrissa-sensitive neurons in capsaicin-treated rats were compared with those of 32 vibrissa-sensitive neurons in control (untreated or vehicle-treated) rats. The use of electronically controlled mechanical stimuli allowed quantitative analysis of response properties of vibrissa-sensitive neurons; these included the number of center- and surround-RF vibrissae within the RF (i.e., those vibrissae which when stimulated elicited >/=1 and <1 action potential per stimulus, respectively), the response magnitude and latency, and the selectivity of responses to stimulation of vibrissae in different directions with emphasis on combining both the response magnitude and direction of vibrissal deflection in a vector analysis. Neonatal capsaicin treatment was associated with significant increases in the total number of vibrissae, in the number of center-RF vibrissae per neuronal RF, and in the percentage of vibrissa-sensitive neurons that also responded to stimulation of other types of orofacial tissues. Compared with control rats, capsaicin-treated rats showed significant increases in the response magnitude to stimulation of surround-RF vibrissae as well as in response latency variability to stimulation of both center- and surround-RF vibrissae. C-fiber depletion also significantly altered the directional selectivity of responses to stimulation of vibrissae. For neurons with multiple center-RF vibrissae, the proportion of center-RF vibrissae with net vector responses oriented toward the same quadrant was significantly less in capsaicin-treated compared with control rats. These changes in the functional properties of principalis vibrissa-sensitive neurons associated with marked depletion of C-fiber afferents are consistent with similarly induced alterations in LTM neurons studied at other levels of the rodent somatosensory system, and indeed may contribute to alterations previously described in the somatosensory cortex of adult rodents. Furthermore, these results provide additional support to the view that C fibers may have an important role in shaping the functional properties of LTM neurons in central somatosensory pathways.

Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury.

Programmed cell death (apoptosis) is a normal process in the developing nervous system. Recent data suggest that certain features seen in the process of programmed cell death may be favored in the developing versus the adult brain in response to different brain injuries. In a well characterized model of neonatal hypoxia-ischemia, we demonstrate marked but delayed cell death in which there is prominent DNA laddering, TUNEL-labeling, and nuclei with condensed chromatin. Caspase activation, which is required in many cases of apoptotic cell death, also followed a delayed time course after hypoxia-ischemia. Administration of boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, was significantly neuroprotective when given by intracerebroventricular injection 3 h after cerebral hypoxia-ischemia. In addition, systemic injections of boc-aspartyl(OMe)-fluoromethylketone also given in a delayed fashion, resulted in significant neuroprotection. These findings suggest that caspase inhibitors may be able to provide benefit over a prolonged therapeutic window after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

Cell death suggestive of apoptosis after spinal cord ischemia in rabbits.

BACKGROUND AND PURPOSE: After spinal cord ischemia, some neurons remain viable after an ischemic insult but may be at risk of dying during reperfusion. We searched for morphological and biochemical features of apoptosis, which is a mechanism of delayed neuronal death, in a rabbit model of spinal cord ischemia. METHODS: The infrarenal aorta of White New Zealand rabbits (n = 24) was occluded for 40 minutes using a loop tourniquet. Rabbits were killed after 12, 24, or 48 hours (n = 8 per group). The loop was placed but never tightened in sham-operated rabbits (n = 6). The lumbar segment of the spinal cord (L5 to L7) was used for morphological studies, including hematoxylin and eosin staining and a modified terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining method. Electron microscopy was used to examine ultrastructural morphology. In addition, lumbar tissue was used for biochemical investigation of DNA laddering by agarose gel electrophoresis. RESULTS: After ischemia, the affected areas contained neurons with positive TUNEL staining. Positive neurons were located in laminae III to IX, although most were concentrated in the intermediate and ventral areas. Adjacent sections stained with hematoxylin and eosin exhibited ischemic cell changes (red and ghost neurons), while apoptotic bodies were also apparent. In addition, electron microscopy of ischemic tissue samples exhibited ultrastructural characteristics of apoptosis, including nuclear condensation and relatively normal organelle morphology. Finally, isolated DNA revealed a ladder on agarose gel electrophoresis, indicating DNA fragmentation into approximately 180 multiples of base pairs. CONCLUSIONS: Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis. These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia.

Postnatal development of terminals and synapses in laminae I and II of the rat medullary dorsal horn.

To better understand developing orofacial nociceptive circuits and to provide a baseline for evaluating injury-induced plasticity, the ultrastructure of the superficial laminae in the rat medullary dorsal horn was examined at birth and at postnatal days 1, 4, 17, and 90. Quantitative features of terminals and synapses were studied with stereological methods. In laminae I and II: 1) Axon terminal density increased significantly from birth to day 4 and again from day 4 to day 90. 2) The density of degenerating profiles increased significantly from birth to day 1 and from birth to day 4 and then decreased from day 4 to day 90. 3) Degenerating profiles were most dense on day 1 and declined steadily thereafter; by day 90, such profiles were rare. 4) Cavitation was by far the most common form of degeneration seen at early postnatal ages. 5) Growth cone-like profiles were most dense at birth and declined steadily during the first 2 postnatal weeks; by day 90, such profiles were absent. 6) Terminals with flat synaptic vesicles were rarely seen before day 90, when they accounted for 7% of the terminal population. 7) The density of synapses increased continuously from birth until day 90. These data suggest that, as in the spinal cord, medullary dorsal horn circuits are very immature at birth. Adult-like quantitative features are not attained until after day 17. Moreover, whereas degenerating profiles are prevalent during early postnatal development, and they have features that resemble naturally occurring degeneration, the total numbers of terminals and synapses continue to increase dramatically and gradually during a protracted postnatal period (to postnatal day 17).

Development of terminals and synapses in laminae I and II of the rat medullary dorsal horn after infraorbital nerve transection at birth.

Infraorbital nerve damage at birth kills neurons and alters anatomical, physiological, and biochemical properties of surviving cells in all portions of the trigeminal brainstem complex, with the exception of laminae I and II of the medullary dorsal horn. The resiliency of laminae I and II may be due to rapid terminal sprouting and reactive synaptogenesis in this region. To test this hypothesis, quantitative electron microscopy revealed the types and numbers of terminals, synapses, and degenerating and growth cone-like profiles in the left laminae I and II at 1, 4, 17, and 90 days after left infraorbital nerve section. Control data were derived from normal newborns and from the right laminae I and II and the left infraorbital nerve of every experimental animal. Deafferented laminae I and II contained a median of 11.7, 8.2, 21.8, and 38.2 synapses/100 microm3 on days 1, 4, 17, and 90, respectively. At corresponding ages, there were 17.1, 19.4, 36.2, and 32 terminals; 14.4, 4.2, 5.1, and 0.3 degenerating profiles; and 4.6, 2.2, 0.1, and 0 growth cone-like profiles/100 microm2. Significant differences from the control right side are: 1) The percentage area occupied by terminals is less on days 1 and 17; 2) terminal density does not increase from day 0 to day 4 as it does on the control side; 3) the density of degenerating profiles is higher on day 17; 4) growth cones are less dense on days 4 and 17; and 5) synapse density is lower on days 1 and 4. Axon number in the infraorbital nerve was highly predictive of terminal and synapse densities in deafferented laminae I and II at all ages. Thus, in laminae I and II, 1) the time course and nature of development are altered by deafferentation at birth; 2) reorganization of terminals and synapses occurs within a day of the lesion; 3) by day 90, there are no remaining lesion effects; and 4) the status of the injured nerve predicts central terminal and synapse densities. These are signs of injury-induced transganglionic degeneration and sprouting. The source of the latter is unknown, although areal fraction data suggest that "replacement" terminals may not be of primary afferent origin.

Slowly triggered excitotoxicity occurs by necrosis in cortical cultures.

This study examined the possibility that the excitotoxin-induced death of cultured cortical neurons might occur by apoptosis, specifically focusing on the slowly triggered death induced by low concentrations of excitotoxin. Cultured murine cortical neurons (days in vitro 10-12) were exposed continuously to N-methyl-D-aspartate (10-15 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (3-100 microM) or kainate (30-60 microM) over 24 h. Within 2 h of exposure onset, neuronal cell body swelling was visible under phase-contrast optics. At this point, transmission electron microscopy revealed disruption of cell membranes and organelles, mitochondrial swelling and scattered chromatin condensation at the periphery of nuclei. By 8 h after exposure onset, many neurons were devoid of cytoplasmic structures, but nuclear membranes remained relatively intact. This excitotoxic degeneration was not blocked by the protein synthesis inhibitor, cycloheximide, or the growth factors, brain-derived neurotrophic factor or insulin-like growth factor-1, agents that did block serum deprivation-induced apoptosis death in other cultures. DNA agarose gel electrophoresis, however, revealed the transient occurrence of internucleosomal DNA fragmentation, appearing 4-8 h after exposure onset, but absent 24 h after exposure onset. The present results suggest that even slowly triggered excitotoxicity occurs by necrosis, and raise a cautionary note in interpreting internucleosomal DNA fragmentation in isolation as evidence for apoptosis.

Peripheral and central predictors of whisker afferent morphology in the rat brainstem.

Prior studies suggest that whisker afferents have but one central projection pattern, despite their association with differing peripheral receptors that predict central morphology in other systems. Target factors in barrelettes are thought to dictate afferent projection patterns; yet, barrelettes differ in their size, shape and development. We tested the hypothesis that whisker afferents have differing morphologies that are predicted by peripheral and central factors. Branching patterns and collaterals of 78 Neurobiotin-stained afferents were compared in rats. Fibers from one whisker had precisely somatotopic projections but highly varied morphologies. For the entire sample, analysis of variance revealed significant intrafiber variance in collateral number and arbor shape that was attributed to the target subnucleus. Significant interfiber variance did not reflect response adaptation rate, direction sensitivity, whisker row origin or parent fiber bifurcation in the trigeminal root. Instead, we found the following. 1) Mandibular fibers had more elongated arbors than maxillary axons. In subnuclei interpolaris and principalis, mandibular fibers had larger arbors with more boutons/collateral than maxillary axons; in oralis and interpolaris, mandibular fibers had fewer collaterals than those of the maxillary division. 2) Upper lip whisker axons had more boutons than those from the B-D row in all subnuclei. 3) Rostral whisker are afferents had larger arbors and more boutons than those from middle or caudal arcs due to significant arc effects in interpolaris and oralis. Thus, whisker afferents are not structurally uniform, and some morphological features are predictable. Intrafiber variance is attributed to the central target; interfiber variance reflects maxillary versus mandibular origin, upper lip origin and whisker rostrocaudal arc.

Central projections of identified trigeminal primary afferents after molar pulp deafferentation in adult rats.

It is known that removal of the tooth pulp from mandibular molar teeth in adult rats alters the mechanoreceptive field properties of many low-threshold mechanoreceptive neurons in the trigeminal brainstem nuclear complex. The present study investigates one possible way that such deafferentation-induced receptive field changes could occur: altered central projections of uninjured trigeminal low-threshold mechanoreceptive primary afferent fibers. Intra-axonal injection of horseradish peroxidase (n = 22) or neurobiotin (n = 44) into characterized fibers was performed ipsilateral to, and 10-32 days after, removal of the coronal pulp from the left mandibular molars in adult rats. Collaterals were reconstructed, quantified, and compared by means of multivariate analyses of variance to equivalent fibers stained in normal adult rats. Stained mechanosensitive fibers from experimental animals were rapidly conducting and responded to light mechanical stimulation of one vibrissa, one tooth, oral mucosa, facial hairy skin, or guard hairs. Their central projections were indistinguishable from those of control axons in all four trigeminal subnuclei. The numbers of collaterals, areas subtended by collateral arbors, numbers of boutons per collateral, and arbor circularity did not differ from those of control afferents. Collateral somatotopy was also unaffected. These data suggest that following pulpotomy, the central collaterals of uninjured trigeminal afferents display normal morphologies and maintain normal somatotopy. Changes in the morphology of low-threshold primary afferents cannot account for the changes that occur in the receptive field properties of trigeminal brainstem neurons after pulp deafferentation.

Trigeminal structure-function relationships: a reevaluation based on long-range staining of a large sample of brainstem a beta fibers.

Prior studies suggest that some classes of thickly myelinated (A beta) afferents have distinct morphologies in the trigeminal (V) brainstem complex, and that single fibers have collaterals with different shapes in the four V subnuclei. However, these conclusions are based upon relatively few and incompletely stained fibers and limited statistical rigor. In the present study, 104 fibers were stained more completely with neurobiotin in rats to provide within-fiber intersubnucleus comparisons, and between-fiber intrasubnucleus comparisons, of collaterals associated with a vibrissa, guard hairs, hairy skin, glabrous skin, or oral structures. Collaterals from all functional categories had similar qualitative features and were distributed somatotopically in the transverse plane according to known maps. Fiber categories were not disproportionately represented at particular sites along the brainstem's rostrocaudal axis, although most fibers adhered to an onion-leaf topography in caudalis. Surprisingly few structure-function relationships were revealed by multivariate analysis of variance and post hoc group comparisons, as follows: Arbors were larger in caudalis than in any other subnucleus; collaterals were most numerous in interpolaris; vibrissa afferents had more collaterals than oral and guard hair afferents; and oral fibers had larger arbors than vibrissa or guard hair afferents in subnucleus oralis. Peripheral receptor association and response adaptation rate failed to predict arbor shapes and terminal bouton numbers in any V subnucleus. These data confirm that the locations of V primary afferent arbors are predicted by their receptive fields. However, collateral number and morphology are predicted only to a very limited extent by the V subnucleus and peripheral receptor affiliation--a conclusion that contrasts with those of most prior studies of somatosensory primary afferents.

Parenchymal microvascular systems and cerebral atrophy in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are hypertensive, hyperactive, and hydrocephalic; furthermore SHR have smaller brain volume and weight than age-matched, normotensive Wistar-Kyoto rats (WKY). At 6-7 months of age, local cerebral glucose is sizably lower in SHR than WKY. The hypothesis that these several abnormalities of SHR lead to variations in cerebral microvascular bed morphology was tested in 6-7-month-old SHR and WKY by quantitating various parameters of small, intermediate, and large parenchymal microvessels (grouped by luminal diameter) in 21 brain areas. Within each rat strain, the microvascular bed properties such as vessel profile frequency (density) varied considerably among the 21 brain areas. In opposition to the hypothesis, mean luminal diameter as well as profile frequency, surface area, and luminal volume of the microvascular beds per unit tissue mass were virtually identical in each brain area of SHR and WKY for the three groups of microvessels. These findings coupled with the reports of less tissue per structure but similar density of neurons throughout the brain of SHR and WKY indicate that there are fewer neurons and less vascular tissue per brain structure in 6-7-month-old SHR than WKY; in addition, they suggest a linkage between the size of parenchymal microvascular beds and the surrounding nervous tissue.