Carlos Avendaño

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P0 in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice

Transgenic mouse lines in which a fluorescent protein is constitutively expressed under the Thy1 gene promoter have become important models in cell biology and pathology studies of specific neuronal populations. As a result of positional insertion and/or copy number effects on the transgene, the populations expressing the fluorescent protein (eYFP+) vary markedly among the different mice lines. However, identification of the eYFP+ subpopulations has remained sketchy and fragmentary even for the most widely used lines such as Thy1-eYFP-H mice (Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman and J.W., Sanes. J.R. 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41-51). Here, we provide a comprehensive mapping of labeled cell types throughout the central nervous system in adult and postnatal (P0-P30) Thy1-eYFP-H mice. Cell type identification was based on somatodendritic morphology, axon trajectories, and, for cortical cells, retrograde labeling with Fast Blue to distinguish between subpopulations with different axonal targets. In the neocortex, eYFP+ cells are layers 5 and 6 pyramidal neurons, whose abundance and sublaminar distribution varies markedly between areas. Labeling is particularly prevalent in the corticospinal cells; as a result, the pyramidal pathway axons are conspicuously labeled down to the spinal cord. Large populations of hippocampal, subicular and amygdaloid projection neurons are eYFP+ as well. Additional eYFP+ cell groups are located in specific brainstem nuclei. Present results provide a comprehensive reference dataset for adult and developmental studies using the Thy1-eYFP-H mice strain, and show that this animal model may be particularly suitable for studies on the cell biology of corticospinal neurons.

Cochlear abnormalities in insulin-like growth factor-1 mouse mutants

Insulin-like growth factor 1 (IGF-1) modulates inner ear cell proliferation, differentiation and survival in culture. Its function in human hearing was first evidenced by a report of a boy with a homozygous deletion of the Igf-1 gene, who showed severe sensorineural deafness [Woods et al., New Engl. J. Med. 335 (1996) 1363-1367]. To better understand the in vivo role of IGF-1 during inner ear differentiation and maturation, we studied the cochleae of Igf-1 gene knockout mice by performing morphometric stereological analyses, immunohistochemistry and electron microscopy on postnatal days 5 (P5), P8 and P20. At P20, but not at P5, the volumes of the cochlea and cochlear ganglion were significantly reduced in mutant mice, although the reduction was less severe than whole body dwarfism. A significant decrease in the number and average size of auditory neurons was also evident at P20. IGF-1-deficient cochlear neurons showed increased apoptosis, along with altered expression of neurofilament 200 kDa and vimentin. The eighth nerve, the cochlear ganglion and the fibers innervating the sensory cells of the organ of Corti of the P20 mouse mutants presented increased expression of vimentin, whereas the expression of neurofilament was decreased. In addition, the myelin sheath was severely affected in ganglion neurons. In conclusion, IGF-1 deficit in mice severely affects postnatal survival, differentiation and maturation of the cochlear ganglion cells.

The nigrostriatal system in the presymptomatic and symptomatic stages in the MPTP monkey model: a PET, histological and biochemical study.

Parkinsons disease (PD) is diagnosed when striatal dopamine (DA) loss exceeds a certain threshold and the cardinal motor features become apparent. The presymptomatic compensatory mechanisms underlying the lack of motor manifestations despite progressive striatal depletion are not well understood. Most animal models of PD involve the induction of a severe dopaminergic deficit in an acute manner, which departs from the typical, chronic evolution of PD in humans. We have used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered to monkeys via a slow intoxication protocol to produce a more gradual development of nigral lesion. Twelve control and 38 MPTP-intoxicated monkeys were divided into four groups. The latter included monkeys who were always asymptomatic, monkeys who recovered after showing mild parkinsonian signs, and monkeys with stable, moderate and severe parkinsonism. We found a close correlation between cell loss in the substantia nigra pars compacta (SNc) and striatal dopaminergic depletion and the four motor states. There was an overall negative correlation between the degree of parkinsonism (Kurlan scale) and in vivo PET ((18)F-DOPA K(i) and (11)C-DTBZ binding potential), as well as with TH-immunoreactive cell counts in SNc, striatal dopaminergic markers (TH, DAT and VMAT2) and striatal DA concentration. This intoxication protocol permits to establish a critical threshold of SNc cell loss and dopaminergic innervation distinguishing between the asymptomatic and symptomatic parkinsonian stages. Compensatory changes in nigrostriatal dopaminergic activity occurred in the recovered and parkinsonian monkeys when DA depletion was at least 88% of control, and accordingly may be considered too late to explain compensatory mechanisms in the early asymptomatic period. Our findings suggest the need for further exploration of the role of non-striatal mechanisms in PD prior to the development of motor features.

Neuron numbers in the sensory trigeminal nuclei of the rat: A GABA- and glycine-immunocytochemical and stereological analysis

The volume, total neuron number, and number of GABA‐ and glycine‐expressing neurons in the sensory trigeminal nuclei of the adult rat were estimated by stereological methods. The mean volume is 1.38 ± 0.13 mm3 (mean ± SD) for the principal nucleus (Vp), 1.59 ± 0.06 for the n. oralis (Vo), 2.63 ± 0.34 for the n. interpolaris (Vip), and 3.73 ± 0.11 for the n. caudalis (Vc). The total neuron numbers are 31,900 ± 2,200 (Vp), 21,100 ± 3,300 (Vo), 61,600 ± 8,300 (Vip), and 159,100 ± 25,300 (Vc). Immunoreactive (‐ir) neurons were classified as strongly stained or weakly stained, depending on qualitative criteria, cross‐checked by a densitometric analysis. GABA‐ir cells are most abundant in Vc, in an increasing rostrocaudal gradient within the nucleus. Lower densities are found in Vip and Vp. The mean total number of strongly labeled GABA‐ir neurons ranges between 1,800 in Vp to 7,800 in Vip and 22,900 in Vc, and varies notably between subjects. Glycine‐ir neurons are more numerous and display more homogeneous densities in all nuclei. Strongly labeled Gly‐ir cells predominate in all nuclei, their total number ranging between 9,400 in Vp to 24,300 in Vip and 34,200 in Vc. A substantial fraction of immunolabeled neurons in all nuclei coexpress GABA and glycine. In general, all neurons strongly immunoreactive for GABA are small, while weakly GABA‐ir cells which coexpress Gly are larger. In Vc, one‐third of all neurons are immunoreactive: 16.6% of them are single‐labeled for GABA and 31.6% are single‐labeled for glycine. The remaining 51.8% express GABA and glycine in different combinations, with those showing strong double labeling accounting for 22.6%. J. Comp. Neurol. 493:538–553, 2005.

Reduction of Infarct Size by Intra-Arterial Nimodipine Administered at Reperfusion in a Rat Model of Partially Reversible Brain Focal Ischemia

BACKGROUND AND PURPOSE When blood flow to a brain region that has undergone an ischemic attack is reestablished, additional injury is to be expected from the reperfusion. The purpose of the study was to determine the effect of the intra-arterial injection of nimodipine at reperfusion on infarct volume in rats subjected to partially reversible focal neocortical ischemia. METHODS Two groups of Long-Evans rats with transient bilateral common carotid artery occlusion and permanent middle cerebral artery occlusion were subjected to retrograde cannulation of the external carotid artery close to the carotid bifurcation to allow the administration of isotonic saline (group 1) or nimodipine solution (group 2) just before and during reperfusion. The estimate for the actual amount of infarcted cortex was calculated by the volume ratio between the spared cortex in the infarcted hemisphere and the total cortex of the contralateral hemisphere by means of a serological method based on the Cavalieri principle. RESULTS The percentage of cortex that was infarcted in control rats was 63.8 +/- 3.1%, whereas nimodipine-treated rats exhibited a significantly smaller (P < .005) percentage of infarct volume (31.3 +/- 12.7%). CONCLUSIONS Our data show that the intra-arterial injection of nimodipine just before and during reperfusion reduced neocortical infarct volume in rats subjected to partially reversible focal cerebral ischemia.

Local anaesthesia induces immediate receptive field changes in nucleus gracilis and cortex.

The reorganization of receptive fields of nucleus gracilis neurones after local anaesthesia, and its relationship to the reorganization of cortical maps were studied in the rat. Cutaneous stimulation was performed using electronically gated air jets. Single unit recordings were obtained in gracilis nucleus and somatosensory cortex. Temporary anaesthesia was induced with lidocaine (2%, 5–15 µl s.c.), which blocked the responses in <2 min and provoked the simultaneous appearance of new overlapping receptive fields in gracilis and cortical neurones in 2–30 min. The present results suggest that the early reorganization of somatosensory cortical maps after temporary anaesthesia may be partly due to the emergence of new receptive fields in nucleus gracilis neurones.

Quantitative stereological evaluation of the gracile and cuneate nuclei and their projection neurons in the rat

Stereological methods were employed to estimate the volume and neuron numbers of the rat dorsal column nuclei (DCN). These methods were applied to Nissl‐stained sections from control animals and cases that received injections of horseradish peroxidase in the thalamus, the cerebellum, or the spinal cord. Additional cases received combinations of fluorescent tracers in the same structures, to examine whether some of the retrogradely labeled neurons sent collaterals to different targets. The mean volume of the DCN is 0.81 mm3 (range 0.65–1.10 mm3), of which 3%, 39%, and 59% correspond, respectively, to the nucleus of Bischoff (Bi), the gracile (Gr), and the cuneate (Cu) nuclei. Within Cu, the middle division (CuM) is the largest (42%), followed by the rostral (CuR; 36%) and caudal (CuC; 22%) divisions. The mean total number of neurons in the DCN is 16,000 (range 12,400–19,500), of which 2.4%, 34.0% and 63.6% correspond, respectively, to Bi, Gr, and Cu. Within Cu, CuM contains 48% of all neurons, and 27% correspond to CuR and 25% to CuC. Interanimal variability is moderate for the whole DCN and Cu but increases when individual nuclei are considered. About 80% of DCN neurons project to the thalamus, 3% to the spinal cord, and 7% to the cerebellum. Thalamic‐projecting cells are more numerous in CuM and Gr (83%), and relatively less common in Bi and CuC (72–74%). Most of the DCN neurons projecting to the spinal cord appear in CuC and CuM. Two‐thirds of the neurons projecting to the cerebellum are located in CuR, 20% in CuM, and 15% in Gr. A small fraction of neurons projects simultaneously to spinal cord and thalamus. J. Comp. Neurol. 463:419–433, 2003.

Thalamic afferents to the motor cortex in the cat. A horseradish peroxidase study

Thalamic projections to the motor cortex have been studied in the cat by injecting small amounts of horseradish peroxidase in different parts of areas 4 and 6. The nuclei which more abundantly project to the motor cortex are the ventral anterior-ventral lateral complex, the ventral medial and the posterior complex. These projections are topographically organized. Other projections arise from the ventral postero-lateral, ventral postero-inferior, central lateral, central medial, medial dorsal, lateral posterior-pulvinar, suprageniculate and centrum medianum-parafascicular nuclei.

Differential growth of axons from sensory and motor neurons through a regenerative electrode: a stereological, retrograde tracer, and functional study in the rat.

Polyimide regenerative electrodes (RE) constitute a promising neural interface to selectively stimulate regenerating fibers in injured nerves. The characteristics of the regeneration through an implanted RE, however, are only beginning to be established. It was recently shown that the number of myelinated fibers distal to the implant reached control values 7 months postimplant; however, the functional recovery remained substantially below normal [J Biomed Mater Res 60 (2002) 517]. In this study we sought to determine the magnitude, and possible selectivity, of axonal regeneration through the RE by counting sensory and motor neurons that were retrogradely labeled from double tracer deposits in the sciatic nerve. Adult rats had their right sciatic nerves transected, and the stumps were placed in silicone tubes; some simply were filled with saline (Tube group), and others held a RE in its center (RE group). Simultaneously, the proximal stump was exposed to Diamidino Yellow. Two months later the nerves were bilaterally excised distal to the implant, and exposed to Fast Blue. Electrophysiological recordings, and skin nociceptive responses confirmed previous findings of partial functional recovery. In controls, an average of 20,000 and 3080 neurons were labeled in L4-L5 dorsal root ganglia (with minor contributions from L3 and/or L6), and in the ventral horn of the lumbar spinal cord, respectively. In the regenerating side, 35% of the DRG neurons were double-labeled, without differences between groups. In contrast, only 7.5% of motoneurons were double-labeled in the RE group, vs. 21% in the Tube group. Moreover, smaller ganglion cells regenerated better than large neurons by a significant 13.8%. These results indicate that the RE is not an obstacle for the re-growth of sensory fibers, but partially hinders fiber regeneration from motoneurons. They also suggest that fine fibers may be at an advantage over large ones to regenerate through the RE.