Perinatal, not adult, hypothyroidism suppresses dopaminergic axon sprouting in the deafferented olfactory tubercle of adult rat

We reported recently that chronic thyroid deficiency in rat, beginning in utero and terminating after maturity, suppresses lesion-induced central catecholaminergic axon sprouting in the adult brain [Gottesfeld et al, 1985]. The present work was undertaken to define the critical period of hypothyroidism on subsequent neuronal sprouting. Thyroid hormones deficiency was induced in rats by methimazole during (a) gestational days 8-21 (20 mg/kg/day in the drinking water); (b) postnatal days 1-15 (0.2 or 0.4 mg/pup/day; i.p.), or (c) in the mature animal for 4 weeks (20 mg/kg/day in the drinking water). The olfactory tubercles (OTs) were used as a model to study sprouting of dopaminergic (DA) nerve terminals, elicited by olfactory bulbectomy. Animals in each group received lesions or sham operations as adults, and sacrificed 3 weeks after the operation. Thus, for each of the above treatments four subgroups were formed: (a) euthyroid/sham-operation, (b) euthyroid/lesion, (c) hypothyroid/sham-operation, and (d) hypothyroid/lesion. Sprouting of DA axon terminals in the OTs was identified by biochemical assays and quantitative immunofluorescent microscopy, using tyrosine hydroxylase (TH) as a marker. Serum thyroxine levels served as an index of the thyroid status. The results demonstrate that lesion-induced sprouting of DA axon terminals in OTs of adult rats is suppressed by hypothyroidism induced prenatally or during the early postnatal period, but not after maturity. Thus, there is a perinatal critical period during which altered thyroid function exerts long-term effects on neuronal plasticity.     

Sympathetic noradrenergic sprouting in response to central cholinergic denervation: A histochemical study of neuronal sprouting in the rat hippocampal formation

An unusual example of neuronal sprouting occurs in the rat brain. Several weeks after fimbrial transection or septal lesions, peripheral sympathetic fibers appear in the dentate and hippocampal gyri. We compared the distribution of normal cholinergic septohippocampal fibers and nerve terminals with the distribution of noradrenergic sympathetic (sympathohippocampal) fibers after septal lesions using anterograde transport of horseradish peroxidase and fluorescence histochemistry. In addition, we destroyed other afferents to the hippocampal formation and examined the effect of subtotal septal lesions on acetylcholinesterase staining and the distribution of sympathohippocampal fibers. The combined results of these experiments suggest that peripheral noradrenergic fibers sprout specifically in response to destruction of central cholinergic fibers after septal lesions. This appears to be the first model of neuronal sprouting in the central nervous system where one identified transmitter system (noradrenergic) sprouts only in response to, and perhaps to replace, another specific transmitter system (cholinergic).     

Prenatal and postnatal methyl mercury exposure in Niigata,Japan: adult outcomes

Methyl mercury (MeHg) poisoning or Minamata disease (MD) from fish consumption is a public health concern throughout the world because all fish contain small amounts. The lowest exposure level needed to impair children's development is controversial. Actual poisoning with MeHg from fish consumption has been reliably reported only two times. It occurred in Minamata, Japan in the 1950s and then in Niigata, Japan in the 1960s. On each occasion, massive industrial pollution led to local fish having mercury levels as high as 40 ppm. In Niigata the pollution was on the Agano River and there were over 2000 commercial fishermen active at that time. We studied adult subjects who had been exposed perinatally to MeHg from fish consumption during the Niigata poisoning to determine the long-term impact of exposure. We identified mothers with elevated levels of exposure during the epidemic and those diagnosed with MeHg poisoning. The subjects of the study were their adult children, born during the epidemic. The evaluation consisted of a questionnaire (administered by interview) focusing on development, symptoms, and current function and a standard medical and neurological examination. The subjects were divided into four groups based upon prenatal levels of mercury in maternal hair or the presence of MD. For Group A the hair mercury levels were 50 ppm or more, for Group B the mercury levels were 25-49 ppm, and for Group C 10-24 ppm. The subjects in Group D were born to mothers diagnosed with MD, but their mercury levels were not measured. Exposure was predominantly prenatal, but some mothers also breast fed their infants. Group A included 13 subjects among whom two were diagnosed with congenital MeHg poisoning and in two others it was suspected. Group B included 10 subjects, of whom three had symptoms compatible with MeHg poisoning. Group C had nine subjects including one with intellectual deficit and another with hearing loss. Group D had eight subjects of whom four had symptoms compatible with MeHg exposure, but only one had abnormal neurological findings. Among the subjects thought to have congenital or childhood MeHg poisoning, intelligence did not appear to have declined over time. More children were affected by prenatal and postnatal MeHg exposure at Niigata than was previously reported.     

Plasticity of spinal systems after unilateral lumbosacral dorsal rhizotomy in the adult rat.

Plasticity of spinal systems in response to lumbosacral deafferentation has previously been described for the cat, by using immunocytochemistry to demonstrate plasticity of tachykinin systems and degeneration methods to demonstrate plasticity of descending systems. In this study, we describe the response to lumbosacral deafferentation in the adult rat. Application of immunocytochemical methods to visualize tachykinins (predominantly substance P magnitude of SP), serotonin (5-HT), and dopamine B-hydroxylase (DBH), the synthesizing enzyme for norepinephrine, permits us to compare the response of SP systems in rat and cat spinal cord and to examine the response of two descending systems, serotoninergic and noradrenergic, to deafferentation. We used image analysis of light microscopic preparations to quantify the immunoreaction product in the spinal cord in order to estimate the magnitude, time course and localization of changes induced by the lesion. The distribution of SP, serotoninergic (5-HT), and noradrenergic staining in the spinal cord of rat is very similar to that of the cat. Unilateral lumbosacral rhizotomy elicits a partial depletion, followed by a partial replacement of tachykinin immunoreactivity in laminae I and II. This response was similar to that described for the cat, although characterized by a longer time course, and, as in the cat, is likely due to plasticity of tachykinin containing interneurons. The same lesion elicits no depletion but a marked and permanent increase in 5-HT immunoreactivity in laminae I and II, which develops more rapidly than the response by the SP system. These results indicate sprouting or increased production of SP and 5-HT in response to deafferentation. No change was seen in DBH immunoreactivity, indicating that the noradrenergic system does not show plasticity in response to deafferentation. Our results demonstrate that dorsal rhizotomy evokes different effects in different systems in the adult spinal cord of the rat and thus suggests that the response of undamaged pathways to partial denervation of their target is regulated rather than random.     

Origin and distribution of noradrenergic innervation in the habenula: A neurochemical study

Retrograde axonal transport of fluorescent dyes was used to demonstrate collateral projections from neurons of the pontine taste area (PTA) to gustatory-responsive areas of the posterior ventromedial thalamic nucleus (VPM), and to the gustatory neocortex (GN) of the rat. Dual-labeled PTA neurons were reliably observed following application of two different fluorescent dyes to the GN and to VPM thalamus. Dye injections into the GN and into thalamic regions surrounding the VPM nucleus, the bed nucleus of stria terminalis or the infralimbic neocortex, did not result in dual-labeled cells within the PTA. This finding suggests that gustatory information may be relayed simultaneously and specifically to VPM thalamus and to the GN via collateral axons of PTA neurons.     

Prenatal experience and postnatal stress modulate the adult neurosteroid and catecholaminergic stress responses

Allopregnanolone (3α-hydroxy-5α-regnan-20-one) is a neuroactive steroid recently shown to be involved in the neurochemical stress response via its positive modulation of the GABA_A receptor complex. This experiment investigated the effects of postnatal stress (daily maternal separation during the first week of life) on the subsequent adult response to a stressor (10 min forced swim) in Long–Evans rats from one of three prenatal treatment groups (alcohol, pair-fed and control). Indices of stress response were allopregnanolone concentrations in plasma, cortex and hippocampus, and dopamine and norepinephrine concentrations in prefrontal cortex, nucleus accumbens and striatum. Females had higher levels of allopregnanolone than males in both plasma and brain. Prenatal alcohol exposure combined with early maternal separation stress resulted in an increase in the endogenous levels of allopregnanolone in the prefrontal cortex and hippocampus of adult offspring in response to a stressor compared to subjects without a prior history of postnatal stress ; this effect was greater in females. This increased allopregnanolone was also associated with decreased dopamine and norepinephrine levels in the prefrontal cortex. In the prenatal alcohol-exposed offspring, postnatal maternal separation blunted the increase in dopamine levels in the striatum seen in both control groups. Postnatal maternal separation increased norepinephrine levels in the nucleus accumbens regardless of prenatal experience, while in the prefrontal cortex only prenatal diet condition (pair-feeding and alcohol) resulted in lower norepinephrine levels. The results of this experiment suggest that experience, both pre- and postnatal, can have long-term consequences for the developing neurochemical responses to stressors.     

Reversible morphological alterations of cortical neurons in juvenile and adult hypothyroidism in the rat

We have studied: (i) whether or not hypothyroidism induced experimentally in the adult rat affects the number and distribution of spines along the apical shaft of pyramidal cells from the visual cortex; and (ii) whether treatment with thyroid hormone would reverse such changes.Two experiments were performed. Experiment A involved the study of rats which were thyroidectomized (T¯) at 40 or at 120 days of age, killed at 120 and 220 days of age, respectively, data being compared to those of adequate sex and age-paired controls. Experiment B involved rats which were T¯ at 40 days of age; they were subdivided into two groups, one of which was left untreated and theother received 0.2 μg of thyroxine/100 g body weight/day for 25 days. Both groups were killed at 90 days of age. We found that: (i) T¯ at 40 and at 120 days of age resulted in a decrease of the number of spines and a deragement of their distribution along the shaft, the observed changes being qualitatively comparable to those previously described^24,25 for rats T¯ at 10 days of age; and (ii) preliminary results from experiment B indicate that the observed changes in neuronal morphology might be reversible.It is suggested that age-independent and reversible alterations of learning capacity and electrocortical activity of hypothyroid rats^7,8 might have age-independent and reversible morphological correlates at the level of the cortex.     

Local cerebral glucose utilization in the adult cretinous rat

Local rates of cerebral glucose utilization were determined in 5-month-old neonatally radiothyroidectomized and control (littermate) rats. Virtually all 48 brain regions examined in the thyroidectomized rats exhibited lower rates of glucose utilization than those of the controls with differences ranging from -24 to -58%. The decreases were particularly large in the cerebral cortex and throughout the auditory system. Altered patterns in the intrastructural distribution of rates of glucose utilization were seen in a number of regions and were particularly prominent in the hippocampus and inferior colliculus. Lesser changes were seen in hypothalamic regions involved in the synthesis of thyrotropin releasing hormone (TRH). The results indicate that the many structural, functional and biochemical abnormalities of cretinism are associated with widespread reductions in energy metabolism throughout the brain.     

Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents

The cholinergic and adrenergic afferents innervating the hippocampal formation in the rat reach the target region via three distinctly separate routes, two dorsal and one ventral one. Partial deafferentation of the hippocampus obtained by destruction of the dorsal routes (through the fimbria-fornix and the supracallosal striae) resulted in removal of 90% and 60% of the cholinergic and adrenergic innervations, respectively, within one month. By 6–10 months after lesion, the remaining cholinergic and adrenergic inputs, reaching the target via the ventral route, had expanded more than two-fold, resulting in a significant recovery in the original cholinergic and adrenergic innervation patterns. Because of its slow and protracted time-course and its ability to re-establish innervation also in initially denervated areas, this compensatory collateral sprouting phenomenon may be of particular interest for the understanding of the long-term, protracted functional recovery that is seen both after experimental brain lesions as well as in patients with severe brain injuries.     

Trafficking of neurokinin-1 receptors in serotonin neurons is controlled by substance P within the rat dorsal raphe nucleus

Substance P (SP) modulates serotonin neurotransmission via neurokinin-1 receptors (NK1rs), and exerts regulatory effects on mood through habenular afferents to the dorsal raphe nucleus (DRN). We have previously demonstrated that, in the caudal DRN of rat, some serotonin neurons are endowed with NK1rs that are mostly cytoplasmic, whereas these receptors are mostly membrane bound in non-serotonin neurons. Here, we first examined by double-labeling immunocytochemistry the relationships between SP axon terminals and these two categories of DRN neurons. Almost half of the SP terminals were synaptic and many were in close contact with serotonin dendrites, but never with non-serotonin dendrites. In additional double-immunolabeling experiments, most if not all dendrites bearing membranous NK1rs appeared to be GABAergic. Treatment with the selective neurokinin-1 antagonist RP67580 modified the subcellular distribution of NK1rs in serotonin neurons. At 1 h after administration of a single dose, the receptor distribution was unchanged in both dendritic types but, after daily administration for 7 or 21 days, the plasma membrane and cytoplasmic density of NK1rs were increased in serotonin dendrites, without any change in non-serotonin dendrites. These treatments also increased NK1r gene expression in the caudal DRN. Lastly, a marked increase in the membrane (but not cytoplasmic) density of NK1rs was measured in serotonin dendrites after bilateral habenular lesion. These results suggest that the trafficking of NK1rs represents a cellular mechanism in control of the modulation of serotonin neuron activity by SP in DRN.     

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 μm³ 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 μm². 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. J. Comp. Neurol. 383:339-348, 1997. © 1997 Wiley-Liss, Inc.     

Induction of ephrin-B1 and EphB receptors during denervation-induced plasticity in the adult mouse hippocampus

Abstract It has been widely demonstrated that Eph receptors and their ephrin ligands play multiple pivotal roles in the development of the nervous system. However, less is known about their roles in the adult brain. Here we reported the expression of ephrin-B1 and its cognate EphB receptors in the adult mouse hippocampus at 3, 7, 15, 30 and 60 days after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent up-regulation of ephrin-B1 in the denervated areas of the hippocampus, which initiated at 3 days postlesion (dpl), reached maximal levels at 7-15 dpl, remained slightly elevated at 30 dpl and recovered to normal levels by 60 dpl. Double labeling of ephrin-B1 and glial fibrillary acidic protein revealed that ephrin-B1-expressing cells in the denervated areas were reactive astrocytes. Furthermore, a ligand-binding assay using ephrin-B1/Fc chimera protein also displayed the up-regulation of EphB receptors in the denervated areas of the hippocampus in a similar manner to that of ephrin-B1. Within the first week postlesion, the EphB receptors were expressed by reactive astrocytes. After 7 dpl, however, EphB receptors were expressed not only by reactive astrocytes but also first by sprouting axons and later by regrowing dendrites. These results suggest that the ephrin-B1/EphB system may participate in the lesion-induced plasticity processes in the adult mouse hippocampus.     

Serotonergic sprouting is induced by dopamine-lesion in substantia nigra of adult rat brain

We have previously extracted a serotonin (5-HT) neurotrophic supernatant from the 5,7-DHT lesioned hippocampus. The current study shows that a new 5-HT neurotrophic signal was monitored in the striatum and nigra after DA-denervation. Such a signal may be involved in the heterotypic sprouting. Dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected directly into the substantia nigra of adult rats. Two months after surgery, immunocytochemical staining showed that tyrosine hydroxylase (TH)-positive cell bodies had mostly disappeared in the substantia nigra, and TH-positive terminals in the striatum were almost completely depleted. Meanwhile, the 5-HT fibers, which exist in the same areas with low density, sprouted in the nigra as well as in the striatum and became dense. Normally 5-HT fibers innervate the striatum sparsely and the globus pallidus densely with sharp delineation (in the control side), and become dense across both areas with no appreciable delineation (in the lesion side). The increase of 5-HT fibers was more prominent in the posterior than in the anterior striatum. A significant increase in 5-HT and 5-HIAA levels was also evident in the posterior striatum when the decrease in DA level exceeded 90% in the nigra and striatum. In addition, we found that induction of 5-HT sprouting requires a greater than 90% decrease of DA level. Current data support that 6-OHDA injection in the substantia nigra of adult rats triggered a trophic signal or removed an inhibition for the growth of 5-HT neurons which responded with sprouting in the nigra as well as in the striatum.     

Central sprouting of uninjured small fiber afferents in the adult rat spinal cord following spinal nerve ligation

Partial nerve injury results in chronic pain that is difficult to treat effectively. To investigate the anatomic basis of this phenomenon we used wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to label the central projections of uninjured small fibers (Adelta and C) in a well-established model of neuropathic pain created by selective spinal nerve ligation in the adult. We found extensive sprouting of uninjured WGA-HRP-labeled afferents into the central termination field in lamina II of dorsal horn normally occupied by L5 afferents whose peripheral axons had been ligated distal to the dorsal root ganglion. The formation of new projections by uninjured fibers into a functionally but not anatomically deafferented field in the adult may play a role in the development of chronic pain.     

In vitro cellular processes sprouting in cortex microexplants of adult rat brains induced by low power laser irradiation

Abstract

The effect of He-Ne low power laser irradiation on the growth of cellular processes of microexplants of the brain cortex of adult rats in tissue culture was studied. Two 8-min doses of direct laser irradiation, 3.6 J/cm² each given on two successive days, caused a significant amount of sprouting of cellular processes outgrowth in microexplants compared to small amounts produced by nonirradiated controls. This preliminary observation suggests that low power laser irradiation applied to the area of an experimentally injured nerve may induce neurite processes sprouting, thereby improving nerve tissue recovery. [Neural Res 1998; 20: 470–472]     

Nerve sprouting and endplate growth induced in normal muscle by contralateral partial denervation of rat plantaris

The incidence of motor nerve and terminal sprouting was quantitatively analyzed in normal unoperated muscles, in homologous muscles contralateral to muscles which have been partially denervated, in partially denervated muscles, and in sham-operated muscles. Muscles were studied by light microscopy after staining motor endplates by a combined silver-cholinesterase stain. In addition, the incidence of endplates containing terminal sprouts, the number of terminal branch points per endplate, and endplate size were also assessed in the various groups examined. We observed that following section of the L₄ spinal nerve, the incidence of sprouting (preterminal and intranodal) in the contralateral muscle exhibited a 2-fold increase over sham-operated controls. We also found a correlation between nerve terminal sprouting, terminal branch point number and endplate size. All of these parameters were significantly increased in the contralateral muscles as compared to the sham-operated control muscles. These findings suggest that normal muscles undergo sprouting which can be enhanced by contraleteral partial denervation. The possible underlying mechanism may be the transneuronal induction of sprouting.     

Plasticity in the rat olfactory cortex

The relationship of age to deafferentation plasticity was studied in the rat olfactory cortex (OC). Ablation of a single olfactory bulb (OB) was performed in each of several rats of selected postnatal (PN) ages: PN2.5, 6, 9, 13, and 21 days and in adults of PN100 days. Following survival times sufficient to remove the resultant degeneration, a cortical lesion was placed in the ipsilateral OC. The patterns of degeneration from the OC lesion were studied and mapped in the adjacent deafferented OC. The results show a spread or sprouting of the usually deep-lying afferents (interrupted by the OC lesion), onto the deafferented superficial dendrites (normally occupied by the OB afferents) in all of the ages. The spread is most striking at PN2.5 to PN9, gradually reduced by PN13 to PN21, and least in the adult (PN100). There is also an apparent increase of afferents to the deeper dendrites nearer the cell bodies in all cases except in the PN 100 group. Shrinkage of layer I is not seen in PN2.5 subjects, is minimal by PN9, but is most marked in the adult PN100 with total OB lesions. Incomplete OB lesions sparing some lateral olfactory tract (LOT) fibers greatly reduce the shrinkage of layer I and the spread of afferents in all ages. Thus, a capacity for reorganization of afferents occurs at least through PN9, with PN13-21 a possible "critical period" after which plasticity is limited and transneuronal effects are more permanent. The association, centrifugal, and olfactory-entorhinal pathways are possible origins for this plasticity. Factors contributing to limitations in this reorganization are discussed.     

Effect of applied electrical fields on sprouting of intact saphenous nerve in adult rat

Saphenous nerve sprouting was measured behaviorally and histologically after chronic sciatic denervation in the adult rat. The effect of electrical stimulation (either weak DC fields, or stronger AC fields) on the rate of sprouting of the intact saphenous was studied. Sprouting was enhanced by DC fields (1 μA) if the cathode was placed distal to the growth tips, but was unaffected by anode stimulation. Sprouting was also enhanced by AC fields (1000 μA per pulse) given at 20 Hz and 0.1 ms duration. In the discussion we postulate that separate mechanisms might mediate the AC and DC results. The DC effects are the first demonstration in mammals of results previously observed in lower vertebrates.