Birthdates of the growth hormone releasing factor cells of the rat hypothalamus: an autoradiographic study of immunocytochemically identified neurons.

Growth hormone releasing factor (GRF) neurons in the arcuate nucleus of the hypothalamus and somatostatin (SRIF) neurons in the anterior periventricular region of the hypothalamus act to control the release of growth hormone from the anterior pituitary. To investigate the possibility that the growth-controlling functions of these cells might be compromised by injuries to the developing brain, it is important to know the details of the production and differentiation of these small, specialized cell groups. The overall pattern of cell production in the hypothalamus is known from autoradiographic studies with general nuclear stains, but no data are available on the birthdates (times of final mitoses) of GRF-producing cells. The present study was undertaken to determine when the GRF cells form. Counts of immunocytochemically identified GRF cells labeled on given days were taken from serial coronal sections through the hypothalamus of adult rats labeled on the 10th-17th days of gestation (day of finding a vaginal plug = day 1). As has been shown for the hypothalamus in general, the GRF cells showed a gradient of production from anterior to posterior. The peak of anterior cell proliferation was on day 13, middle cells on day 14, and posterior cells on day 15. These dates are 1 or 2 days earlier than those of GRF-negative cells in the same regions. No lateral to medial gradient of formation was seen in GRF cells. Rather, the laterally placed cells along the base of the brain and those surrounding the ventromedial nucleus formed simultaneously with the GRF cells of the arcuate nucleus. The birthdating results presented here are in agreement with the results of studies of teratogens which suggest that rat postnatal growth is reduced most severely by exposure to neurotoxic agents on days 12 or 13 of gestation. On the basis of data for the whole hypothalamus, such treatments would appear to be too early to interfere with cell production for the arcuate nucleus, but the timing fits the period of vulnerability as defined by the birthdates determined in the present study for the subpopulation of cells destined to produce GRF.

Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei.

The underlying brain injury that leads to autism has been difficult to identify. The diagnostic criteria of the disease are not readily associated with any brain region or system, nor are they mimicked by vascular accidents, tumors, or degenerative neurological diseases occurring in adults. Fortuitously, a recent report of autism induced by thalidomide exposure provides evidence that the disease originates by an injury at the time of closure of the neural tube. The human data suggest that the initiating lesion includes the motor cranial nerve nuclei. To test this hypothesis, we first examined motor nuclei in the brainstem of a human autistic case. The autopsy brain exhibited near-complete absence of the facial nucleus and superior olive along with shortening of the brainstem between the trapezoid body and the inferior olive. A similar deficit has been reported in Hoxa-1 gene knockout mice in which pattern formation of the hindbrain is disrupted during neurulation. Alternatively, exposure to antimitotic agents just after neural tube closure could produce the observed pattern of deficits. Thus, the lesions observed in the autopsy case appear to match those predicted by the thalidomide cases in both time of origin and central nervous system (CNS) location. To produce similar brain lesions experimentally, we exposed rat embryos to valproic acid, a second teratogen newly linked to autism. Dams received 350 mg/kg of valproic acid (VPA) on day 11.5 (the day of neural tube closure), day 12, or day 12.5 gestation. Each treatment significantly reduced the number of motor neurons counted in matched sections of the earliest-forming motor nuclei (V, XII), and progressively later exposures affected the VIth and IIIrd cranial nerve nuclei. All treatments spared the facial nucleus, which forms still later. Counts from the mesencephalic nucleus of trigeminal, the dorsal motor nucleus of the vagus, and the locus ceruleus were not affected by exposure to VPA, even though these nuclei form during the period when exposure occurred. Despite its effects on the motor nuclei, valproic acid exposure did not alter the further development of the brain in any obvious way. Treated animals were robust and had no external malformations. The autopsy data and experimental data from rats confirm that CNS injuries occurring during or just after neural tube closure can lead to a selective loss of neurons derived from the basal plate of the rhombencephalon. The results add two new lines of evidence that place the initiating injury for autism around the time of neural tube closure.

Developing brain as a target of toxicity.

The human brain forms over an unusually long period compared to other organs. While most of the basic structure is laid down before birth, neuron proliferation and migration continue in the postnatal period. The blood-brain barrier is not fully developed until the middle of the first year of life. The number of synaptic connections between neurons reaches a peak around age two and is then trimmed back by about half. Similarly, there is great postnatal activity in the development of receptors and transmitter systems as well as in the production of myelin. Many of the toxic agents known to damage the developing brain interfere with one or more of these developmental processes. Those with antimitotic action, such as X-ray and methyl mercury, have distinctly different effects on structure depending on which neurons are forming at the time of exposure. Vulnerability to agents that interfere with cell production decreases rapidly over the early postnatal period. Other toxic substances, such as psychoactive drugs and agents that alter hormone levels, are especially hazardous during synaptogenesis and the development of transmitter systems, and thus continue to be damaging for years after birth. Still other toxic substances such as lead, seem to have their greatest effects during even later stages of brain development, perhaps by interfering with the trimming back of connections. Guidelines designed to protect human populations from developmental neurotoxicity need to take into account the changing sensitivity of the brain as it passes through different developmental stages, as well as the fundamental differences in the effects of toxicants on the mature and the developing brain.

Vulnerable periods and processes during central nervous system development.

The developing central nervous system (CNS) is the organ system most frequently observed to exhibit congenital abnormalities. While the developing CNS lacks a blood brain barrier, the characteristics of known teratogens indicate that differential doses to the developing vs mature brain are not the major factor in differential sensitivity. Instead, most agents seem to act on processes that occur only during development. Thus, it appears that the susceptibility of the developing brain compared to the mature one depends to a great extent on the presence of processes sensitive to disruption. Yet cell proliferation, migration, and differentiation characterize many other developing organs, so the difference between CNS and other organs must depend on other properties of the developing CNS. The most important of these is probably the fact that nervous system development takes much longer than development of other organs, making it subject to injury over a longer period.

Critical periods for behavioral anomalies in mice.

While mice have been used less frequently than rats in behavioral research, there use has some advantages in teratological studies. The development of the mouse CNS has been investigated more extensively than that of the rat. Since time of insult has been found to be an important factor in effects on both anatomy and behavior, data on the sequence of events in CNS development are valuable in planning and interpreting behavioral assessments of potential teratogens. A comparison of studies in mice and rats suggests that behavioral effects of teratogens are similar in the two species and demonstrates that mice can be used successfully in a variety of behavioral evaluations.

Reduction of axonal transport in the rat optic system after direct application of methylmercury.

Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation counts of protein-bound radioactivity along the visual pathway after an intraocular injection of [3H]proline. In control rats the label traveled at a rate of about 60 mm/day, reaching the optic chiasm at 4 h and the lateral geniculate body at 8 h postinjection. When methylmercury was injected simultaneously with [3H]proline, the label traveled at a rate of about 30 mm/day. At 8 h postinjection, the labeled protein had reached the optic chiasm, but the more distal pathway was unlabeled. The same pattern was observed histologically by emulsion autoradiography of the pathway. Some label was detected in the lateral geniculate of methylmercury-treated animals at 8 h, but this may have resulted from local incorporation, as judged by a similar level of labeling in the contralateral visual pathway. Alternatively, it may be the case that a small fraction of the axons in the treated pathway continued to transport proteins in a normal fashion. The very heavy label observed throughout the pathway in controls was present only in the proximal half of the pathway in methylmercury-treated rats. Methylmercury significantly reduced incorporation of [3H]proline in the rat retina, but this reduction was not as great as the effect in the optic nerve. In contrast, cycloheximide, a potent protein synthesis inhibitor, reduced labeled protein in the optic nerve only to the same extent as it reduced incorporation. These results suggest that methylmercury's effect on transport is not dependent solely on its effects on protein synthesis, but represents a separate mechanism of neurotoxicity.

Increased axonal transport in the rat optic system after systemic exposure to methylmercury: differential effects in local vs systemic exposure conditions.

Axonal transport was studied by several techniques in the optic system of adult female Long-Evans rats following systemic exposure to methylmercury in 5 mM Na2CO3. Control rats were treated with the buffer alone. Four mg Hg/kg body weight for 4-6 days, or for 12 days, induced significant changes in the rate of protein synthesis in the retinal cells and in the rate of propagation of protein-bound radioactivity along the ganglion cell axons. Axonal transport of particulate material in both groups treated with methylmercury increased to a rate of 147 mm/day compared to 93 mm/day in controls. Methylmercury was distributed evenly throughout the retinogeniculate system. No clinical neuropathy was evident in either mercury-treated group. It is proposed that the increased rates of transport may represent an adaptive compensatory response to distal axonopathy caused by methylmercury. To investigate why systemic dosing produced effects opposite to those observed with local application of MeHg, various doses of MeHg were tested in the local and systemic paradigms, including doses which yielded equal concentrations of Hg in the retina. The results indicate that the differential response between the two treatment conditions is not a function of local dose, per se. Local and systemic application produce different dose-effect curves, which do not coincide at any dose.

Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure.

Developing animals have long been believed to be more sensitive to methylmercury toxicity than adults, but the reasons for differential effects are not well understood. In the present study, 2-day-old mice received a single per os dose of 4 mg Hg/kg methylmercury and were sacrificed 24 h or 19 days later. This resulted in a mean brain concentration of 1.8 micrograms Hg/g tissue on day 3 and less than 0.1 micrograms Hg/g on day 21. Compared to littermate vehicle controls, the methylmercury-treated mice exhibited a significant reduction in cell numbers in 1 of 4 regions of the developing cerebellar external granular layer 24 h after treatment. Although the mitotic index over the same 4 regions was not significantly altered by methylmercury treatment, the total number of mitotic figures per section of cerebellum was significantly reduced in the treated group. The ratio of late mitotic figures to total mitotic figures was significantly reduced, indicating mitotic arrest. Both of these antimitotic effects were greater in males than females. Cerebellar structure was also examined 19 days after methylmercury treatment. The number of cells in the molecular layer and thickness of the molecular layer and internal granular layer were significantly reduced in males; the number of Purkinje cells in both sexes and all measures in females remained unaltered. This suggests that early cell loss results in persistent reductions in cell number. Although the basis for the differential effect in males and females is not known, the antimitotic effect of methylmercury is most likely the mechanism underlying the reduced cellularity in treated animals.

Linking etiologies in humans and animal models: studies of autism.

Thalidomide has been shown to lead to a high rate of autism when exposure occurs during the 20th to 24th d of gestation. Both the critical period and the neurological deficits of the autistic cases indicate that they have sustained injuries to the cranial nerve motor nuclei. To determine whether such lesions characterize other cases of autism, the brain stem of an autistic case was compared to that of a control. The autopsy case showed abnormalities predicted by the thalidomide cases and evidence of shortening of the brain stem, a defect that could have occurred only during neural tube closure. To test whether animals can be similarly injured but remain viable, rats were treated with 350 mg/kg of valproic acid on day 11.5, 12, or 12.5 of gestation. Neuron counts showed reductions of cell numbers in the cranial nerve motor nuclei. Rats with motor neuron deficits also had cerebellar anomalies like those reported in studies of autistic cases, supporting the idea that these animals may be a useful model of the developmental injury that initiates autism.

A comparison of hypothalamic cell numbers in dwarf and normal weight rats exposed prenatally to methylazoxymethanol (MAM).

Growth deficiencies follow MAM exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, while animals exposed slightly later in gestation when the inhibitors of growth hormone release are forming, exhibit giantism. Counts of sample regions of the hypothalamus have shown that rats treated in utero on the 14th day of gestation have reductions in the number of GRF cells, increases in the number of SRIF (somatotropin release inhibiting factor) cells, and alterations of pituitary structure. These effects occurred in all treated subjects, even though obvious effects on body size were present in a small fraction of the treated animals. The present study was designed to examine the effect of 20 mg/kg MAM on the 13th day of gestation (a peak period for production of GRF cells) on GRF and SRIF cell numbers, in a large sample of dwarf-treated rats, normal weight-treated rats, and controls. The results of total counts of hypothalamic cells identified by immunocytochemistry demonstrated significant reductions in GRF cells in both dwarf and normal weight rats exposed to MAM, compared to controls, with no difference between the two treated groups. Like pituitary weights, the neuron counts were significantly correlated with body weight only in dwarf animals. SRIF cell numbers were equivalent to those in controls, suggesting that the increase reported earlier may have been due to a rebound effect in proliferation rather than some response of SRIF cells to GRF cell reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylmercury developmental neurotoxicity: a comparison of effects in humans and animals.

A qualitative and quantitative comparison of the neuropathological and neurobehavioral effects of early methylmercury (MeHg) exposure is presented. The focus of the qualitative comparison is the examination of how specific end-points (and categories of behavioral functions) compare across species. The focus of the quantitative comparison is the investigation of the relationship between MeHg exposure, target-organ dose and effects in humans and animals. The results of the comparisons are discussed in the context of the adequacy of the proposed EPA neurotoxicity battery to characterize the risk of MeHg to humans. The comparisons reveal several qualitative and quantitative similarities in the neuropathological effects of MeHg on humans and animals at high levels of exposure. Reports of neuropathological effects at lower levels are available for animals only, precluding any comparison. At high levels of exposure, specific neurobehavioral end-points affected across species are also similar. Effects at lower levels of exposure are similar if categories of neurobehavioral functioning are compared. Changes in the EPA test battery consistent with the results of the comparisons are discussed.

The relationship of rat brain weight and pituitary weight to postnatal growth after prenatal exposure to methylazoxymethanol.

Teratogens can affect body weight in various ways, but the association of brain damage with postnatal growth abnormalities suggests a role for neuroendocrine growth-controlling systems. Growth deficiencies follow methylazoxymethanol (MAM) exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, and the pattern of growth of the animals is like that of animals deficient in growth hormone. The present studies were designed to examine the growth, body proportions, brain weight, and pituitary weight of animals treated with 20 mg/kg MAM on the 13th day of gestation, a peak period for production of GRF neurons. Among the offspring, this treatment produced about 25% dwarfs (animals smaller than the smallest control of the same sex). Significantly more females than males were categorized as dwarfs. The weight effect occurred long after birth, as is characteristic of animals and humans with growth hormone deficiency. Analyses of weights over the course of development indicated that prenatal factors, rather than factors operating between birth and weaning, predicted the adult body weight of dwarfs, while both sets of factors were significant in other animals. The growth reduction was symmetrical, as would be expected if the animals were growth hormone deficient, with an 18% reduction in weight reflecting a 6% reduction in bone length. The remaining treated animals were similar to controls in absolute weight, body proportions, and rate of growth. Neither pituitary weight nor brain weight appears to play the key role in determining which animals will exhibit growth deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism.

Abnormalities in anatomy and function of the cranial nerve motor nuclei have been demonstrated in some people with autism and can be modeled in rats by exposure to valproic acid during neural tube closure. Reductions in Purkinje cell number and cerebellar volume, particularly of the posterior lobe, have also been reported in people with autism. Thus, a stereological examination of cerebellar morphology was undertaken in valproate-exposed rats. Compared to controls, rats exposed to a single dose of 600-mg/kg sodium valproate on embryonic day 12.5 had significantly fewer Purkinje cells in the cerebellar vermis and a reduction short of significant in the hemispheres. The diminished cell numbers reflect reductions in tissue volume throughout the cerebellum, rather than cell density, which was unaffected in all regions. Within the vermis, the reduction in volume was significantly greater in the posterior lobe than in the anterior lobe. The results parallel those reported for human cases of autism.

Evidence for multiple loci from a genome scan of autism kindreds.

We performed a genome-wide linkage scan using highly polymorphic microsatellite markers. To minimize genetic heterogeneity, we focused on sibpairs meeting the strict diagnosis of autism. In our primary analyses, we observed a strong linkage signal (P=0.0006, 133.16 cM) on chromosome 7q at a location coincident with other linkage studies. When a more relaxed diagnostic criteria was used, linkage evidence at this location was weaker (P=0.01). The sample was stratified into families with only male affected subjects (MO) and families with at least one female affected subject (FC). The strongest signal unique to the MO group was on chromosome 11 (P=0.0009, 83.82 cM), and for the FC group on chromosome 4 (P=0.002, 111.41 cM). We also divided the sample into regression positive and regression negative families. The regression-positive group showed modest linkage signals on chromosomes 10 (P=0.003, 0 cM) and 14 (P=0.005, 104.2 cM). More significant peaks were seen in the regression negative group on chromosomes 3 (P=0.0002, 140.06 cM) and 4 (P=0.0005, 111.41 cM). Finally, we used language acquisition data as a quantitative trait in our linkage analysis and observed a chromosome 9 signal (149.01 cM) of P=0.00006 and an empirical P-value of 0.0008 at the same location. Our work provides strong conformation for an autism locus on 7q and suggestive evidence for several other chromosomal locations. Diagnostic specificity and detailed analysis of the autism phenotype is critical for identifying autism loci.

Autism and the serotonin transporter: the long and short of it.

Autism is a neurodevelopmental disorder manifesting early in childhood. Some symptoms of autism are alleviated by treatment with selective serotonin reuptake inhibitors, which are known to interact with the serotonin transporter. Moreover, variation in the gene that encodes the transporter (SLC6A4), especially the HTTLPR locus, is known to modulate its expression. It is natural, therefore, to evaluate whether this variation plays a role in liability to autism. We investigated the impact of alleles at HTTLPR and three other loci in SLC6A4 by using a large, independent family-based sample (390 families, 1528 individuals) from the NIH Collaborative Programs of Excellence in Autism (CPEA) network. Allele transmissions to individuals diagnosed with autism were biased only for HTTLPR, both for the narrow diagnosis of autism (P=0.035) and for the broader diagnosis of autism spectrum (P=0.007). The short allele of HTTLPR was significantly overtransmitted. Investigation of haplotype transmissions suggested that, in our data, biased transmission was only due to HTTLPR. With respect to this locus, there are now seven of 12 studies reporting significant transmission bias of HTTLPR alleles, a noteworthy result in itself. However, the studies with significant findings are almost equally divided between overtransmission of short and overtransmission of long alleles. We place our results within this extremely heterogeneous field of studies. Determining the factors influencing the relationship between autism phenotypes and HTTLPR variation, as well as other loci in SLC6A4, could be an important advance in our understanding of this complex disorder.

Chronology of neuron development: animal studies and their clinical implications.

Because different parts of the central nervous system form at different stages of development, there is not one critical period but many critical periods. Some neurons are formed around the time of closure of the neural tube: these include the motor horn cells of the spinal cord and some motor nuclei of the brain stem. Other neurons, most notably the granule cells of the cerebellum, olfactory bulb and hippocampus, are produced in great numbers after birth. This review focuses on the mouse, the species for which the most data on neurogenesis are available, and draws parallels with other species. The clinical significance of the chronology of neuron formation is discussed in the context of recent studies of experimentally-induced congenital brain damage.