Dystrophin levels and clinical severity in Becker muscular dystrophy patients.

OBJECTIVE: Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45-47 deletion. METHODS: Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration. RESULTS: 33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%-78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45-47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45-47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients' age. CONCLUSIONS: Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45-47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.

Identification of genomic deletions spanning the PCDH19 gene in two unrelated girls with intellectual disability and seizures.

Recently, missense and truncating mutations in the gene PCDH19 have been reported to cause female-restricted epilepsy with mental retardation (EFMR). EFMR (MIM#300088) is an X-linked disorder characterized by early onset seizures and intellectual disability (ID). Interestingly, unlike typical X-linked mode of inheritance, the phenotype is restricted to females, and males are unaffected carriers. PCDH19 is highly expressed in brain, and the encoded protein belongs to the cadherin superfamily. Here we report two unrelated female patients with deletions spanning PCDH19 identified by copy number variation (CNV) analysis and validated by qPCR. In one, we have identified a 3 Mb interstitial deletion at Xq21.33-q22.1 which spans PCDH19, LOC442459 & TNMD. This patient had her first seizure at 8 months old, and also has ID and aggressive behavior. In another female patient we identified a de novo 603 kb heterozygous deletion in a female patient with fits (since 1 year of age), ID, hyperactivity and aggressive behavior. The deletion spans the entire PCDH19 gene (also TNMD, SRPX2, TSPAN6 and SYTL4). In conclusion, our results suggest that deletions at PCDH19 also cause EFMR.

Autoimmune polyglandular syndrome type 2 shows the same HLA class II pattern as type 1 diabetes.

Autoimmune polyglandular syndrome (APS) type 2 is defined by the manifestation of at least two autoimmune endocrine diseases. Only few data exist on genetic associations of APS type 2. In this controlled study, 98 patients with APS type 2, 96 patients with type 1 diabetes (T1D), and 92 patients with autoimmune thyroid disease, both as a single autoimmune endocrinopathy, were tested for association with alleles of the human leukocyte antigen (HLA) class II loci DRB1, DQA1, and DQB1. Patients with APS type 2 had significantly more often the alleles DRB1*03 (P(c) < 0.0001), DRB1*04 (P(c) < 0.000005), DQA1*03 (P(c) < 0.0001), and DQB1*02 (P(c) < 0.05), when compared with controls. Less frequent in APS were DRB1*15 (P(c) < 0.05), DQA1*01 (P(c) < 0.0005), and DQB1*05 (P(c) < 0.005). With regard to frequency and linkage of these alleles, the susceptible haplotypes DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*0401/04-DQA1*0301-DQB1*0302 were deduced. Protective haplotypes in this study were DRB1*1501-DQA1*0102-DQB1*0602 and DRB1*0101-DQA1*0101-DQB1*0501. Comparing APS patients with vs without AD, no significant differences regarding HLA class II alleles were noted in our collective. Patients with T1D as a singular disease had the same susceptible and protective HLA alleles and haplotypes. The prevalence of DRB1*03 and DRB1*04 in APS patients was not because of the presence of diabetes, as the APS type 2 patients without diabetes had the same allele distribution. In conclusion, these data suggest a common immunogenetic pathomechanism for T1D and APS type 2, which might be different from the immunogenetic pathomechanism of other autoimmune endocrine disease.

Selective activation of central thalamic fiber pathway facilitates behavioral performance in healthy non-human primates.

Central thalamic deep brain stimulation (CT-DBS) is an investigational therapy to treat enduring cognitive dysfunctions in structurally brain injured (SBI) patients. However, the mechanisms of CT-DBS that promote restoration of cognitive functions are unknown, and the heterogeneous etiology and recovery profiles of SBI patients contribute to variable outcomes when using conventional DBS strategies,which may result in off-target effects due to activation of multiple pathways. To disambiguate the effects of stimulation of two adjacent thalamic pathways, we modeled and experimentally compared conventional and novel 'field-shaping' methods of CT-DBS within the central thalamus of healthy non-human primates (NHP) as they performed visuomotor tasks. We show that selective activation of the medial dorsal thalamic tegmental tract (DTTm), but not of the adjacent centromedian-parafascicularis (CM-Pf) pathway, results in robust behavioral facilitation. Our predictive modeling approach in healthy NHPs directly informs ongoing and future clinical investigations of conventional and novel methods of CT-DBS for treating cognitive dysfunctions in SBI patients, for whom no therapy currently exists.

Low dystrophin variability between muscles and stable expression over time in Becker muscular dystrophy using capillary Western immunoassay.

Becker muscular dystrophy (BMD) is the milder allelic variant of Duchenne muscular dystrophy, with higher dystrophin levels. To anticipate on results of interventions targeting dystrophin expression it is important to know the natural variation of dystrophin expression between different muscles and over time. Dystrophin was quantified using capillary Western immunoassay (Wes) in the anterior tibial (TA) muscle of 37 BMD patients. Variability was studied using two samples from the same TA biopsy site in nine patients, assessing nine longitudinal TA biopsies, and eight simultaneously obtained vastus lateralis (VL) muscle biopsies. Measurements were performed in duplicate with two primary antibodies. Baseline dystrophin levels were correlated to longitudinal muscle strength and functional outcomes. Results showed low technical variability and high precision for both antibodies. Dystrophin TA levels ranged from 4.8 to 97.7%, remained stable over a 3-5 year period, and did not correlate with changes in longitudinal muscle function. Dystrophin levels were comparable between TA and VL muscles. Intra-muscle biopsy variability was low (5.2% and 11.4% of the total variability of the two antibodies). These observations are relevant for the design of clinical trials targeting dystrophin production, and may urge the need for other biomarkers or surrogate endpoints.

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

A Mycobacterium leprae-specific human T cell epitope cross-reactive with an HLA-DR2 peptide.

Mycobacterium leprae induces T cell reactivity and protective immunity in the majority of exposed individuals, but the minority that develop leprosy exhibit various types of immunopathology. Thus, the definition of epitopes on M. leprae antigens that are recognized by T cells from different individuals might result in the development of an effective vaccine against leprosy. A sequence from the 65-kD protein of this organism was recognized by two HLA-DR2-restricted, M. leprae-specific helper T cell clones that were derived from a tuberculoid leprosy patient. Synthetic peptides were used to define this epitope as Leu-Gln-Ala-Ala-Pro-Ala-Leu-Asp-Lys-Leu. A similar peptide that was derived from the third hypervariable region of the HLA-DR2 chain, Glu-Gln-Ala-Arg-Ala-Ala-Val-Asp-Thr-Tyr, also activated the same clones. The unexpected cross-reactivity of this M. leprae-specific DR2-restricted T cell epitope with a DR2 peptide may have to be considered in the design of subunit vaccines against leprosy.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Prevalence of obesity in Sweden.

Although the prevalence of obesity in Sweden still is low in an international perspective, the development during the last decades is alarming in adults, adolescents and children alike. The prevalence of obesity [body mass index (BMI) > 30 kg m-2] in adults has doubled during the last two decades and is now approximately 10% in both men and women, according to estimates based on self-reported BMI from repeated random samples of the population. However, prevalence estimates based on measured BMI from the WHO MONICA study indicate that the self-reported data result in underestimates. In military conscripts, the prevalence of obesity (BMI > 30 kg m-2) almost quadrupled to 3.2% from 1971 to 1995, while the overweight fraction (BMI > 25 kg m-2) more than doubled to 16.3%. The development in younger age groups seems to be similar; the prevalence of overweight [International Obesity Task Force (IOTF)/Cole] in children aged 10 years in Gothenburg has doubled to 18% (2.9% obese) during the last decade, and similar figures have been reported in other studies. However, most reports on childhood overweight stem from the larger metropolitan areas, and hence may be underestimates because of the urban-rural influence on obesity-status. Recent data from non-urban areas in the northern part of Sweden estimate the prevalence of overweight (BMI > 20 kg m-2) in 10-year-olds to above 30%. In the most comprehensive study in children, including both rural and urban areas, BMI was measured among all children aged 10 years (n = 5517; 92.7% of the population) in the county of Ostergotland, and the prevalence of overweight (IOTF/Cole) was 22% in both boys and girls, of which 4% and 5% were obese respectively.

Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

SmiNet-2: Description of an internet-based surveillance system for communicable diseases in Sweden.

Electronic systems for communicable diseases surveillance enhance quality by simplifying reporting, improving completeness, and increasing timeliness. In this article we outline the ideas and technologies behind SmiNet-2, a new comprehensive regional/national system for communicable disease surveillance in Sweden. The system allows for reporting from physicians (web form) and laboratories (direct from lab data system) over the internet. Using a unique personal identification number, SmiNet-2 automatically merges clinical and laboratory notifications to case records. Privileged users, at national and county level, work against a common central server containing all notifications and case records. In addition, SmiNet-2 has separate county servers with tools for outbreak investigations, contact tracing and case management. SmiNet-2 was first used in September 2004. Individual counties receive up to 90% of all notifications electronically. In its first year, SmiNet-2 received 54 980 clinical notifications and 32 765 laboratory notifications, which generated 58 891 case records. Since most clinicians in Sweden have easy access to the internet, a general web-based reporting has been feasible, and it is anticipated that within a few years all reporting to SmiNet-2 will be over the internet. In this context, some of the major advantages of SmiNet-2 when compared with other systems are timeliness in the dataflow (up to national level), the full integration of clinical and laboratory notifications, and the capability to handle more than 50 diseases with tailor-made notification forms within one single system.

Recent changes in estuarine benthic and suprabenthic communities resulting from the development of harbour infrastructure.

Using a Before/During/After sampling protocol, the effects of the Le Havre harbour extension, which was started at the end of 2001, on the macrobenthic and suprabenthic communities in the eastern Bay of Seine (English Channel) were examined. As the construction phase has not yet been completed, the results presented here reflect only the data collected before and during the operations (September 2000 and 2002 for benthos sampling and March 2001, September 2001, October 2002 and March 2003 for suprabenthos sampling). Although bio-sedimentary changes did occur at the mouth of the Seine river, an analysis of benthic assemblages reveals that the dredging and construction operations do not seem to have influenced assemblage structure or the spatial distribution of organisms. Comparisons of the suprabenthic assemblages at each sampling date indicate that seasonal dynamics was mainly responsible for determining species distribution. We conclude that, 1 year into the harbour management plan, the observed changes in benthic and suprabenthic assemblage abundance do not exceed the range of spatial variability that exists naturally in the Seine estuary. Despite this compensatory actions designed to protect the aquatic habitats and to preserve a sustainable and healthy ecosystem have been added to the infrastructure development plan.

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury.

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.

Effects of stimulatory and inhibitory thyrotropin receptor antibodies on lipolysis in infant adipocytes.

TSH is a potent lipolytic hormone for isolated human adipocytes from neonatal subjects. Crude immunoglobulin fractions from sera of patients with Graves' disease, containing stimulatory TSH receptor (TSHR) autoantibodies, significantly increased lipolysis in fat cells from infants, whereas immunoglobulin fraction from a patient with inhibitory TSHR autoantibodies (TBab) blocked TSH-induced lipolysis in a dose-dependent manner. Although TBab totally blocked the maximum lipolysis induced by TSH (10(5) mU/L), no effect was seen on isoprenaline-induced lipolysis. The maximum lipolytic response to TSH was similar to that seen with the beta-adrenoceptor agonist isoprenaline, and there was a similar cAMP increase in response to both stimulators. From these results, it is concluded that the TSHR in infant adipocytes is likely to be coupled to the adenylate cyclase system, and the lipolytic effect of TSH can be simulated by stimulatory TSHR autoantibodies or inhibited by TBab.

Nimodipine promotes regeneration and functional recovery after intracranial facial nerve crush.

The calcium flow inhibitor, nimodipine, has been shown to promote motor neuron survival in the facial nucleus after intracranial facial nerve transection. However, it has not been known whether the neuroprotective effects primarily involve survival of nerve cell bodies or outgrowth and/or myelination of nerve fibers. Here, we studied the effects of nimodipine in a different injury model in which the facial nerve was unilaterally crushed intracranially. This lesion caused complete anterograde degeneration and partial retrograde degeneration that were studied with a combination of several stereological methods. Nimodipine did not attenuate the modest lesion-induced neuronal loss (13%) but accelerated the time course of functional recovery and axonal regrowth, inducing increased numbers and sizes of myelinated axons in the facial nerve. It is interesting to note that nimodipine also enlarged the axons and the myelin sheaths in the nonlesioned facial nerve, which points to the possibility of using this substance for new clinical applications to promote axonal growth and remyelination.

Quantitative study of neurofilament-positive fiber length in rat spinal cord lesions using isotropic virtual planes.

Spontaneous reocurrence of neurofilament (NF)-positive fibers has been described after spinal cord lesions in rats. However, previously introduced methods to evaluate the lesion and the regenerative fiber outgrowth suffer from several biases, why a new concept of quantitative, morphological analysis after spinal cord injury is needed. Length quantification of the putatively spontaneously regenerating fibers has been difficult until recently, when two length estimators based on sampling with isotropic virtual planes within thick physical sections were introduced. The applicability of these techniques to estimate the total length of NF-positive fibers was evaluated in photochemically induced ischemic lesions of thoracic spinal cords in young rats 6 weeks postlesion. Fiber length was found to be the most consistent measure with a mean of 3.71 m (coefficient of variation, CV = 0.16) in the 0.90 mm3 (CV = 0.26) large lesions. Whether or not the NF-positive fibers observed inside the lesion represent spontaneously regenerating axons needs to be confirmed in longitudinal, functional, and ultrastructural studies.

Chronic nicotine treatment counteracts nigral cell loss induced by a partial mesodiencephalic hemitransection: an analysis of the total number and mean volume of neurons and glia in substantia nigra of the male rat.

This study combines immunocytochemical and stereological methods for the first time to obtain unbiased estimates of the number of cells in the entire substantia nigra and their respective mean volume. Nicotine, delivered by subcutaneously implanted osmotic pumps (0.125 mg/kg/h, 14 days) to male Sprague-Dawley rats with a partial unilateral mesodiencephalic lesion, caused a significant counteraction of the lesion-induced reduction in total number of nigral tyrosine hydroxylase-like immunoreactive neurons counterstained with Cresyl Violet compared with saline treated control animals. The number of Nissl stained neurons without tyrosine hydroxylase-like immunoreactivity was not affected by the lesion nor by nicotine. The numbers of non-neuronal glial fibrillary acidic protein-like immunoreactive cells counterstained with Cresyl Violet and smaller cells seen after Cresyl Violet staining alone, possibly representing microglia, were increased by the lesion but not affected by nicotine. No nicotine-induced effects were found on the number of nigral cells located contralateral to the lesion. The lesion-induced reduction in the mean volume of the nigral cells showing tyrosine hydroxylase-like immunoreactivity, as determined with the stereological rotator method, was not affected by nicotine. These findings suggest that continuous nicotine infusion exerts protective effects on lesioned nigroneostriatal dopamine systems and that these protective effects are selective for the nigral dopamine neurons not affecting other populations of neurons or non-neuronal cells. This neuroprotective effect might lead to new therapeutic strategies in clinical neurodegenerative disorders such as Parkinson's Disease.

Studies of protective actions of nicotine on neuronal and vascular functions in the brain of rats: comparison between sympathetic noradrenergic and mesostriatal dopaminergic fiber systems, and the effect of a dopamine agonist.

Neuroprotective and possible trophic actions of nicotine were studied in two types of experimental models: (1) one in which the meso-striatal dopamine system was subjected to partial hemitransection, and regional glucose utilization (using 2-[3H]deoxyglucose) and blood flow (using [14C]iodoantipyrine) were measured by computer-assisted quantitative autoradiography based on a double-isotope technique; and (2) another where the sympathetic cranial nervous system supplying the brain vasculature was subjected to decentralization, axotomy, and partial or complete ganglionectomy, and the neuronal survival and fiber regeneration were elucidated by fluorescence histochemistry of noradrenaline, tyrosine hydroxylase, and neuropeptide Y. Continuous nicotine infusion for 4 weeks failed to significantly affect the neuronal response to the surgical interference of the sympathetic noradrenergic system. The same nicotine treatment for 2 weeks significantly improved glucose utilization and blood flow in caudate-putamen on the side in which the meso-striatal dopamine system had been transected, thus eliminating the 16% side-to-side asymmetry in the metabolism caused by the axotomy. The dopamine agonist, EMD 23,448, was without significant effect on this asymmetry. The hemitransection produced marked reduction in metabolism and flow also in the ventro-lateral thalamus. In substantia nigra, glucose utilization was markedly elevated--perhaps as a consequence of a regenerative increase in protein synthesis--opposite to a considerable reduction in nigral blood flow. Little or no effect of the hemitransection was seen in hippocampus or nucleus accumbens. In neither of these four regions did nicotine (or EMD 23,448) have any overt influence on glucose metabolism or blood flow. It is concluded that nicotine, mainly through its protective action on the meso-striatal dopaminergic system, is able to improve striatal glucose utilization and associated blood flow, probably reflecting a tendency to amelioration of neurotransmission function of surviving terminals belonging to the nigro-striatal dopamine neurons.

Protective effects of chronic nicotine treatment on lesioned nigrostriatal dopamine neurons in the male rat.

The present results demonstrate that chronic nicotine treatment can in part protect against mechanically-induced and neurotoxin-induced degeneration of nigrostriatal DA neurons. These results indicate that in sufficient doses chronic treatment with nicotine may be considered in the pharmacological treatment of Parkinson's disease. It remains to be demonstrated whether these protective actions can be extended to include also other injured neurons such as the cholinergic neurons, known to be severely affected in Alzheimer's disease.

Medianosomes as integrative units in the external layer of the median eminence. Studies on grf/catecholamine and somatostatin/catecholamine interactions in the hypothalamus of the male rat.

Somatostatin/catecholamine as well as growth hormone releasing factor/catecholamine interactions have been characterized in the hypothalamus and the preoptic area using morphometrical and quantitative histofluorimetrical analyses. The combined results of the present and previous studies have led us to put forward the medianosome concept. The medianosome is defined as an integrative unit, which consists of well defined aggregates of transmitter identified nerve terminals interacting with one another in the external layer of the median eminence. Our present data indicate the existence of putative medianosomes consisting predominantly of growth hormone releasing factor nerve terminals costoring dopamine as well as of somatostatin and dopamine nerve terminals, which interact locally to control growth hormone secretion. A complementary control of growth hormone secretion may be exerted by noradrenaline mechanisms in the subependymal layer, in the ventral zone and/or in the suprachiasmatic preoptic nucleus. However, further analyses in view of the differential effects seen with the present doses of rat hypothalamic and human pancreatic growth hormone releasing factor have to be done. The results also indicate the possible existence of growth hormone releasing factor receptors in the median eminence which may participate in the feedback control of the growth hormone releasing factor immunoreactive neurons in the ventral zone of the hypothalamus.