Seizures induce widespread upregulation of cystatin B, the gene mutated in progressive myoclonus epilepsy, in rat forebrain neurons

Loss of function mutations in the gene encoding the cysteine protease inhibitor, cystatin B (CSTB), are responsible for the primary defect in human progressive myoclonus epilepsy (EPM1). CSTB inhibits the cathepsins B, H, L and S by tight reversible binding, but little is known regarding its localization and physiological function in the brain and the relation between the depletion of the CSTB protein and the clinical symptoms in EPM1. We have analysed the expression of mRNA and protein for CSTB in the adult rat brain using in situ hybridization and immunocytochemistry. In the control brains, the CSTB gene was differentially expressed with the highest levels in the hippocampal formation and reticular thalamic nucleus, and moderate levels in amygdala, thalamus, hypothalamus and cortical areas. Detectable levels of CSTB were found in virtually all forebrain neurons but not in glial cells. Following 40 rapidly recurring seizures evoked by hippocampal kindling stimulations, CSTB mRNA expression showed marked bilateral increases in the dentate granule cell layer, CA1 and CA4 pyramidal layers, amygdala, and piriform and parietal cortices. Maximum levels were detected at 6 or 24 h, and expression had reached control values at 1 week post-seizures. The changes of mRNA expression were accompanied by transient elevations (at 6-24 h) of CSTB protein in the same brain areas. These findings demonstrate that seizure activity leads to rapid and widespread increases of the synthesis of CSTB in forebrain neurons. We propose that the upregulation of CSTB following seizures may counteract apoptosis by binding cysteine proteases.     

Opioid peptide gene expression in rat trigeminal nucleus caudalis neurons: normal distribution and effects of trigeminal deafferentation

Preproenkephalin (preproenkephalin A) and preprodynorphin (preproenkephalin B) are the opioid peptide genes expressed in neurons of the nucleus caudalis of the trigeminal nuclear complex. We have used recently developed techniques for quantitative in situ hybridization to identify the neurons in laminae I and II of the nucleus caudalis that display the mRNA products of each of these genes. The specificity of these hybridization patterns is supported by several biochemical features, and by qualitative and quantitative parallels with previous immunohistochemical results. In animals killed 4 days after unilateral lesions of the trigeminal ganglion, neuronal expression of both preproenkephalin and preprodynorphin is altered in the nucleus caudalis. Decreases in preproenkephalin mRNA are due to a decline in the number of neurons that appear to express this gene. Conversely, preprodynorphin mRNA increases by adding a significant population of expressing neurons. These deafferentation-induced changes in gene expression may provide clues to the role of primary afferent information in modulating the functions of nucleus caudalis neurons containing opioid peptides.     

Amphetamine-induced c-fos mRNA expression is altered in rats with neonatal ventral hippocampal damage

To further characterize the mechanisms underlying enhanced dopamine-related behaviors expressed during adulthood in rats with neonatal excitotoxic ventral hippocampal (VH) damage, we studied the expression of c-fos mRNA in these rats after a single saline or amphetamine (AMPH) (10 mg/kg, i.p.) injection using in situ hybridization. The VH of rat pups was lesioned with ibotenic acid on postnatal day 7 (PD7). At the age of 90 days, rats were challenged with AMPH or saline, and the expression of c-fos mRNA using an oligonucleotide probe was assessed 30, 90, and 180 min later. AMPH significantly increased c-fos mRNA expression in medial prefrontal cortex, piriform cortex, cingulate cortex, septal region, and dorsolateral and ventromedial striatum in control and lesioned rats. However, this response to AMPH was attenuated 30 min after AMPH injection in all of these regions in the lesioned as compared to the sham-operated rats. No significant changes were seen at other time points. These results indicate that the neonatal VH lesion alters time-dependent intracellular signal transduction mechanisms measured by AMPH-induced c-fos mRNA expression in cortical and subcortical brain regions. Changes in c-fos mRNA expression in this putative animal model of schizophrenia may have implications for long-term alterations in cellular pheno type because of altered regulation of certain target genes. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Activation of oxytocin-containing neurons of the paraventricular nucleus (PVN) following generalized seizures

Due to the complex nature of generalized limbic seizures, marked disturbances in physiological homeostasis occur. Accompanying the motor manifestations which characteristically are associated with generalized limbic seizures, alterations in neuroendocrine, behavioral, and autonomic functions may be observed. The paraventricular nucleus (PVN) of the hypothalamus is known to play a significant role in such neuronal responses to stressful stimuli; however, the effect of seizures on hypothalamic neurons is unknown. We have used the immunocytochemical detection of the Fos protein to anatomically identify neurons in the PVN which are activated following generalized limbic seizures. To induce seizures, rats received intraperitoneal injections of kainic acid or were kindled from the entorhinal cortex. We have demonstrated that elicitation of generalized limbic seizures induces a dramatic number of neurons in the PVN to express the Fos protein. Numerous Fos-immunolabeled neurons were identified in both the parvicellular and magnocellular component of the PVN. In the latter, this study clearly reveals a preferential and selective activation of oxytocin-containing neurons, and it extends and supports the hypothesis that oxytocin plays a role in the body's response to specific stress paradigms. Data suggest that an activation of the oxytocin neuronal system may be part of the adaptive mechanism that enables the hypothalamus to modulate and maintain an adequate response to stressors (e.g., generalized seizures) to regain homeostasis. © 1996 Wiley-Liss, Inc.     

Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation

The mRNAs for two forms of glutamic acid decarboxylase (GAD65 and GAD67) were localized in the rat hippocampal formation by nonradioactive in situ hybridization methods with digoxigeninlabeled cRNA probes. Some neurons in all layers of the hippocampus and dentate gyrus were readily labeled for each GAD mRNA, and the patterns of labeling for GAD65 and GAD67 mRNAs were very similar. All major groups of previously described GAD-and GABA-containing neurons appeared to be labeled for each GAD mRNA. Such findings suggest that most GABA neurons in the hippocampal formation contain both GAD mRNAs. When the labeling of neurons in the hippocampal formation and cerebral cortex was compared in the same sections, the intensity of neuronal labeling for GAD67 mRNA was generally similar in the two regions. However, the intensity of labeling for GAD65 mRNA was generally stronger for many neurons in the hippocampal formation than for most neurons in the cerebral cortex. Neurons in the hilus of the dentate gyrus were particularly well labeled for GAD65. The nonradioactive labeling for the GAD mRNAs was confined to the cytoplasm of neuronal cell bodies, and this allowed a clear visualization of the relative number and location of labeled neurons. Several distinct patterns of GAD mRNA-containing neurons were observed among different regions of the hippocampal formation. In the hilus of the dentate gyrus, GAD mRNA-containing neurons were numerous in the regions deep to the granule cell layer as well as in more central parts of the hilus. Within CA3, the densities (quantities) of labeled neurons varied among the regions. In the inner or hilar segment of CA3, the density of labeled neurons was often lower than that in the outer part of CA3 where numerous labeled neurons were distributed throughout all layers. In CA1, GAD mRNA-labeled neurons were distributed in a relatively laminar pattern with the highest density in stratum pyramidale and moderate densities in stratum oriens and at the interface between strata radiatum and lacunosum-moleculare. Lower densities were found within the latter two layers. The prominent localization of the two GAD mRNAs in the hippocampal formation suggests that dual system for GABA synthesis is necessary for normal GABAergic function in this brain region. Most putative GABA neurons contain relatively high levels of GAD67 mRNA as might be expected if this GAD form is responsible for the synthesis of GABA for metabolic and baseline synaptic function. The relatively high levels of GAD65 mRNA in many hippocampal neurons, particularly those of the dentate hilus, may indicate that these neurons have a well-developed reserve system for GAD and GABA synthesis. © 1994 Wiley-Liss, Inc.     

Neuropeptide Y activates urocortin 1 neurons in the nonpreganglionic Edinger-Westphal nucleus

Central regulatory pathways promoting stress adaptation utilize various neurotransmitters/neuropeptides, such as urocortin 1 (Ucn1) and neuropeptide Y (NPY). Ucn1 is abundantly expressed in the nonpreganglionic Edinger-Westphal nucleus (npEW), where it is codistributed with NPY-immunoreactive (ir) terminals. A special role for both neuropeptides has been postulated in stress adaptation. Using double-labeling immunohistochemistry, we observed close appositions between NPY-ir terminals and neurons immunoreactive for Ucn1 in the rat, as well as in the human npEW. Therefore, we hypothesized that NPY might control the activity of Ucn1-positive neurons in the npEW. To test this hypothesis, NPY was injected into the lateral cerebral ventricle of rats, resulting in a strong activation of npEW Ucn1 neurons as revealed by Fos immunohistochemistry. Ucn1 mRNA was also upregulated in the npEW 2 hours after the injection of NPY. In a search for the type of NPY receptor that mediates this NPY-induced recruitment of npEW-Ucn1 cells, we found that the great majority of Ucn1 cells exhibited NPY Y5 receptor immunoreactivity, and only a few of the Ucn1 cells coexpressed the Y1 receptor. We concluded that NPY, via NPY Y5 and to a lesser extent via the Y1 receptors, exerts a stimulatory action on Ucn1 cells in the npEW. Further studies are currently in progress to elucidate the significance of this NPY-Ucn1 interaction in the npEW.     

Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization.

The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10.     

实验性癫癇大鼠海马内GDNFmRNA表达的变化

目的 :为探讨癫活动对大鼠GDNF基因表达的影响。方法 :应用同位素标记的原位杂交研究海人藻酸致后大鼠海马区GDNFmRNA表达时相的变化。结果 :正常大鼠海马未见GDNFmRNA表达 ,而癫大鼠海马神经元在致后 4h以后出现GDNFmRNA表达强烈的上调反应 ,12h达高峰 ,且各区均有表达 ,此后逐渐衰减 ,2 4h恢复正常。结论 :癫后内源性GDNFmRNA表达上调很可能是神经元对抗兴奋性损害的一种保护效应     

Chondroitin sulfate proteoglycans in the developing central nervous system. I. Cellular sites of synthesis of neurocan and phosphacan

We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13–16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5′-leader of neurocan may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing central nervous system. © 1996 Wiley-Liss, Inc.     

Activation of metabotropic glutamate receptor 5 is associated with effect of amphetamine on brain neurons

The role of metabotropic glutamate receptor 5 (mGluR5) was explored in mechanisms underlying the action of amphetamine (AMPH). The activity of mGluR5 was monitored by measuring the level of [3H]inositol monophosphates in brain neurons, in response to stimulation of 2-choloro-5-hydroxyphenylglycine (CHPG), a selective agonist of mGluR5. Treatment with 1 microM of AMPH for 1 h or 7 days increased the CHPG (1 mM, 30 min)-evoked phosphoinositide turnover by 46% or 92% and 26% or 84% in cultured cortical and hippocampal neurons, respectively, from that of CHPG-only treated cells. When AMPH was present during CHPG application post-1 h or 7 day AMPH incubation, the rate of phosphoinositide hydrolysis in cortical neurons became 121% or 142% higher than that treated with CHPG only. The postnatal day (P) 21 (juvenile) and P60 (adult) rats received three intraperitoneal injections of 5 mg/kg of AMPH or saline daily for 6 days. They were challenged on the eighth day with one dosage and sacrificed 3 h later. Reversible 3H-glutamate binding detected increases of 22-89% in the binding levels of cortex and hippocampus of both ages following the AMPH injections. Increases of 13-18% in the levels of mGluR5 mRNA were seen in the juvenile pyramidal neurons of hippocampal CA1-4, granular cells of dentate gyrus, and ventral thalamic nuclei, as shown by in situ hybridization. The AMPH-induced altered activity of mGluR5 is probably associated with changes in the expression of the glutamate receptors, including mGluR5. AMPH may modify the sensitivity of mGluR5 or interact with the receptor itself.     

Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat.

The neurotransmitter gamma-aminobutyric acid (GABA) is present in efferent neurons of the striatum and of the pallidum, one of the main striatal target areas. Dopaminergic nigrostriatal neurons play a critical role in the regulation of GABAergic neurotransmission in the striatum. In the present study, we investigated their role in the regulation of glutamate-decarboxylase (GAD) mRNA expression in two divisions of the pallidum in rats: the globus pallidus and entopeduncular nucleus, equivalent to the external and internal pallidum, respectively, of primates. Dopaminergic neurons were lesioned by unilateral injections of 6-hydroxydopamine (6-OHDA) in the substantia nigra of adult rats. Two or 3 weeks after the lesion, frontal cryostat-cut sections of the brain were processed for in situ hybridization histochemistry with 35S-labeled RNA probes synthesized from cDNAs encoding two distinct isoforms of GAD of respective molecular weight 67,000 (GAD67) and 65,000 (GAD65). The number of labeled cells was determined, and intensity of labeling in individual cells was analyzed by computerized image analysis on emulsion radioautographs. In the globus pallidus, the number of labeled neurons and intensity of labeling per cell were increased on the side ipsilateral to the lesion as compared with control rats in sections hybridized with the GAD67 RNA probe. No changes were detected on the side contralateral to the lesion or in the levels of labeling for GAD65 mRNA. Confirming previous data, the level of labeling for GAD65 mRNA was much higher than for GAD67 mRNA in the entopeduncular nucleus of control rats. In rats with a 6-OHDA lesion, labeling for both GAD67 and GAD65 mRNAs was decreased on the side contralateral, but not ipsilateral, to the lesion, as compared with control rats. The results show that lesions of the nigrostriatal pathway in rats affect the levels of mRNAs encoding two distinct isoforms of GAD in neurons of the globus pallidus and entopeduncular nucleus differently. In addition, results in the entopeduncular nucleus further support a bilateral effect of unilateral dopaminergic lesions.     

Dopamine receptor-mediated regulation of corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus

The present study examined the effects of intraperitoneal administration of selective D1 (SKF 38393) and D2 (quinelorane) dopaminergic receptor agonists on Fos-like immunoreactivity (Fos-LI) and levels of corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (cAMY). Ninety minutes after administration of the D1 agonist SKF 38393, Fos-LI was increased in both the PVN and cAMY. Administration of SCH 39166, a selective D1 antagonist, blocked and attenuated the SKF 38393-induced increase in Fos-LI in the PVN and cAMY, respectively. Similarly, 90 minutes after intraperitoneal injection of the D2 agonist quinelorane, Fos-LI was increased in both PVN and cAMY. Administration of the selective D2 antagonist raclopride prevented the ability of quinelorane to increase Fos-LI in the PVN and cAMY. Both SKF 38393 and quinelorane stimulated the expression of CRH mRNA in the PVN, but failed to alter its expression in the cAMY. Taken together, these results indicate that stimulation of either D1 or D2 dopaminergic receptors activates CRH neurons in the PVN. Stimulation of either D1 or D2 receptors activates neurons in the cAMY, but these changes do not appear to be occurring in CRH neurons.     

Neuropeptide expression in rat paraventricular hypothalamic neurons that project to the spinal cord

The paraventricular hypothalamic nucleus (PVH) exerts many of its regulatory functions through projections to spinal cord neurons that control autonomic and sensory functions. By using in situ hybridization histochemistry in combination with retrograde tract tracing, we analyzed the peptide expression among neurons in the rat PVH that send axons to the spinal cord. Projection neurons were labeled by immunohistochemical detection of retrogradely transported cholera toxin subunit B, and radiolabeled long riboprobes were used to identify neurons containing dynorphin, enkephalin, or oxytocin mRNA. Of the spinally projecting neurons in the PVH, approximately 40% expressed dynorphin mRNA, 40% expressed oxytocin mRNA, and 20% expressed enkephalin mRNA. Taken together with our previous findings on the distribution of vasopressin-expressing neurons in the PVH (Hallbeck and Blomqvist [1999] J. Comp. Neurol. 411:201-211), the results demonstrated that the different PVH subdivisions display distinct peptide expression patterns among the spinal cord-projecting neurons. Thus, the lateral parvocellular subdivision contained large numbers of spinal cord-projecting neurons that express any of the four investigated peptides, whereas the ventral part of the medial parvocellular subdivision displayed a strong preponderance for dynorphin- and vasopressin-expressing cells. The dorsal parvocellular subdivision almost exclusively contained dynorphin- and oxytocin-expressing spinal cord-projecting neurons. This parcellation of the peptide-expressing neurons suggested a functional diversity among the spinal cord-projecting subdivisions of the PVH that provide an anatomic basis for its various and distinct influences on autonomic and sensory processing at the spinal level.     

Coexistence of corticotropin-releasing factor and enkephalin in the paraventricular nucleus of the rat

Corticotropin-releasing factor and enkephalin-containing neurons are found in the paraventricular nucleus of the rat hypothalamus. Their immunocytochemical distribution suggests that a subpopulation of neurons of the paraventricular nucleus might contain both peptides. The present study describes the coexistence of corticotropin-releasing factor and enkephalin in parvicellular neurons of the paraventricular nucleus. Immunocytochemical labeling of peptides was combined with in situ hybridization of mRNA to visualize peptide and mRNA labeling in the same tissue section. This resulted in several observations: (1) neurons labeling for the peptide corticotropin-releasing factor also contain the mRNA to synthesize corticotropin-releasing factor, (2) neurons labeling for the peptide enkephalin also contain the mRNA to synthesize the peptide enkephalin, (3) a subpopulation of corticotropin-releasing factor neurons contains the mRNA to synthesize enkephalin, and (4) a subpopulation of enkephalin neurons contains the mRNA to synthesize corticotropin-releasing factor. A large percentage of enkephalin immunoreactive neurons contains corticotropin-releasing factor mRNA, where as a smaller percentage of corticotropin-releasing factor immunoreactive neurons contains enkephalin mRNA. These double-labeled neurons are present throughout the rostral-caudal extent of the paraventricular nucleus; the majority of these neurons is present in the medial parvicellular paraventricular nucleus.