Hr Postlesion Research Articles

NAD(P)H oxidase contributes to neurotoxicity in an excitotoxic/prooxidant model of Huntington's disease in rats: Protective role of apocynin

Intrastriatal injection of quinolinic acid (QUIN) to rodents reproduces some biochemical, morphological, and behavioral characteristics of Huntington's disease. NAD(P)H oxidase is an enzymatic complex that catalyzes superoxide anion (O(2).(-)) production from O(2) and NADPH. The present study evaluated the role of NAD(P)H oxidase in the striatal damage induced by QUIN (240 nmol/microl) in adult male Wistar rats by means of apocynin (APO; 5 mg/kg i.p.), a specific NAD(P)H oxidase inhibitor. Rats were given APO 30 min before and 1 hr after QUIN injection or only 30 min after QUIN injection. NAD(P)H oxidase activity was measured in striatal homogenates by O2(*)(-) production. QUIN infusion to rats significantly increased striatal NAD(P)H oxidase activity (2 hr postlesion), whereas APO treatments decreased the QUIN-induced enzyme activity (2 hr postlesion), lipid peroxidation (3 hr postlesion), circling behavior (6 days postlesion), and histological damage (7 days postlesion). The addition of NADH to striatal homogenates increased NAD(P)H oxidase activity in striata from QUIN-treated animals but not from sham rats. Interestingly, O2(*)(-) production in QUIN-lesioned striata was unaffected by the addition of substrates for intramitochondrial O2(*)(-) production, xanthine oxidase and nitric oxide synthase, suggesting that NAD(P)H oxidase may be the main source of O2(*)(-) in QUIN-treated rats. Moreover, the administration of MK-801 to rats as a pretreatment resulted in a complete prevention of the QUIN-induced NAD(P)H activation, suggesting that this toxic event is completely dependent on N-methyl-D-aspartate receptor overactivation. Our results also suggest that NAD(P)H oxidase is involved in the pathogenic events linked to excitotoxic/prooxidant conditions.

Distinct pattern of microglial response, cyclooxygenase‐2, and inducible nitric oxide synthase expression in the aged rat brain after excitotoxic damage

Microglial and inflammatory responses to acute damage in aging are still poorly understood, although the aged brain responds differently to injury, showing poor lesion outcome. In this study, excitotoxicity was induced by intrastriatal injection of N-methyl-D-aspartate in adult (3-4 months) and aged (22-24 months) rats. Cryostat brain sections were processed for the analysis of microglial response by lectin histochemistry and cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) expression by immunohistochemistry and confocal analysis. Aged injured animals showed more widespread area of microglial response at 12 hr postlesion (hpl) and greater microglia/macrophage density at 3 days postlesion (dpl). However, aged reactive microglia showed prevalence of ramified morphologies and fewer amoeboid/round forms. Aged injured animals presented a diminished area of COX2 expression, but a significantly larger density of COX2(+) cells, with higher numbers of COX2(+) neurons during the first 24 hpl and COX2(+) microglia/macrophages later. In contrast, the amount of COX2(+) neutrophils was diminished in the aged. iNOS was more rapidly induced in the aged injured striatum, with higher cell density at 12 hpl, when expression was mainly neuronal. From 1 dpl, both the iNOS(+) area and the density of iNOS(+) cells were reduced in the aged, with lower numbers of iNOS(+) neurons, microglia/macrophages, neutrophils, and astrocytes. In conclusion, excitotoxic damage in aging induces a distinct pattern of microglia/macrophage response and expression of inflammatory enzymes, which may account for the changes in lesion outcome in the aged, and highlight the importance of using aged animals for the study of acute age-related insults.

Distinct spatial and temporal activation of caspase pathways in neurons and glial cells after excitotoxic damage to the immature rat brain

Although cleaved caspase-3 is known to be involved in apoptotic cell death mechanisms in neurons, it can also be involved in a nonapoptotic role in astrocytes after postnatal excitotoxic injury. Here we evaluate participation of upstream pathways activating caspase-3 in neurons and glial cells, by studying the intrinsic pathway via caspase-9, the extrinsic pathway via caspase-8, and activation of the p53-dependent pathway. N-methyl-D-aspartate (NMDA) was injected intracortically in 9-day-old postnatal rats, which were sacrificed at several survival times between 4 hr postlesion (pl) and 7 days pl. We analyzed temporal and spatial expression of caspase-8, caspase-9, and p53 and correlation with neuronal and glial markers and caspase-3 activation. Caspase-9 was significantly activated at 10 hpl, strongly correlating with caspase-3. It was present mainly in damaged cortical and hippocampal neurons but was also seen in astrocytes and oligodendrocytes in layer VI and corpus callosum (cc). Caspase-8 showed a diminished correlation with caspase-3. It was present in cortical neurons at 10-72 hpl, showing layer specificity, and also in astroglial and microglial nuclei, mainly in layer VI and cc. p53 Expression increased at 10-72 hpl but did not correlate with caspase-3. p53 Was seen in neurons of the degenerating cortex and in some astrocytes and microglial cells of layer VI and cc. In conclusion, after neonatal excitotoxicity, mainly the mitochondrial intrinsic pathway mediates neuronal caspase-3 and cell death. In astrocytes, caspase-3 is not widely correlated with caspase-8, caspase-9, or p53, except in layer VI-cc astrocytes, where activation of upstream cascades occurs.

Differential Activation of c‐fos Immunoreactivity After Hypophysectomy in Developing and Adult Rats

The aim of the present study is to compare c-fos expression in identified hypothalamic vasopressin (AVP) and oxytocin (OT) neurons in developing (PN7 and PN14) and adult rats following hypophysectomy using dual-labeled immunostaining. Our results showed that hypophysectomy induced c-fos expression in supraoptic (SON) and paraventricular (PVN) nuclei in both the developing and adult rats. Few or no positive cells were observed in the same nuclei in sham-operated animals. Quantitative analysis for c-fos and either of the above named neuropeptides revealed that almost all AVP and OT neurons in the adult and PN14 groups expressed c-fos in response to hypophysectomy. In PN7, hypophysectomy also induced all AVP neurons to express c-fos in SON and PVN. However, few OT neurons in the SON and PVN produced c-fos after hypophysectomy. In addition, the time course of c-fos expression was different in the developing and adult rats after hypophysectomy. The c-fos expression in the developing rats exhibited a more prolonged induction in which staining for c-fos persisted for at least 3 days after hypophysectomy compared with that in the adult in which c-fos immunoreactivity disappeared within 24 hr post-lesion. This study demonstrates that c-fos expression after hypophysectomy is regulated differently during development.

Vimentin-Dependent Spatial Translocation of an Activated MAP Kinase in Injured Nerve

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain.

It is well established that after adult brain damage the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play an important role in the inflammatory processes and oxidative stress, which are considered to be the leading factors contributing to delayed cell death. The contribution of these enzymes to postnatal brain damage, however, is poorly understood. In our study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in the cortex of postnatal day 9 rats. After different survival times ranging from 4 hr to 7 days post-lesion, brain sections were processed for the immunocytochemical demonstration of COX-2 and iNOS and double labeling with neuronal, glial and neutrophil markers. First and maximal de novo induction of iNOS and COX-2 expression was found at 10 hr post-lesion. Expression of both enzymes started to diminish at 24 hr, reaching basal levels at day 3. iNOS-expressing cells were mainly identified as infiltrated neutrophils as well as highly ramified protoplasmic astrocytes closely associated with blood vessels. Moreover, scattered iNOS-positive neurons were found at the lesion borders. In contrast, COX-2 was mainly observed in reactive microglial cells and neuronal cells. COX-2-positive neurons were found within the degenerating area at 10 hr and at the borders of the lesion later on. This study shows that maximal iNOS and COX-2 expression precedes the period of massive neuronal death observed at 24 hr post-lesion, and may therefore contribute to the evolution of the inflammatory response and the neurodegenerative process after an excitotoxic lesion to the postnatal brain.

Deafferentation Causes Apoptosis in Cortical Sensory Neurons in the Adult Rat

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Regulation of α7Nicotinic Acetylcholine Receptors in the Developing Rat Somatosensory Cortex by Thalamocortical Afferents

Distributions of alpha7 nicotinic acetylcholine receptor (nAChR) mRNA and [125]alpha-bungarotoxin (alpha-BTX) binding sites in the developing rat somatosensory cortex were characterized in relation to acetylcholinesterase (AChE) histochemical staining of thalamocortical terminals to investigate the role of this receptor in cortical development. Using quantitative in situ hybridization and receptor autoradiography, elevated levels of mRNA and binding-site expression were first detected at post-natal day 1 (P1) in deep and superficial layers, just beneath the AChE-stained thalamocortical terminals. Onset of expression occurred approximately 1 d after ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and binding-site expression exhibited a disjunctive, barrel-like pattern in layer IV and, more clearly, in layer VI. The mRNA and binding-site expressions peaked at approximately 1 week postnatal and then declined to adult levels. Unilateral electrolytic or cytochemical lesions placed in the thalamic ventrobasal complex at P0 (just as thalamocortical afferents are innervating the cortex) and at P6 (when the somatotopic map is well established) resulted in a marked reduction of alpha7 nAChR mRNA and [125]alpha-BTX binding-site levels in layers IV and VI, indicating their regulation by thalamocortical afferents. With P6 lesions, this reduction was observed as early as 6 hr postlesion. These results suggest that alpha7 nAChRs are localized primarily on cortical cells in rat somatosensory cortex and provide further evidence for thalamocortical influence on cortical ontogeny. These data also suggest a role for cholinergic systems during a critical period of cortical synaptogenesis.

At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats

Removal of the superior colliculus (SC) in neonatal Wistar rats results in a rapid loss of retinal ganglion cells (RGCs). There is an early twofold increase in RGC death 4-8 hr postlesion (PL) followed by a later 10-11-fold increase in pyknosis about 24 hr PL. We have now used neurotrophic factors (BDNF, NT-4/5, NT-3, NGF, LIF), glutamate receptor antagonists (MK-801, DNQX, CNQX), an antioxidant (N-ace-tyl-L-cysteine), and an NOS inhibitor (L-NAME) to determine whether the early and late phases of lesion-induced RGC death involved similar or different mechanisms. Normal and pyknotic nuclei of tectally projecting RGCs were visualized by injecting the left s.c. of 2 d old rats with diamidino yellow (DY). Two days later the injection site was removed. In most rats, right eyes were injected with factors immediately after the s.c. ablation. Rats were perfused either 6 or 24 hr PL. In the latter group a second intravitreal injection of the appropriate factor was sometimes made 12 hr PL. NT- 4/5 and BDNF significantly decreased RGC pyknosis 6 and 24 hr PL, whereas NT-3 was only protective 6 hr PL. LIF slightly reduced RGC death 24 hr PL, but NGF had no influence on RGC survival at either time point. NT-4/5 also reduced the rate of naturally occurring RGC death. MK-801, DNQX, CNQX, N-acetylcysteine, and L-NAME all prevented the early lesion-induced increase in RGC death but had no significant effect on RGC death measured 24 hr PL; none of these factors significantly reduced the rate of naturally occuring RGC death. Cycloheximide, shown previously to reduce RGC pyknosis 24 hr PL, did not prevent RGC death 6 hr PL. The data indicate that there are at least two mechanisms involved in RGC death after neonatal target ablation. The early increase is related to excitotoxic effects mediated by glutamate receptors and involves NOS and the production of free radicals. We found no evidence that RGC death measured 24 hr PL is dependent on these processes, but the later death does require protein synthesis and, most likely, the activation of an endogenous suicide program. NT-4/5 and BDNF protected RGCs from both types of lesion-induced death.

Cortical hypometabolism in injured brain: new correlations with the noradrenergic and serotonergic systems and with behavioral deficits.

Changes with time after injury in behavioral deficits, as determined by the Morris swim test, and the in vivo specific binding of HEAT, a selective alpha 1-adrenoreceptor ligand, were compared with the time-course of development of cortical hypometabolism in rats with focal freezing lesions. In our trauma model, cortical hypometabolism was detectable in the lesioned hemisphere at 4 hr, became maximal (50% of normal) at 3 days and diminished towards normal on days 5 and 10 post-injury. Progressive impairment of acquisition of the Morris water maze task was demonstrated up to day 3 post-lesion with improvement thereafter. On day 3 the latency to reach criterion was 60% longer in lesioned animals than in corresponding sham-operated ones. An increase in the volume of distribution of HEAT, limited to cortical areas of the lesioned hemisphere, was demonstrable at 4 hr post-lesion and reached its maximum on day 3 (200% of normal) with subsequent return toward normal on days 5 and 10. Several types of drugs were shown previously to modify the cortical hypometabolism associated with cerebral injury. The present data indicate that the same drugs also modify the in vivo binding of HEAT and the behavioral deficits induced by brain lesions. Ibuprofen, a non-steroidal anti-inflammatory drug, p-chlorophenylalanine, an inhibitor of serotonin synthesis, ketanserin, a specific 5HT2-receptor antagonist, and prazosin, an alpha 1-adrenergic receptor blocker all normalized the in vivo binding of HEAT in the cortical areas of the lesioned hemisphere. All groups of animals treated with these drugs also showed subtle, but statistically highly significant improvements in latency to locate the platform in the Morris water maze. Taken together these results show good correlation between behavioral deficits, changes in alpha 1-noradrenergic receptor binding and cortical hypometabolism in injured brain. This supports the hypothesis that post-injury cortical hypometabolism is a reflection of cortical functional depression in which both the serotonergic and noradrenergic neurotransmitter systems play a role, compatible with their inhibitory effects in the cortex and their postulated involvement in cortical information processing.

Chronic elevation of secreted amyloid precursor protein in subcortically lesioned rats, and its exacerbation in aged rats

Subcortically lesioned rats were used as an animal model of some of the neurochemical and behavioral deficits of Alzheimer's disease (AD) to investigate the in vivo expression and metabolism of amyloid precursor protein (APP). Previously, the rapid and persistent induction of APP was described in cerebral cortices after disruption of its cholinergic, serotonergic, or noradrenergic afferents. In the present study, this induction was found to lead to the elevated secretion of APP into the cerebrospinal fluid of lesioned animals. Lesions of the forebrain cholinergic system in aged rats caused an even greater increase in the CSF levels of secreted APP. Antibodies to the extracellular domain of APP detected the protein whereas antibodies to the cytoplasmic region did not, indicating that the APP present in CSF was of the soluble form. Immunoprecipitation with an A beta sequence-specific antibody followed by immunoblot analysis indicated that a significant portion of secreted APP was of the species that contains at least the first 28 amino acids of the A beta sequence (APP gamma or APPA beta). By contrast, very low levels of A beta peptide were detected in CSF. The secretion was accompanied by an elevation of cellular C-terminal fragments of the APP in the lesioned cortex. Consistent with our previous results, this increased APP secretion was caused by lesions of subcortical cholinergic and serotonergic systems. The postlesion time course of APP secretion showed an initial reduction of APP (1 hr postlesion) in CSF followed by an eventual twofold elevation 1-6 weeks later. These results indicate that the induction of APP in response to loss of subcortical innervation leads to elevated secretion of a soluble form of cortically derived APP that contains significant portions of the A beta sequence.

Apoptotic cell death induced by optic nerve lesion in the neonatal rat

Cell death can be ascribed to one of two distinct modes of degeneration: apoptosis (programmed or active cell death) or necrosis (passive degeneration). While apoptosis is generally assumed to occur in physiological conditions such as normal development or tissue turnover, necrotic cell degeneration is induced in pathological situations. Here we report that also in a pathological situation, such as after axotomy in the CNS, apoptotic type of cell death comes into play: following intracranial transection of the optic nerve in the neonatal rat in vivo, retinal ganglion cells undergo an active, apoptotic cell death. In fact, the administration of protein synthesis inhibitors (actinomycin D and cycloheximide) prevents the appearance of pyknotic nuclei as well as of fragmented DNA of ganglion cells at 24 hr postlesion. Correspondingly, the number of surviving cells after actinomycin D and cycloheximide treatment is comparable to normal, unlesioned retinas. In addition, cycloheximide decreases the number of pyknotic ganglion cells during spontaneous cell death.

Increased expression of the NG2 chondroitin-sulfate proteoglycan after brain injury

Injury to the adult mammalian CNS results in reactive changes among the glial cells surrounding the site of damage. Recently, an unusual class of glial cells has been identified within the intact adult rat cerebellum on the basis of the expression of the NG2 chondroitin-sulfate proteoglycan (Levine and Card, 1987). To determine whether the cells that express the NG2 proteoglycan show reactive changes after injury, small puncture lesions were made into the cerebelli of adult rats, and changes among astrocytes, microglia and NG2-positive cells were examined using immunohistochemical staining with cell type-specific marker antibodies. Beginning at 24 hr after lesion, NG2-positive cells immediately adjacent to the lesion site bound the anti-NG2 antibodies more heavily than cells within the undamaged areas of the cerebellum. This increase in anti-NG2 immunoreactivity was transient, reaching a maximum at 7 d postlesion and declining slowly thereafter. The increase in anti-NG2 immunoreactivity was accompanied by an increase in the levels of mRNA encoding the NG2 core protein as demonstrated by in situ hybridization. NG2-positive cells adjacent to the lesion site incorporated 3H-thymidine into their nuclei beginning at 24 hr postlesion and increased in number. Concurrent with these changes, microglia became activated and increased in number, monocytes invaded the damaged tissue, and an astrocytic scar formed. These observations demonstrate that the cells that express the NG2 proteoglycan are a reactive cell type that responds to brain injury. The increased expression of the NG2 chondroitin-sulfate proteoglycan may contribute to the failure of damaged CNS axons to regenerate successfully.

The process of reinnervation in the dentate gyrus of adult rats: time course of increases in mRNA for glial fibrillary acidic protein

The present study evaluates the time course of increased expression of the mRNA for glial fibrillary acidic protein (GFAP) within the dentate gyrus and hippocampus after unilateral lesions of the entorhinal cortex. Levels of GFAP mRNA were evaluated by dot blot hybridization of mRNA isolated from the hippocampus and quantitative in situ hybridization. For dot blot hybridization, RNA was isolated from pooled hippocampi obtained from animals killed at 12 hr, 1, 2, 4, 6, 8, 10, 14, and 30 d postlesion. A separate set of animals killed at 2, 4, 6, 8, 10, 12, 14, and 32 d were prepared for in situ hybridization. The dot blot analyses of mRNA isolated from the hippocampus revealed that on the side ipsilateral to the lesion, the levels of GFAP mRNA increased rapidly, reaching a peak at 2 d postlesion. The increases were not evident by 12 hr postlesion, but by 24 hr, levels of GFAP mRNA were 5-fold higher than control, and by 48 hr, the levels were over 6-fold higher than control. The levels of GFAP mRNA decreased after 2 d postlesion. At 4 and 6 d postlesion the levels were about 2-fold higher than control. At later postlesion intervals, mRNA levels were comparable to the control. At 2 d postlesion, the levels of GFAP mRNA were also increased about 2-fold over control levels on the contralateral side. After 2 d, the levels of GFAP mRNA on the contralateral side were comparable to the control. In situ hybridization revealed a complex pattern of changes in the levels of GFAP. At 2 d postlesion, the levels of GFAP mRNA increased dramatically throughout the hippocampus bilaterally. The increases were most pronounced in the denervated portions of the neuropil; however, the levels of GFAP mRNA were also substantially elevated in laminae that do not receive direct projections from the entorhinal cortex. GFAP mRNA levels were also increased in other areas that receive projections from the entorhinal cortex, including the septum, lateral-dorsal thalamus, and entorhinal cortex contralateral to the lesion. In addition, GFAP mRNA levels were increased in regions bordering the ventricles throughout the brain, and over the surface of the tectum. After 2 d postlesion, the increases in the levels of GFAP mRNA were for the most part restricted to areas containing terminal degeneration. The generalized increases throughout the hippocampus were no longer apparent. Areas bordering the ventricles continued to exhibit higher labeling than in control animals, but this effect was not as prominent as at 2 d postlesion.(ABSTRACT TRUNCATED AT 400 WORDS)

Focal hippocampal lesions induce seizures and long-lasting changes in mossy fiber enkephalin and CCK immunoreactivity

Electrolytic lesions of the dentate gyrus hilus have been demonstrated to induce behavioral seizure activity and to result in perturbations in the amount of enkephalin, cholecystokinin, and dynorphin immunoreactivity in the hippocampal mossy fiber system. In the present study, electroencephalographic (EEG) recordings, made from hippocampus contralateral to a hilus lesion in mouse, demonstrate the presence of recurrent hippocampal seizure activity which begins approximately one hour postlesion and continues for several hours thereafter. Behavioral seizures were found to correspond to periods of epileptiform hippocampal EEG. Immunocytochemical analyses of enkephalin-(ENK-I) and cholecystokinin-immunoreactivity (CCK-I) in contralateral hippocampus of animals sacrificed at various postlesion intervals revealed that both ENK-I and CCK-I were depleted from the mossy fibers at 6 and 12 hr postlesion, and that ENK-I rebounded to supranormal levels by 27 hr. In two animals sacrificed 60 days following lesions which induced extreme behavioral seizure activity, ENK-I was still elevated while CCK-I was completely absent from the mossy fiber system. These data suggest that heightened physiological activity, in the form of recurrent limbic seizures, induces long-lasting but quite different alterations in enkephalin and CCK concentration in the hippocampal mossy fiber system.

Effects of CDP-choline on phospholipase A 2 and cholinephosphotransferase activities following a cryogenic brain injury in the rabbit

Within the tissue surrounding the necrotic lesion, following a cryogenic injury of the brain, there is a definite activation of phospholipase A 2 (at 2 and 4 hr post lesion) that accounts, at least in part, for the phospholipid breakdown. There is also an activation of cholinephosphotransferase (at 2 hr post lesion) that may correspond to an early process of phospholipid resynthesis. Oral CDP-choline in this model is able to completely inhibit the activation of phospholipase A 2,but has no detectable effect on cholinephosphotransferase activity. The beneficial effect of CDP-choline might be explained by a prevention of destruction rather than by an enhancement of reconstruction of phospholipids.

Increased gonadotropin levels in goldfish do not result in alterations in circulating thyroid hormone levels

To determine whether increases in gonadotropin levels are capable of altering thyroid function in goldfish, plasma thyroid hormone levels were measured following induced changes in endogenous gonadotropin secretion and injection of carp gonadotropin. Radio-frequency lesions placed in the nucleus preopticus periventricularis or monosodium- l-glutamate-induced lesions of the posterior nucleus lateralis tuberis (NLT) of the hypothalamus were capable of stimulating significant increases in plasma gonadotropin levels, but were without effect on plasma triiodothyronine (T 3) or thyroxine (T 4) at time intervals ranging from 5 hr to 10 days. Likewise, injections of a superactive analog of gonadotropin-releasing hormone resulted in profound increases in gonadotropin levels without associated changes in thyroid hormones. No changes in the circulating levels of T 4 or T 3 were observed in response to injection of purified carp gonadotropins whereas injection of bovine thyrotropin or carp pituitary extracts stimulated significant increases in T 4. Radiofrequency lesions of the pituitary stalk or of the anterior NLT also resulted in significant increases in circulating levels of T 4, but not of T 3, at 10 and 30 hr postlesion. These results demonstrate that direct acute stimulation of circulating thyroid hormone levels is not an intrinsic action of endogenous goldfish gonadotropin and that activation of the reproductive system, leading to ovulation in some cases, is without effect on blood total thyroid hormone levels. Additionally, these results confirm that hypothalamic inhibition of the pituitary-thyroid axis exists in this teleost fish and demonstrate that interruption of this inhibition results in a time-dependent, high-magnitude increase in circulating thyroxine levels.

Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus

The biosynthesis and posttranslational processing of proenkephalin and the level of preproenkephalin mRNA were investigated in the mossy fiber system of the granule cells of the hippocampus in the presence or absence of a unilateral lesion of the hilus, a procedure that produces an episode of recurrent bilateral hippocampal seizures lasting several hours. Both immunocytochemistry and radioimmunoassay (RIA) have demonstrated that the hilus lesion leads to large bilateral increases in enkephalin immunoreactivity in the mossy fiber system. In the present study, RIA data indicate that following an initial decline in immunoreactivity, enkephalin content within the mossy fibers first begins to increase between 18 and 24 hr after lesioning. Using the technique of in vivo radiolabeling and high-performance liquid chromatographic purification of identified radiolabeled peptides, we observed a 14-fold increase in incorporation of radiolabeled methionine into Met5-enkephalin at 24-30 hr postlesion, as compared with control animals, when Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the posttranslational proteolytic processing of proenkephalin, the biosynthesis of 5 additional Met5-enkephalin-containing peptides was also examined. We determined that the posttranslational processing of proenkephalin did not yield exclusively penta-, hepta-, and octapeptides but larger opioid peptides as well in both control and lesioned animals, and that the ratio of the enkephalin peptides was not altered following the lesion. Measurement of preproenkephalin messenger RNA levels in the granule cells by Northern analysis revealed a marked increase following the lesion. Compared with the control animals, preproenkephalin mRNA was 8.5-fold higher in the contralateral dentate gyrus at 12 hr postlesion and 14- to 15-fold higher by 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of sympathectomy on the lateral hypothalamic lesion syndrome.

Sympathetic involvement in the lateral hypothalamic (LH) lesion syndrome was examined. Male rats were surgically or chemically sympathectomized and then given LH lesions. At 24 hr postlesion, lesion-induced hyperglycemia but not hyperthermia was attenuated by splanchnicectomy and celiac ganglionectomy. Hyperthermia but not hyperglycemia was attenuated by adrenal demedullation, adrenalectomy, and neonatal guanethidine treatment. Guanethidine-sympathectomized rats also displayed lower basal temperatures, more perilesion chromatolysis, and severer external symptoms than their controls. No form of sympathectomy affected lesion-induced gastric pathology, plasma gastrin concentrations, or body weight loss. Nor did any sympathectomy influence the recovery of ingestive behavior, daily food intake, the feeding response to 2-deoxy-D-glucose, or body weight maintenance in recovered LH-lesion subjects. These results suggest that sympathetic hyperactivity contributes to some aspects of the acute LH syndrome: Hyperglycemia results from sympathetic outflow to the abdomen, whereas hyperthermia is determined by circulating catecholamines and extraabdominal sympathetic innervation. The results fail to support the hypothesis that chronic increases in sympathetic tone are responsible for the reduced food intake and body weight of the LH-lesion rat.

Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus

The response of the astroglial population of the dentate gyrus molecular layer to removal of that region's primary afferent was investigated using Cajal's gold sublimate method. Deafferentation caused the astrocytes to hypertrophy, an effect which was detectable at 24 hr and maximal at 72–96 hr post-lesion. Following this, the astroglia entered a lengthy period of gradual atrophy. Counts of the astrocytes in the various sublayers of the molecular layer led to the conclusion that these cells migrate into denervated dendritic areas from neighboring, nondeafferented zones.