Psychosis: pathological activation of limbic thalamocortical circuits by psychomimetics and schizophrenia?

Non-competitive NMDA receptor antagonists, such as phencyclidine, ketamine and MK801, produce psychosis in humans. These drugs also produce injury to cingulate-retrosplenial cortex in adult rodents that can be prevented by GABA-receptor agonists and antipsychotics such as haloperidol and clozapine. MK801 injections into anterior thalamus reproduce limbic cortex injury, and GABA-receptor agonist injections into anterior thalamus prevent injury produced by systemic MK801. Inhibition of NMDA receptors on GABAergic thalamic reticular nucleus neurons might activate thalamocortical 'injury' circuits in animals. Pathological activation of thalamocortical circuits might also mediate the psychosis produced by NMDA-receptor antagonists in humans, and might contribute to psychosis in schizophrenia.

Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia-ischemia.

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia-ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90-150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90-150 min of hypoxia-ischemia was associated with intense immunofluorescence in the hemisphere ipsilateral to the carotid occlusion, with a pattern similar to that reported previously for the histological damage seen in this model. This study provides a sensitive method for the evaluation of the level of oxygen depletion in brain tissue after neonatal hypoxia-ischemia at times much earlier than any method demonstrates apoptotic or necrotic cell death Since the level of in vivo formation of macromolecular adducts of EF5 depends on the degree of oxygen depletion in a tissue, intracellular EF5 binding may serve as a useful marker of regional cellular vulnerability and redox state after brain injury resulting from hypoxia-ischemia.

Expression of zinc finger immediate early genes in rat brain after permanent middle cerebral artery occlusion.

The prolonged expression of the leucine zipper fos/jun immediate early genes (IEG) has been correlated with neuronal death after cerebral ischemia. In this study, the expression of six zinc finger IEG was examined using in situ hybridization in adult rats after middle cerebral artery occlusion (MCAO) with the suture model. NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 mRNA were all induced throughout the ipsilateral cortex at 1 hour to 12 hours after MCAO. The cortical induction for most of the genes was greatest in the anterior cingulate and the anterior cerebral artery (ACA) and middle cerebral artery (MCA) transition zone. All of the zinc finger IEG were induced at 1 hour in all regions of hippocampus. NGFI-A and NGFI-B were induced in ipsilateral thalamus. Within areas of infarction, the basal IEG mRNA expression, and expression of the housekeeping gene cyclophilin A mRNA, decreased below control levels by 12 hours after the ischemia. Immediate early gene expression outside areas of infarction returned to control levels in most brain regions by 24 hours except for egr-3, which continued to be induced in the MCA/ ACA transition zone for 24 hours, and NGFI-A, which continued to be expressed in specific regions of the thalamus for 72 hours. The induction of these IEG in the cortex is likely caused by ischemia-induced cortical spreading depression, with the hippocampal and thalamic IEG induction being caused by activation of efferent cortical pathways to these regions. The prominent induction of NGFI-B, NGFI-C, egr-2, and egr-3 in the anterior cingulate cortex, the ACA/MCA transition zone, and medial striatum could reflect the ischemic regions around MCA infarcts. The prolonged NGFI-A expression observed in thalamus in this study, and in CA1 of hippocampus after global ischemia in the gerbil in a previous study, suggests that the prolonged NGFI-A, expression could be the result of or the cause of the delayed cell death. Prolonged NGFI-A expression, like c-fos and c-jun, seems to provide a marker for slowly dying neurons.

Anatomic patterns of Fos immunostaining in rat brain following systemic endotoxin administration.

To identify brain neurons that participate in the acute phase response, rat brains were examined immunocytochemically for Fos protein following the intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS). Two to three hours after the injection of LPS, 150 micrograms/kg body weight, to adult male Long-Evans rats, a consistent anatomic pattern of Fos immunostained cell nuclei is seen. In the brain stem, prominant Fos immunostaining is induced in tyrosine hydroxylase immunoreactive neurons of the caudal ventral-lateral medulla (the A1 cell group), in both tyrosine hydroxylase positive and negative neurons of nu. tractus solitarius, in the parabrachial nu., and in a few neurons of the locus ceruleus. In the hypothalamus, endotoxin induces Fos expression in magnocellular neurons of the paraventricular and supraoptic nuclei and internuclear cell groups. A higher percentage of oxytocin-immunoreactive cells is double labeled for Fos nuclear immunostaining than vasopressin-immunoreactive cells. A minority of somatostatin immunoreactive periventricular hypothalamic neurons are Fos positive. Other hypothalamic nuclei that contain endotoxin-induced Fos nuclear immunostaining include the parvocellular neurons of the paraventricular nu., the dorsomedial and arcuate nuclei, the lateral hypothalamus, the dorsal hypothalamic area (zona incerta), and the median nucleus of the preoptic area. LPS induces numerous Fos-positive neurons in regions known to respond to a variety of stressful stimuli; these regions include the preoptic area, bed nucleus of the stria terminalis, lateral septum, and the central and medial nuclei of the amygdala. Moreover, Fos nuclear immunostaining is seen in neurons of circumventricular organs: the organum vasculosum of the lamina terminalis, the subfornical organ, and the area postrema. The maximum intensity of Fos nuclear immunostaining occurs 2-3 h after endotoxin administration and declines thereafter. It is attenuated by pretreatment with indomethacin, 25 mg/kg body weight Sc, or dexamethasone, 1 mg/kg IP. These observations are consistent with the participation of a variety of brain neuronal systems in the acute phase response and elucidate the functional neuroanatomy of that response at the cellular level.

MK-801 inhibits the induction of immediate early genes in cerebral cortex, thalamus, and hippocampus, but not in substantia nigra following middle cerebral artery occlusion.

Middle cerebral artery (MCA) occlusion in rats induced c-fos and junB mRNA 4h later in all ipsilateral cortex outside the MCA distribution and in many subcortical structures: medial striatum; most of thalamus including medial and lateral geniculate nuclei: substantia nigra; and hippocampus. The N-methyl-D-aspartate (NMDA) antagonist, MK-801 (4 mg/kg, i.p.) inhibited c-fos and junB mRNA induction in the cortex, striatum, thalamus, and hippocampus but not in the substantia nigra. These data show that c-fos and junB mRNA induction in cortex, striatum, thalamus, hippocampus involves the activation of NMDA receptors whereas different receptors must be implicated in the induction in substantia nigra.

Whisker stimulation metabolically activates thalamus following cortical transplantation but not following cortical ablation.

Local cerebral glucose utilization was assessed during whisker stimulation by 2-deoxyglucose autoradiography. Whisker stimulation increased local cerebral glucose utilization in brainstem, thalamus and whisker sensory cortex in normal rats. Whereas whisker stimulation increased glucose metabolism in brainstem, whisker stimulation failed to increase glucose metabolism in thalamus of rats that had whisker sensory cortex ablated 5 h to five weeks previously. The failure of whisker stimulation to activate thalamus after cortical ablations was probably not due to decreased cortical input to thalamus because whisker stimulation activated thalamus after large cortical tetrodotoxin injections. Failure of whisker stimulation to activate thalamus at early times (5 h and one day) after cortical ablations was not due to thalamic neuronal death, since it takes days to weeks for axotomized thalamic neurons to die. The failure of whisker stimulation to activate thalamus at early times after cortical ablations was likely due to the failure of trigeminal brainstem neurons that project to thalamus to activate axotomized thalamic neurons. This might occur because of synaptic retraction, glial stripping or inhibition of trigeminal brainstem synapses onto thalamic neurons. The thalamic neuronal death that occurs over the days and weeks following cortical ablations was associated with thalamic hypometabolism. This is consistent with the idea that the thalamic neurons die because of the absence of a cortically derived trophic factor, since the excitotoxic thalamic cell death that occurs following cortical kainate injections is associated with thalamic hypermetabolism. The glucose metabolism of parts of the host thalamus was higher and the glucose metabolism in surrounding nuclei lower than the normal side of thalamus in rats that sat quietly and had fetal cortex transplants placed into cavities in whisker sensory cortex five to 16 weeks previously. Whisker stimulation in these subjects activated the contralateral host thalamus and fetal cortical transplants. This was accomplished using a double-label 2-deoxyglucose method to assess brain glucose metabolism in the same rat while it was resting and during whisker stimulation. The high glucose metabolism of parts of host thalamus ipsilateral to the fetal cortical transplants is consistent with prolonged survival of some axotomized thalamic neurons. The finding that whisker stimulation activates portions of host thalamus further suggests that the cortical transplants maintained survival of the host thalamic neurons and that synaptic connections between whisker brainstem and thalamic neurons were functional.

c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress.

The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.

Marked neurotrophic effects of diffusible substances released from non-target cerebellar cells on thalamic neurons in culture.

A primary culture of thalamic cells from 6-day-old postnatal rats was co-cultured for 6 days with neocortical or cerebellar cells (neurons and astrocytes) from the same litter using a Transwell mesh system. The survival of thalamic neurons grown on the lower well, which were affected by substances released from cells grown on the upper wells, was remarkably promoted by both neocortical co-cultures (target for thalamic projection neurons) and cerebellar co-cultures (non-target). When the cells were seeded on mesh at lower density, the neurotrophic effects of neocortical co-cultures on thalamic neurons (204% of control) were significantly greater than those of cerebellar co-cultures (138%). When the cells were seeded on mesh at higher density, the effects of cerebellar co-cultures increased dramatically (517% of control), while the neurotrophic effects of neocortical co-cultures did not change. Morphologically, the survival of multipolar-shaped thalamic neurons was remarkably improved, as compared to the survival of monopolar, bipolar, and tripolar-shaped thalamic neurons. Basic fibroblast growth factor slightly promoted thalamic neuronal survival (136%), whereas nerve growth factor had no effect. These results suggest that neocortical and cerebellar cells release diffusible factor(s) that promote the survival of specific subpopulation of thalamic neurons, and that at least one of the non-target cerebellar cell-derived factor(s) might be more potent than those released from target neocortical cells.

Induction of fos-like immunoreactivity in hypothalamic corticotropin-releasing factor neurons after adrenalectomy in the rat.

To identify brain sites responding to the removal of corticosterone feedback by adrenalectomy (ADX), rat brains were processed for fos immunocytochemistry 1, 3, and 7 days after ADX, sham-ADX, or no surgery using a polyclonal antiserum to fos residues 132-154. Compared to SHAM, ADX rats exhibited strong fos-like immunoreactivity (FLI) only in the parvocellular neurons of the paraventricular hypothalamic nuclei (PVN) 1, 3, and 7 days after surgery. Replacement with a corticosterone pellet at the time of adrenalectomy (ADX + B) prevented this increase in PVN FLI in three of four rats at 1 day, all rats at 3 days, and two of seven rats 7 days after surgery; 100 micrograms/ml corticosterone in the drinking water for 2 days before perfusion reversed ADX-induced increases in PVN FLI in 7-day ADX rats. Providing 25 micrograms/ml corticosterone in the drinking water to ADX rats for 5 days after surgery did not prevent expression of PVN FLI, even though this dose has been shown to normalize morning basal ACTH levels in ADX rats. Virtually all parvocellular PVN neurons expressing FLI after ADX costained for CRF. Some parvocellular neurons also expressed both fos and vasopressin. In all rats, many brain regions expressed FLI that was not related to adrenalectomy. We conclude that the changes in neuronal FLI correlate with demonstrated changes in neuroendocrine activity after ADX; however, suppression of ADX-induced FLI may require higher replacement levels of corticosterone than inhibition of ADX-induced ACTH secretion.

Expression of c-fos protein in brain: metabolic mapping at the cellular level.

The proto-oncogene c-fos is expressed in neurons in response to direct stimulation by growth factors and neurotransmitters. In order to determine whether the c-fos protein (Fos) and Fos-related proteins can be induced in response to polysynaptic activation, rat hindlimb motor/sensory cortex was stimulated electrically and Fos expression examined immunohistochemically. Three hours after the onset of stimulation, focal nuclear Fos staining was seen in motor and sensory thalamus, pontine nuclei, globus pallidus, and cerebellum. Moreover, 24-hour water deprivation resulted in Fos expression in paraventricular and supraoptic nuclei. Fos immunohistochemistry therefore provides a cellular method to label polysynaptically activated neurons and thereby map functional pathways.

Common fur and mystacial vibrissae parallel sensory pathways: 14 C 2-deoxyglucose and WGA-HRP studies in the rat.

Stimulation of mystacial vibrissae in rows A,B, and C increased (14C) 2-deoxyglucose (2DG) uptake in spinal trigeminal nucleus pars caudalis (Sp5c) mostly in ventral portions of laminae III-IV with less activation of II and V. Stimulation of common fur above the whiskers mainly activated lamina II, with less activation in deeper layers. The patterns of activation were compatible with an inverted head, onion skin Sp5c somatotopy. Wheatgerm Agglutinin-Horseradish Peroxidase (WGA-HRP) injections into common fur between mystacial vibrissae rows A-B and B-C led to anterograde transganglionic labeling only of Sp5c, mainly of lamina II with less label in layer V, and very sparse label in III and IV. WGA-HRP skin injections appear to primarily label small fibers, which along with larger fibers, were metabolically activated during common fur stimulation. Mystacial vibrissae stimulation increased 2DG uptake in ventral ipsilateral spinal trigeminal nuclei pars interpolaris (Sp5i) and oralis (Sp5o) and principal trigeminal sensory nucleus (Pr5). Common fur stimulation above the whiskers slightly increased 2DG uptake in ventral Sp5i, Sp5o, and possibly Pr5. The most dorsal aspect of the ventroposteromedial (VPM) nucleus of thalamus was activated contralateral to whisker stimulation. Stimulation of the common fur dorsal to the whiskers activated a region of dorsal VPM caudal to the VPM region activated during whisker stimulation. This is consistent with previous data showing that ventral whiskers and portions of the face are represented rostrally in VPM, and more dorsal whiskers and dorsal portions of the face are represented progressively more caudally in VPM. Mystacial vibrissae stimulation activated the contralateral primary sensory SI barrelfield cortex and a separate region in the second somatosensory SII cortex. Common fur stimulation above the whiskers activated a cortical region between the SI and SII whisker activated regions of cortex. It is proposed that this region represented the combined SI and SII common fur regions of somatosensory neocortex. Both whisker and common fur stimulation activated all layers of cortex, with layer IV being most activated followed by II-III, V, and VI. These data indicate that sensory input from the mystacial vibrissae in the adult rat is processed in brainstem, thalamic, and cortical pathways which are predominantly parallel to those which process information from the neighboring common fur sensory receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats: reciprocal connections with host thalamus demonstrated with WGA-HRP.

Fetal frontal cortex was transplanted into lesion cavities formed in host motor/sensory cortex of adult rats. Eight to twenty-eight weeks later wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into host thalamus and the brain was sectioned and reacted using a sensitive TMB procedure. A large amount of fine granular WGA-HRP was detected in most transplants. This could represent anterograde transport demonstrating that injured adult host thalamic neurons sprouted axons into fetal cortical transplants. Conversely, none or very few retrogradely labeled pyramidal neurons were present in the transplants. This indicates that pyramidal neurons in transplants either did not sprout into adult host brain or sprouted such short distances that they did not pick up the WGA-HRP. These results are compatible with the hypothesis that high trophic/growth factor levels in newborn or fetal brain and low levels in adults determine the more extensive connections seen in newborn hosts compared with those in adult transplanted hosts. The data are also consistent with the proposal that adult host brains impair axonal growth. Functionally, the data suggest that although corticofugal effects of fetal cortical transplants in adult host brains are likely to be limited, transplants could exert beneficial trophic effects on adult host thalamic neurons.

Decreases of cortical and thalamic glucose metabolism produced by parietal cortex stimulation in the rat.

Parietal cortex stimulation elicited focal decreases as well as increases of brain glucose metabolism in ipsilateral cortex, ipsilateral thalamus, and contralateral cortex of rats in a pattern resembling 'surround inhibition'. It is proposed that parietal stimulation activated inhibitory circuits which decreased cortical and thalamic glucose metabolism. This decrease of cerebral glucose metabolism is important for interpreting brain glucose metabolic studies particularly when metabolic changes do not correlate with changes of neuronal activity.

Vibrissae tactile stimulation: (14C) 2-deoxyglucose uptake in rat brainstem, thalamus, and cortex.

The right mystacial vibrissae of awake, adult rats were stroked at 4-6 times/second and brain regions which increased (14C) 2-deoxyglucose (2DG) uptake were mapped autoradiographically. The ventral parts of the ipsilateral spinal trigeminal nuclei pars caudalis (Sp5c), pars interpolaris (Sp5i), pars oralis (Sp5o), and the principal trigeminal sensory (Pr5) nuclei were activated. The lateral part of the ipsilateral facial (VII) nucleus (the region which innervates the vibrissae muscles) was also activated possibly via excitatory, trigeminal (Sp5c, Sp5i, Sp5o, Pr5) sensory afferents. A number of regions were activated contralateral to the sensory stimulus. Discrete patches of (14C) 2DG uptake occurred in deep layers of the superior colliculus (SCsgp). Dorsolateral and dorsomedial parts of the ventrobasal nucleus (VB), and posterior, dorsolateral parts of the reticular nucleus (R) of thalamus were activated, along with broad portions of the primary somatosensory cortex (SI) and second somatosensory cortex (SII). Though all layers of SI and SII cortex increased 2DG uptake, VB thalamic afferents to layers IV and Vc-Vla presumably accounted for the greater activation of these cortical layers during repetitive sensory stimulation of the vibrissae (RSSV). Activation of the above structures fits well with known anatomical data. However, the pattern of activation during RSSV was very different from that previously described during vibrissae motor cortex stimulation (VMIS). RSSV and VMIS both produced similar repetitive movements of all the mystacial vibrissae. However, only a few overlapping brain regions were activated during both RSSV and VMIS. These RSSV-VMIS overlap zones included Sp5o; rostral Sp5i; lateral VII; SCsgp; ventrobasal-posteromedial and ventrobasal-ventrolateral zones in thalamus; and a rostral region of SI probably anterior to the Woolsey vibrissae barrelfield in the dysgranular somatosensory (SI) cortex. Since RSSV and VMIS would both be expected to activate vibrissae proprioceptors, we have hypothesized that vibrissae proprioceptive input was processed in part in the RSSV-VMIS overlap zones. Convergence of motor-sensory inputs and other types of processing could have also occurred in these overlap zones.

Cochlear deoxyglucose uptake: relationship to stimulus intensity.

The relationship between stimulus intensity and the uptake of 2-deoxyglucose (2-DG) in the cochlea of the gerbil was studied using an autoradiographic technique. In silence, incorporation of labeled 2-DG into stria vascularis and the spiral ligament was significantly higher than for other inner ear structures. With increasing intensity of noise exposure, 2-DG uptake in the spiral ganglion and VIIIth nerve increased dramatically when compared to the lateral wall structures. In contrast, relative 2-DG uptake in the organ of Corti was much less affected by noise exposure. Only at 105 dB SPL, the highest intensity tested, was a modest but statistically significant increase observed in the sensory epithelium. The small change in relative 2-DG uptake observed in the organ of Corti during acoustic stimulation is consistent with Davis' (1965: Quant. Biol. 30, 181-190) battery model of the cochlear transduction process. Alternatively, a larger change may have occurred, but been restricted to a small portion of the epithelium, such as one or both populations of hair cells.