Arg3.1/Arc mRNA induction by Ca2+ and cAMP requires protein kinase A and mitogen-activated protein kinase/extracellular regulated kinase activation.

Long-term potentiation (LTP) is a cellular model for persistent synaptic plasticity in the mammalian brain. Like several forms of memory, long-lasting LTP requires cAMP-mediated activation of protein kinase A (PKA) and is dependent on gene transcription. Consequently, activity-dependent genes such as c-fos that contain cAMP response elements (CREs) in their 5' regulatory region have been studied intensely. More recently, arg3.1/arc became of interest, because after synaptic stimulation, arg3.1/arc mRNA is rapidly induced and distributed to dendritic processes and may be locally translated there to facilitate synapse-specific modifications. However, to date nothing is known about the signaling mechanisms involved in the induction of this gene. Here we report that arg3.1/arc is robustly induced with LTP stimulation even at intensities that are not sufficient to activate c-fos expression. Unlike c-fos, the 5' regulatory region of arg3.1/arc does not contain a CRE consensus sequence and arg3.1/arc is unresponsive to cAMP in NIH3T3 and Neuro2a cells. However, in PC12 cells and primary cultures of hippocampal neurons, arg3.1/arc can be induced by cAMP and calcium. This induction requires the activity of PKA and mitogen-activated protein kinase, suggesting a neuron-specific pathway for the activation of arg3.1/arc expression.

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms.

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons.

We monitored developmental alterations in the morphology of dendritic spines in primary cultures of hippocampal neurons using confocal laser scanning microscopy (CLSM) and the fluorescent marker Dil. Dissociated rat hippocampal neurons were plated on polylysine-coated glass cover slips and grown in culture for 1-4 weeks. Fixed cultures were stained with Dil and visualized with the CLSM. Spine density, spine length, and diameters of spine heads and necks were measured. Some cultures were immunostained for synaptophysin and others prepared for EM analysis. In the 1-3 week cultures, 92-95% of the neurons contained spiny dendrites. Two subpopulations of spine morphologies were distinguished. At 1 week in culture, "headless" spines constituted 50% of the spine population and were equal in length to the spines with heads. At 2, 3, and 4 weeks in culture headless spines constituted a progressively smaller fraction of the population and were, on average, shorter than spines with heads. Spines with heads had narrower necks than headless spines. At 3 weeks in culture, spines were associated with synaptophysin-immunoreactive labeling, resembling synaptic terminals. At 4 weeks in culture, only 70% of the Dil-filled cells had spiny dendrites, and the density of spines decreased. Ultrastructurally, the majority of dendritic spine-like structures at 1 week resembled long filopodia without synaptic contacts. The majority of axospinous synapses were on short "stubby" spines. At 3 weeks in culture, the spines were characteristic of those seen in vivo. They contained no microtubules or polyribosomes, were filled with a characteristic, filamentous material, and formed asymmetric synapses. These studies provide the basis for further analysis of the rules governing the formation, development, and plasticity of dendritic spines under controlled, in vitro conditions.

Ultrastructural plasticity of the dentate gyrus granule cells following recurrent limbic seizures: I. Increase in somatic spines.

Various paradigms have been used to assess the capacity of the adult brain to undergo activity-dependent morphological plasticity. In this report we have employed recurrent limbic seizures as a means of studying the effects of this form of enhanced neuronal activity on cellular morphology and, in particular, on the incidence of somatic spines on the dentate gyrus granule cells. Seizure activity was induced by the placement of focal, unilateral electrolytic lesions in the dentate gyrus hilus of adult rats. At various intervals postlesion, rats with behaviorally verified seizures were sacrificed, and the hippocampi contralateral to the lesions were removed and prepared for electron microscopy. Quantitative analysis showed that as early as 5 hours postlesion there was a dramatic increase in the density and morphological complexity of spines on the perikarya of the granule cells in rats that received seizure-producing hilus lesions when compared to granule cells from control rats. Many of the somatic spines received asymmetric synapses. The increase in somatic spines was dependent on seizure activity and persisted for at least 1 month following a single recurrent seizure episode. CA1 pyramidal neurons, which exhibit changes in gene expression in response to hilus lesion-induced seizures but do not normally possess somatic spines, did not exhibit an activity-dependent elaboration of somatic spines. Thus, the seizure-induced elaboration of somatic spines represents an amplification of an existing feature of the granule cells and not an effect occurring throughout hippocampus. These data provide evidence for very rapid and long-lasting structural plasticity in response to brief episodes of seizure activity in the adult brain.

Ultrastructural plasticity of the dentate gyrus granule cells following recurrent limbic seizures: II. Alterations in somatic synapses.

Hilus lesion-induced recurrent limbic seizures cause a dramatic increase in the numbers of somatic spines on dentate gyrus granule cells in the adult rat. Somatic spines are maximally increased 3 h after the initiation of seizures at which time many of these spines form synapses. The present quantitative electron microscopic study assessed the numbers and types of synapses present on the granule cell perikarya and somatic spines of control and experimental seizure rats with the goal of determining if newly elaborated somatic spines arise at the site of pre-existing synapses or are associated with new innervation. Experimental rats were sacrificed 5 h after hilar lesion placement (or 3 h after seizure onset). In both control and hilus-lesioned (HL) rats, 15-20% of the somatic spines could be seen to form synaptic contacts within a single plane of section; these synapses were almost exclusively of the asymmetric type. With the increased incidence of spines in experimental-seizure rats, there was a 6.25-fold greater number of spine synapses in HL versus control rats. There was, in addition, a 60% decrease in the number of asymmetric synapses occurring directly on the granule cell perikarya but no change in the total (spine plus somatic) number of asymmetric synapses. Although few asymmetric synapses were associated with spines in control tissue, 60-70% of asymmetric synapses were associated with spines in experimental-seizure tissue. In addition, in hilus lesion rats symmetric somatic synapses were increased by 20% on cells in deep stratum granulosum resulting in a dissolution of the superficial-to-deep innervation gradient present in the untreated rat. These findings support the conclusion that spines induced by seizure activity form at the site of pre-existing asymmetric synapses on the granule cells and demonstrate that brief seizure episodes can rapidly induce marked changes in innervation patterns in the adult brain.

Seizures and the regulation of neurotrophic factor and neuropeptide gene expression in brain.

Seizure-induced plasticity, in the form of either changes in cellular morphology or changes in neurochemistry, could have a profound impact upon regional excitability in brain. There is now ample evidence that in genetically 'normal' animals, seizure activity stimulates alterations in neuronal gene expression which could lead to changes in levels of excitability and, hence, to changes in the susceptibility for further seizures. Here we describe the influence of limbic seizures upon the expression of nerve growth factor (NGF), 2 related neurotrophic factors, brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), and several neuropeptides (enkephalin, dynorphin, and neuropeptide Y) in the rat forebrain. Using 35S-labeled riboprobes and in situ hybridization methods, the effects of recurrent limbic seizures and of individual hippocampal paroxysmal discharges have been evaluated. Recurrent seizures are found to increase levels of mRNAs for NGF and BDNF and to decrease levels of mRNA for NT3 within select hippocampal neurons. Temporally distinct increases in the expression of mRNAs for NGF and BDNF are also observed across broad fields of neocortex, paleocortex (entorhinal, piriform, and cingulate cortices), and the amygdala. As little as one 20-sec paroxysmal discharge is sufficient to stimulate large changes in neurotrophic factor mRNA content of hippocampal neurons. The time courses and cellular specificities of these alterations in neurotrophic factor expression are discussed and contrasted with seizure-induced changes in neuropeptide expression. Mechanisms by which seizure-induced increases in hippocampal neuropeptide and neurotrophic factor synthesis could lead to both short- and long-term changes in regional excitability, and thereby could contribute to susceptibility for further seizure activity, are considered.

Redistribution of lipofuscin in aged neurons induced by colchicine.

The effect of a single, 40 micrograms, intracerebroventricular injection of colchicine on the distribution of neuronal lysosomes and lipofuscin granules in aged mice was studied. At the light microscope level we observed that colchicine induced a redistribution of dipeptidyl aminopeptidase II (Dpp II), a lysosomal and lipofuscin granule marker enzyme, from the cell bodies of neurons to the dendrites; cell bodies became depleted of Dpp II while dendrites became enriched with this enzyme. Quantitation of this phenomenon at the electron microscope level demonstrated that colchicine induced a rapid and significant decrease in the density of lysosomes and lipofuscin granules from the somata of neurons whereas in dendrites we observed a significant increase in the density of these organelles.

Neuronal localization of pseudocholinesterase in the rat cerebellum: sagittal bands of Purkinje cells in the nodulus and uvula.

The histochemical distribution of pseudocholinesterase was studied in the rat cerebellum using Koelle's copper-thiocholine method. Throughout the cerebellum, pseudocholinesterase is uniformly localized in the endothelial cells of blood vessels and in the cell bodies and processes of the Bergmann glia. In addition, we demonstrate that exclusively in the ventral uvula and in the nodulus (lobules IXc and X of Larsell) pseudocholinesterase is localized in a small subpopulation of Purkinje cells. The cell bodies and dendrites of these labeled Purkinje cells form at least 4 distinct parallel bands extending along the sagittal plane of each of the lobules. Two broad bands on either side of the midline, approximately 800-900 microns wide and composed of 15-20 Purkinje cells have been designated as A bands. Two narrower bands, approximately 160-300 microns wide and composed of 5-7 Purkinje cells, on the lateral aspects of the lobules have been designated as B bands. The unique distribution of pseudocholinesterase in a small and anatomically restricted population of neurons suggests that in the cerebellum this enzyme may play a role in the metabolism of neuroactive substances.

Dendritic transport. I. Colchicine stimulates the transport of lysosomal enzymes from cell bodies to dendrites.

Injection of colchicine into the lateral cerebral ventricle of the rat was found to induce a paradoxical translocation of two lysosomal enzymes, dipeptidyl peptidase II (Dpp II) and acid phosphatase, from the soma of neurons to the dendrites. Following a single injection of colchicine, neuronal somata, which normally contain the bulk of these lysosomal enzymatic activities, become depleted of these enzymes, whereas dendrites become abnormally enriched. All neurons which contained these enzymes, except those of the mesencephalic nucleus of the trigeminal nerve, displayed this phenomenon. Lysosomal enzyme translocation into dendrites was observed in the mitral cell layer within 1 hr after a colchicine injection and could be induced in most neuronal populations by injections of colchicine as low as 25 micrograms. Five days after a 100-micrograms colchicine injection, a normal pattern of enzyme distribution was observed, indicating that the effect of colchicine was reversible. Enzyme translocation was not accompanied by gross changes in cell morphology, nor did it result in the specific loss of neuronal cell bodies which contained these enzymes. The results indicate that colchicine, under conditions known to inhibit axoplasmic transport, stimulates the transport of lysosomal enzymes from the cell body to the dendrites.

In vivo increase in hypothalamic cyclic AMP following 5-hydroxytryptophan administration in rats.

The administration of 5-hydroxytryptophan (5-HTP, 100 mg/kg, i.p.) consistently increased hypothalamic cyclic AMP levels in rats treated 10 days earlier with the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), to produce 5-HT receptor supersensitivity. However 5-HTP (100 mg/kg), failed to cause an increase in hypothalamic cyclic AMP in rats not pretreated with 5,7-DHT. The 5-HTP-induced increase in cyclic AMP was blocked by the decarboxylase inhibitor, benserazide (RO 4-4602, 800 mg/kg) and by the 5-HT antagonist metergoline (5 mg/kg). Other treatments that caused a significant elevation of hypothalamic cyclic AMP included: (a) L-Tryptophan plus the monoamine oxidase inhibitor, tranylcypromine, and (b) the serotonin agonist, 1-(m-trifluromethylphenyl)-1-piperazine. The 5-HT antagonist, methysergide, blocked the serotonin receptor mediated behavioral syndrome, but failed to prevent the increase in hypothalamic cyclic AMP. Moreover, the 5-HT agonist, 5-methoxy-N, N-dimethyltryptamine, (5-Me-DMT), induced a strong behavioral syndrome but failed to significantly increase hypothalamic cyclic AMP. These findings suggest that activation of 5-HT receptors somewhere in the brain causes an increase in hypothalamic cyclic AMP, but further studies will be needed to determine whether this is a direct result of activation of the 5-HT receptors in the hypothalamus.

Effect of midbrain and pontine tegmental lesions on the maximal electroshock seizure pattern in rats.

A bilaterally induced mechanical lesion of the midbrain was highly effective in abolishing the hindlimb extensor (HLE) component of the maximal electroshock seizures (MES) in rats. Although these lesions produced damage to a variety of midbrain structures, correlations between different lesion placements and effects in the MES test provided evidence that damage to superior cerebellar peduncle (PCS) and/or reticular formation (RF) was responsible for inhibition of hindlimb extension. Moreover, discretely placed electrolytic lesions disrupting either the PCS or the RF were found to abolish the hindlimb extensor component of the MES test. These findings are consistent with the work of other investigators showing that total cerebellectomy abolishes the HLE component of MES and suggest that activity in the cerebellum and the midbrain reticular formation plays a major role in regulating the tonic phase of electroshock induced seizures.

Micromolar calcium stimulates proteolysis and glutamate binding in rat brain synaptic membranes.

Incubation of cortical synaptic membranes with low concentrations of calcium resulted in a decrease in the amount of a high-molecular-weight doublet protein and an increase in the sodium-independent binding of glutamate. Both effects were blocked by the thiol protease inhibitor leupeptin. These results suggest that calcium-induced proteolysis of membrane components regulates the number of glutamate receptors in neuronal membranes.

Failure to produce blood pressure changes following pharmacological or surgical depletion of brain serotonin in the spontaneously hypertensive rat.

Spontaneously hypertensive (SH) and normotensive (Wistar-Kyoto, WKY) rats were examined for blood pressure changes following depletion of CNS serotonin (5-HT) by 3 separate techniques: (1) p-chlorophenylalanine, (2) 5,7-dihydroxytryptamine, and (3) a lesion of the dorsal and median raphe nuclei. All of these procedures failed to alter blood pressure in either hypertensive or normotensive rats, despite marked reductions (75-85%) in forebrain 5-HT. Moreover, treatment of 10 day-old hypertensive rat pups with intracisternal injections of 5,7-DHT (10 microgram) failed to alter the development of hypertension despite a 75-80% decrease in spinal cord 5-HT. These findings, which show that 5-HT depletion does not alter blood pressure in the SH or the WKY rat, do not lend support to the idea that 5-HT is involved in the regulation of blood pressure or in the development and maintenance of hypertension in the SH rat.

Metabolism of histones in avian erythroid cells.

The synthesis and enzymatic modifications of histones by phosphorylation, acetylation, and methylation during erythroid cell maturation have been studied. All newly synthesized histones, H1, H5, H2a, h2b, h3, and H4 undergo phosphorylation; histones H2a, H2b, H3, and H4, are acetylated and histones H3 and H4 are methylated. This type of histone metabolism is common to all dividing cells and therefore may be related to the assembly of histones into chromatin subunits. In the nondividing reticulocytes, the synthesis of histone H5 continues, while all the other histones show negligible incorporation of [3H]amino acids. Furthermore, the reticulocytes show a unique pattern of enzymatic modification: phosphorylation of histone H2b, acetylation of histones H2a, H2b, H3, and H4, and methylation of histones H3 and H4. These "differentiation-linked" modifications are not dependent on histone synthesis, nor related to RNA synthesis, but may be related to the reorganization of chromatin in preparation for genomic inactivation.