GABA withdrawal modifies network activity in cultured hippocampal neurons.

Dissociated hippocampal neurons, grown in culture for 2 to 3 weeks, tended to fire bursts of synaptic currents at fairly regular intervals, representing network activity. A brief exposure of cultured neurons to GABA caused a total suppression of the spontaneous network activity. Following a washout of GABA, the activity was no longer clustered in bursts and instead, the cells fired at a high rate tonic manner. The effect of removing GABA could be seen as long as 1 to 2 days after GABA withdrawal and is expressed as an increase in the number of active cells in a network, as well as in their firing rates. Such striking effects of GABA removal may underlie part of the GABA withdrawal syndrome seen elsewhere.

Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons.

We have previously shown that estradiol causes a twofold rise in dendritic spine density in cultured rat hippocampal neurons, as it does in vivo. More recently, estrogen receptors have been localized to aspiny inhibitory hippocampal interneurons, indicating that their effect on spiny pyramidal neurons may be indirect. We therefore examined the possibility that estradiol affects spine density by regulating inhibition in cultured hippocampal interneurons. Immunocytochemically, estrogen receptors were found to be co-localized with glutamate decarboxylase (GAD)-positive neurons (approximately 21% of total neurons in the culture). Exposure of cultures to estradiol for 1 d caused a marked decrease (up to 80%) in the GAD content of the interneurons, measured both by immunohistochemistry and Western blotting. Also, the number of GAD-positive neurons in the cultures decreased to 12% of the total cell population. Moreover, GABAergic miniature IPSCs were reduced in both size and frequency by estradiol, whereas miniature EPSCs increased in frequency. We then mimicked the proposed effects of estradiol by blocking GABA synthesis with mercaptopropionic acid (MA). Cultures treated with MA expressed a dose-dependent decrease in GABA immunostaining that mimicked that seen with estradiol. MA-treated cultures displayed a significant 50% increase in dendritic spine density over controls, similar to that produced by estradiol. These results indicate that estradiol decreases GABAergic inhibition in the hippocampus, which appears to effectively increase the excitatory drive on pyramidal cells, and thus may provide a mechanism for formation of new dendritic spines.

The effects of raphe grafts on hippocampal electrophysiology in aged rats.

In previous studies we have demonstrated that raphe grafts, implanted into serotonin-depleted rat hippocampus can restore behavioral and physiological functions impaired by serotonin depletion. Since aging is associated with a reduction in serotonergic functions, we explored the possibility that grafting embryonic raphe tissue will ameliorate age-associated reduction of serotonergic functions in the hippocampus. Aged rats were implanted with E14 embryonic neural tissue, containing the raphe, or part of the parietal cerebral cortex. Three months later, the rats were anesthetized, and the responses of the dentate gyrus to perforant-path stimulation were measured. Serotonin-containing neurons were found in the raphe-grafted hippocampi. No differences were found between the two groups in the volume of the graft in the host brain. Raphe-grafted rats were not different from the cortex-grafted rats in reactivity to perforant path stimulation or in the response to a second of a pair of stimuli to the perforant path. They did, however, express a pronounced commissural inhibition, unlike the cortex-grafted rats. These results are similar to those found previously with a pharmacological enhancement of serotonergic neurotransmission. It is suggested that a graft of serotonergic neurons can ameliorate age-associated reduction in serotonergic functions in the hippocampus.

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.

The effects of general and restricted serotonergic lesions on hippocampal electrophysiology and behavior.

Depletion of the forebrain serotonergic system was found in previous studies to induce an increased excitability of the dentate gyrus (DG) granule cells and, when combined with a cholinergic deficiency, to impair spatial learning. We now compared the effects of general forebrain serotonergic lesions induced by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), to those of a more restricted injection of 5,7-DHT into fornix-fimbria and cingulum, to eliminate hippocampal serotonergic innervation. Control and lesioned rats were injected with atropine and tested in the spatial learning water-maze task. Following the behavioral tests, rats were anesthetized and the responsiveness of the DG to perforant path (PP) stimulation was measured. To assess the lesions functionally, responses to application of the serotonin releasing drug fenfluramine (FFA) were measured. Finally, the reduction, in the hippocampus, of serotonergic innervation was evaluated by [3H]imipramine binding. The effects of the lesions on the responsiveness to FFA confirmed that the ICV lesions were functionally more general than the FF lesions. [3H]Imipramine binding indicated that both lesions reduced the serotonergic innervation of the hippocampus significantly. Behaviorally, both lesioned groups were impaired in the water-maze. Electrophysiologically, in both DG excitability was higher than in controls and in both hyperexcitability was associated with an increase in feed-forward inhibition. The results suggest that the serotonergic innervation of the hippocampus proper is involved in cognitive functions associated with the hippocampus.

Changes in cytosolic sodium caused by a toxic glutamate treatment of cultured hippocampal neurons.

Changes in cytosolic Na+ ([Na+]i) caused by a toxic glutamate (GLU) or NMDA treatment of cultured hippocampal neurons were monitored by using SBFI fluorescent probe and imaging microscopy. Both GLU and NMDA (50 or 100 microM in Mg(2+)-free solution, 15 min) induced a marked increase in [Na+]i (from 6-8 to 30-45 mM) which persisted after the termination of a treatment. The competitive NMDA antagonist, APV (100 microM) when applied in the post-NMDA period failed to decrease the elevated [Na+]i. The results obtained strongly suggest that the main reason for an impairment of Na+/Ca2+ exchange in the post-glutamate period (see Febs Letters 1993, 324, 271-273) is a reduction of the transmembrane Na+ gradient caused apparently by inhibition of Na(+)-K+ pump.

Regional specificity of raphe graft-induced recovery of behavioral functions impaired by combined serotonergic/cholinergic lesions.

We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex on the ability of rats to perform a spatial memory water-maze task. Serotonin depletion or partial cholinergic lesion of the hippocampus (by injection of colchicine into the septum) did not affect the ability of rats to perform the task, but the combined treatment did. Double-lesioned rats, with raphe grafts in the hippocampus, but not in the entorhinal cortex, performed similar to control or single-lesioned rats. The results suggest that the functioning of the serotonergic innervation of the hippocampus, and not of its afferents, is crucial for the ability of rats to perform spatial memory tasks, especially when the septohippocampal cholinergic connection is disrupted.

Acidic amino acids evoke a smaller [Ca2+]i rise in GABAergic than non-GABAergic hippocampal neurons.

Changes in intracellular calcium concentration ([Ca2+]i) in response to topical application of the glutamate agonists N-methyl-D-aspartate (NMDA), or amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were measured in cultured rat hippocampal neurons loaded with Fluo-3 and visualized in a confocal laser scanning microscope. These neurons were subsequently stained for the presence of the enzyme marker for gamma-amino butyric acid (GABA), glutamate decarboxylase (GAD). GAD-positive, putative interneurons were less responsive to NMDA and AMPA than GAD-negative neurons. The time course of the rise and decay of [Ca2+]i was similar in the two groups of neurons. Also, there was no clear difference in the shape and size of these two neuron groups indicating that the difference between them is not due to diffusion distances. These data indicate that interneurons are probably more able to handle a calcium load than other neurons, a difference that may underly their resistance to treatments which cause degeneration of other neurons in the hippocampus.

Septal transplants ameliorate spatial deficits and restore cholinergic functions in rats with a damaged septo-hippocampal connection.

Behavioral effects of septal lesion and fornix-fimbria transection were compared in absence and presence of a septal transplant in the hippocampus. The transplant grew in the hippocampus and projected acetylcholinesterase (AChE)-containing fibers throughout the extent of the denervated hippocampus. There were no differences in graft size or AChE reinnervation pattern after septal lesion or fornix transection. An increase in the density of M1 binding sites seen in hippocampal CA3 region after a cholinergic lesion, was restored back to normal after reinnervation of the hippocampus by the graft. Fornix-transected rats were more impaired in water maze acquisition than septal-lesioned rats which were impaired compared to controls. Septal-grafted rats were not different from lesioned rats in the behavioral tasks. However, an injection of physostigmine improved their performance relative to lesioned non-grafted rats. These experiments indicate that grafts can ameliorate behavioral deficits when the efficacy of acetylcholine of graft origin is enhanced.

Aging and brain cholinergic muscarinic receptor subtypes: an autoradiographic study in the rat.

Cholinergic M1 and M2 muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated QNB in the presence of pirenzepine or carbachol. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most thalamic and hypothalamic nuclei. M1 and M2 receptors showed small but significant decreases in cortical regions and in the striatum. The largest effects were seen in M2 receptors of the ventral forebrain cholinergic nuclei where binding was reduced by up to 40%. No similar reductions were seen in the M1 receptor population in these regions. The results suggest that both muscarinic receptor subtypes show an anatomically selective pattern of decrease with age, with the M2 receptor subtype in the basal forebrain nuclei being specially vulnerable to the effects of aging.

Presynaptic cholinergic action in the hippocampus.

The hippocampus is among the regions in the brain richest in M1 cholinergic receptors. Topical application of acetylcholine (ACh) onto hippocampal slices produces a characteristic complex response consisting of a depolarization, an increase in input resistance especially upon depolarization and a blockade of a slow afterhyperpolarization (AHP). The first two of these responses can be recorded also in non-cholinergic septal neurons in an area which contains about 8% of the M1 muscarinic receptors found in the hippocampus. The responses of hippocampal but not septal neurons to ACh involve an increase in the spontaneous synaptic activity and a decrease in evoked responses to afferent stimulation. The dissociated hippocampal culture was used to study these presynaptic effects. The neurons in culture possess muscarinic receptors which develop gradually over a period of several weeks after plating. ACh rarely depolarizes hippocampal neurons in culture. Instead, it causes an increase in spontaneous discharge of small postsynaptic currents (PSC's) and a marked decrease of large, evoked PSC's. In some cultured hippocampal cells ACh reduced ICa without affecting any of several outward K currents studied. It is suggested that ACh reduces evoked activity by reducing Ca currents at presynaptic terminals.

Aging and brain cholinergic muscarinic receptors: an autoradiographic study in the rat.

Cholinergic muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated quinuclidinyl benzilate. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most thalamic and hypothalamic nuclei. Small but significant decreases were found in cortical regions and in the striatum. The largest effects were seen in ventral forebrain cholinergic nuclei, where 40-60% depletions were found in the diagonal band, nucleus basalis magnocellularis, ventral pallidum, and substantia innominata.

A correlation between regional acetylcholinesterase activity in rat brain and performance in a spatial task.

The acquisition and retention of a water maze task was examined in 12 intact, young Wistar rats. Acetylcholinesterase activity in 43 discrete brain regions was then measured in the same rats by quantitative histochemistry. Individual learning and retention indices were found to be significantly correlated with acetylcholinesterase (AChE) levels in specific regions, e.g. cholinergic nuclei; the ventral pallidum and nucleus basalis; and in the dentate gyrus of the hippocampus. High levels of AChE in these regions predicted poor performance in the water maze. Thus cholinergic activity in selected regions of the rat brain might be involved in the performance of spatial memory tasks.

Quantitative histochemistry of brain acetylcholinesterase and learning rate in the aged rat.

Using a newly developed method for quantitative acetylcholinesterase (AChE) histochemistry we find a substantial decrease in AChE content in aged rats compared to young controls in the cholinergic cell body regions (the ventral pallidum and the medial septal nuclei) and, to a smaller extent in the projection areas (the cortex and hippocampus). In the same group of aged rats we find a severe deficit in the acquisition of a water maze task. Quantitative histochemistry is a potent method for detecting localized changes in AChE content in otherwise intact, learning-impaired aged rats.

Cyclic AMP-generating systems in rat hippocampal slices.

Properties of the norepinephrine (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3--4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 sec but maximal stimulation was reached only after 5--10 min of incubation. Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12--15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation. It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.