Resistance to hypoxia in the rat hippocampal slice

Unilateral microinjection of GABA agonists into the pedunculopontine nucleus (PPN) of the rat resulted in contraversive postural asymmetry and circling behavior; GABA antagonists caused ipsiversive asymmetry and circling when applied to the PPN. A hemitransection was placed immediately caudal to substantia nigra (SN) and rostral to PPN in order to interrupt all connections between the PPN and ipsilateral forebrain nuclei. After hemitransection, microinjection of GABAergic drugs into the PPN on the hemitransected side produced postural asymmetry and circling identical to that observed in intact rats. The hemitransection resulted in a loss of glutamic acid decarboxylase activity in PPN (25%) not substantially greater than that observed in animals with unilateral destruction of SN, indicating that a major proportion of GABA terminals in PPN are derived from hindbrain sources. It appears that forebrain (that is, nigrotegmental) GABAergic projections are not essential for the GABA-mediated asymmetry elicited from PPN.     

Action and location of neuropeptide tyrosine (Y) on hippocampal neurons of the rat in slice preparations

The action of bath applied NPY (1-1,000 nM) was investigated on hippocampal slices of the rat with extra- and intracellular recording. Neuropeptide Y (NPY) at 10-1,000 nM caused a concentration-dependent, long-lasting reduction of excitatory postsynaptic potentials (EPSPs) in the hippocampal subfield CA1 and the area dentata, and an even stronger reduction of population spikes. Paired pulse experiments with low intensity, stimulation-evoked PSPs showed a marked increase in facilitation in the presence of NPY, indicating a presynaptic action. Spontaneous burst firing of CA1 pyramidal cells in low calcium, high magnesium medium was reduced, indicating a partially postsynaptic inhibitory action of NPY on their dendrites. Intracellular recording from CA1 somata during NPY administration revealed a reduction of the amplitudes of excitatory-inhibitory postsynaptic potential (EPSP-IPSP) sequences in the absence of changes in membrane potential and conductance. Accommodation of firing during long depolarizing pulses and afterhyperpolarizations were unchanged. The innervation pattern of NPY immunoreactive fibers in the same regions was studied in slices adjacent to the ones used for electrophysiology by using antisera against NPY and light and electron microscopy. There is a dense innervation of CA1 by NPY-immunoreactive axons and terminals, particularly in the stratum moleculare. NPY-immunoreactive neurons are present in the stratum oriens and pyramidale. The NPY labeled axons of the stratum moleculare participate in numerous synaptic contacts with the smaller dendritic elements in this layer, many of which belong to pyramidal neurons. These observations provide evidence for a dendritic NPY-immunoreactive innervation of CA1 neurons, which is in keeping with the electrophysiological effects of NPY on pyramidal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters enhance transmitter release in rat hippocampal slices

Phorbol esters enhance synaptic transmission in the rat hippocampal slice preparation most likely by acting at a presynaptic locus. To more directly examine the actions of phorbol esters on neurotransmitter release we have measured their effects on the occurrence of spontaneous postsynaptic potentials as well as on the potassium stimulated release of endogenous glutamate. Both measures of transmitter release were increased by phorbol esters suggesting a functional or regulatory role for protein kinase C in controlling the release of neurotransmitter in the mammalian CNS.     

Selective vulnerability of hippocampal CA3 neurons to hypoxia after mild concussion in the rat

Immunohistochemical staining for microtubule-associated protein 2 (MAP2) and synaptophysin was used to investigate the effect of hypoxia on hippocampal neurons after mild concussion in the rat. Male Sprague-Dawley rats were divided into four groups: Group 1 (n = 3) was subjected to a mild impact-acceleration closed head injury group 2 (n = 3) was subjected to 30 min of moderate hypoxia, group 3 (n = 5) was subjected to head trauma followed by 30 min of moderate hypoxiaand group 4 (n = 3) comprised sham-operated controls. All rats were fixed by transcardial perfusion 24 h after insult. No damage was observed in CA1 or CA2 neurons in any of the rats. However, rats in group 3 showed selective damage of hippocampal CA3 neurons manifested by a pycnosis and a marked decrease in MAP2 immunoreactivity. Presynaptic terminals visualized by synaptophysin immunostaining showed no differences among groups. The loss of immunoreactivity for the post-synaptic somal and dendritic protein marker MAP2 from the CA3 subfield 24 h after combined insults indicates an increased vulnerability of pyramidal cells in this brain area. [Neurol Res 1995; 17: 455-460]     

Pre- and postsynaptic effects of baclofen in the rat hippocampal slice

CA1 pyramidal cells responded to baclofen with a hyperpolarization. This response was found in the apical and basal dendrites and, like the hyperpolarizing response of the dendrites to GABA, appeared to be Ca2+-dependent since it was blocked or reduced by intracellular injection of EGTA or extracellular application of cadmium. Baclofen also reduced the excitatory and inhibitory postsynaptic potentials produced by stimulation of the Schaffer collaterals. The pre- and postsynaptic effects on the synaptic waveform could be distinguished.     

Calcium dependance of synaptic transmission in the hippocampal slice

'Population' afferent spike and 'population' EPSP were recorded with extracellular microelectrodes in slices of hippocampal tissue maintained in vitro. Calcium concentration was changed in the bathing solution, and calcium activity ([Ca2+]0) was measured in interstitial fluid of the slice with ion-selective microelectrodes. Synaptic transfer was a non-linear continuous function of [Ca2+]0. Deviation of [Ca2+]0 by 0.1 mM from the 1.2 mM control level caused a change of approximately 15% in the slope of the input-output function.     

Evidence for a bicuculline-insensitive long-lasting inhibition in the CA3 region of the rat hippocampal slice

Long-lasting inhibition (up to 2 s) of the commissurally-evoked response in the CA3 region of hippocampal slices was observed following a mossy fibre or commissural conditioning stimulus. Bicuculline applied iontophoretically or by superfusion (1-5 X 10(-6) M) blocked the early phase (20-40 ms) of the post-stimulus inhibition but either had no effect or potentiated the later inhibition.     

Effects of tissue preincubation and hypoxia on CA3 hippocampal neurons in the in vitro slice preparation

Using the in vitro hippocampal slice preparation, we studied the electrophysiological properties of pyramidal cells in tissue that was 'preincubated' (2-6 h in a large, static volume of oxygenated bathing medium) before being placed in an interface chamber for study. Striking differences were found in 'preincubated' vs 'non-preincubated' CA3 cells. The preincubated cells had more negative resting potentials, higher input resistance, lower threshold for stimulus-evoked burst discharge and larger hyperpolarizing afterpotentials. Cells in the preincubated CA3 region were also more likely to show spontaneous synchronized burst discharge, but were relatively resistant to hypoxia-induced spreading depression. CA1 cells were less dramatically affected by preincubation, showing little difference from their non-preincubated counterparts. Possible mechanisms involved in the CA3 preincubation effect, including glial buffering alterations and changes in Na+, K+-ATPase activity, are discussed.     

Calcium-dependent mechanisms involved in presynaptic long-term depression at the hippocampal mossy fibre-CA3 synapse

Long-term potentiation (LTP) and long-term depression (LTD) are induced presynaptically at the hippocampal mossy fibre-CA3 synapse. Activation of presynaptic metabotropic glutamate receptors (mGluRs) is necessary, but not sufficient for the LTD induction. Using mouse hippocampal slices, we attempted to identify additional presynaptic factors involved in the induction of mossy fibre LTD. Suppression of a rise in the presynaptic intracellular Ca2+ concentration ([Ca2+]i) with a membrane-permeable Ca2+ chelator, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), reduced the magnitude of LTD, whereas an increase in Ca2+ influx induced LTD, suggesting that an elevation of presynaptic [Ca2+]i is crucial for the LTD induction. A broad-spectrum protein kinase inhibitor, H-7, blocked LTD without affecting a presynaptic inhibition induced by an mGluR agonist. Furthermore, LTD was reduced by an inhibitor of calmodulin or Ca2+/calmodulin-dependent protein kinases. Thus, we conclude that mossy fibre LTD requires an increase in presynaptic [Ca2+]i and subsequent activation of Ca2+/calmodulin-dependent protein kinases. Because mossy fibre LTP may also require a rise in presynaptic [Ca2+]i, bidirectional long-term plasticity at the mossy fibre synapse is likely to be regulated by presynaptic Ca2+-dependent processes.     

Phorbol ester-induced neuritic alterations in the rat neocortex

In order to explore the effect of aberrant sprouting in the CNS, phorbol 12-myristate 13-acetate (PMA) was administered into the neocortex of adult rats. PMA is a growth-promoting agent that activates and eventually downregulates protein kinase C (PKC), and induces in the rat the expression of several genes, including amyloid precursor protein (APP). We found that multiple injections of 100 nM PMA into the rat neocortex promote, in the first week postinjection, a widespread vacuolization of the neuropil with a subsequent disruption of the synapses in the injection site, followed, at d 15, by the formation of abnormally distended clusters of neurites that resembled aberrant, sprouting axons. At d 30, fewer aberrant sprouts were observed, and many degenerating neurites were found. At the ultrastructural level, the PMA-induced abnormal neurites at d 7–15 resembled growth cones, whereas the dystrophic neurites at d 30 contained abundant dense and laminated bodies. Immunohistochemical analysis indicated that the abnormal neurites in the areas of denervation and PMA administration were positive with antisynaptophysin and antigrowth-associated protein 43 (GAP-43), with an increased APP immunoreactivity surrounding them. APP immunoreactivity around the injection site was mostly associated with pyramidal neurons and glial cells. Control experiments, where saline alone or 4α-phorbol 12, 13-didecanoate (PDD, an inactive phorbol derivative) was injected, failed to show aberrant sprouting neurites. Further immunohistochemical analysis showed that the PMA-treated animals presented increased amyloidΒ immunoreactivity in the pyramidal cells at the site of injection, when compared with control injections. These findings suggest that aberrant sprouting induced by overstimulation could be followed by neurodegeneration. Alternatively, PKC downregulation could directly induce the neurodegeneration, with a secondary sprouting response.     

Effect of adenosine on bicuculline-resistant paired-pulse inhibition in the rat hippocampal slice

This study extends previous investigations into the effect of adenosine on bicuculline-resistant paired-pulse inhibition between field potentials evoked 300 ms apart in the CA1 area of the rat hippocampal slice. A direct assessment of the effect of adenosine on paired-pulse inhibition is complicated by the facts that adenosine directly depresses evoked potentials and bicuculline-resistant paired-pulse inhibition is greather between pairs of small potentials than between pairs of larger potentials. Adenosine increased biculline-resistant paired-pulse inhibition when stimulus strength was constant between adenosine and control but paired-pulse inhibition of responses in adenosine was markedly less than paired-pulse inhibition of control responses of the same size. Futhermore, adenosine decreased the size of conditioned potentials to a significantly lesser extent than unparired potentials of the same initial size. Taken together the results indicate that adenosine can decrease bicuculline-resistant paired-pulse inhibition in the hippocampus. A possible mechanism for this effect is that adenosine is suppressing transmission at excitatory terminals onto interneurones which would suggest that these receptors are more sensitive to adenosine than those on the Schaffer collateral/CA1 pyramidal cell synapses. In this case adenosin should reduce paired-pulse inhibition at lower concentrations than are required for depression of single evoked potentials. A comparison of the concentration-response relationships for the effects of adenosine on paired-pulse inhibition and on single evoked potentials ruled out greater sensitivity of adenosine receptors at excitatory terminals onto interneurones as an explanation for adenosine's action on bicuculline-resistant paired-pulse inhibition. Adenosine was less effective at reducing inhibition evoked by large supramaximal conditioning stimuli than by submaximal for evoked potential size, although control paired-pulse inhibiton is larger in the later case. This finding is consistent with adenosine reducing bicuculline-resistant paired-pulse inhibition by causing an increase in simultaneous paired-pulse facilitation. © 1995 Wiley-Liss, Inc.     

Changes in protein kinase C isozymes in the rat hippocampal formation following hippocampal lesion

The cellular and regional distribution of the four protein kinase C (PKC) isoforms in the rat hippocampal formation and the response of PKC to lesions were determined by employing immunohistochemical and immunochemical techniques with antibodies specific to PKC (α), -(β I), -(β II), and -(γ). PKC (α) intensely stained the periphery of the pyramidal cell in the stratum pyramidale. The granule cells, glial cells, and mossy fibers were anti-PKC (α) negative. The cytoplasm, axons, and dendrites of basket cells and interneurons in the hilus were labeled with anti-PKC (α). Anti-PKC (β I) immunoreactivity was localized on the periphery of pyramidal cells and interneurons of the hilus, as well as the oriens, radiatum, and molecular layers of the CA regions. Anti-PKC (β II) immunoreactivity was mainly cytoplasmic, extending into the dendrites in the hippocampal pyramidal cells and the dentate granule cells, and also in some glial cells. In the stratum radiatum of the CA1, anti-PKC (γ) immunoreactivity localized to the pyramidal cell cytoplasm, extending into the dendrites. Following fimbria-fornix (FF) lesions, the anti-PKC (α) and -(β I) staining of the pyramidal cell periphery was markedly reduced. The anti-PKC (γ) staining of the pyramidal and granular cells of the dentate gyrus was reduced whereas the interneuron staining in the hilus was increased. In the FF-lesioned hippocampus, anti-PKC (α) and antiαPKC (β II) labeled reactive glial cells, whereas anti-PKC (β I) and -(γ) did not. Quantitative Western blot analysis revealed a dramatic increase in the particulate/total PKC for all isozyme forms, although the total levels of PKC, except PKC (γ), did not change following FF lesions. The PKC (γ) concentration doubled after FF lesions. Perforant path lesions resulted in a marked alteration in the neuronal staining in dentate gyrus with anti-PKC (α) and -(β I) and in increased numbers of anti-PKC (α)- and anti-PKC (β II)-positive glial cells. Anti-PKC (γ) staining did not change noticeably. The total PKC concentration did not change for isozymes α, β I, and γ, but PKC (βII) concentration increased by 48% following perforant path lesions as detected by Western blot analysis. The particulate/total PKC decreased for all four isozymes although the reduction in PKC (β I) concentration was not statistically significant. This change in PKC compartmentalization is in marked contrast to an increased level of particulate PKC following FF lesions. Thus, the effects of deefferentation and deafferentation for each PKC isoform were different.     

Modulation of an inhibitory circuit by adenosine and AMP in the hippocampus

The action of adenosine or AMP on the efficacy of a recurrent inhibitory loop was examined utilizing the hippocampal slice preparation. A reversible attenuation of paired-pulse inhibition was produced by micromolar concentrations of these compounds. Thus, in addition to its well-described capacity for reducing the strength of excitatory circuitry, adenosine appears capable of attenuating inhibitory circuitry.     

Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units/ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 microM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures.     

Quantitative relationship between post-tetanic biochemical and electrophysiological changes in rat hippocampal slices

Slices of rat brain hippocampus were tetanized in the perforant path fibers. In individual slices long-term changes of the electrophysiological parameters were determined simultaneously with post hoc endogeneous phosphorylation of proteins in a lyzed crude mitochondrial/synaptosomal fraction. A quantitative relation between the electrophysiological parameters and the degree of phosphorylation of a 52K protein was found to follow a non-linear function.     

Time course of protein changes following in vitro ischemia in the rat hippocampal slice

Following 5 min in vitro ischemia, total protein synthesis is dramatically and persistently inhibited in neurons in the rat hippocampal slice. This model system was used to explore the responses of individual proteins to this irreversible insult. In vitro ischemia inhibited new protein synthesis of most proteins analyzed; however, the synthesis of a 68/70kDa protein was substantially stimulated for the first hour after ischemia. By 3 h postischemia, its synthesis rates were depressed to 60% of control rates. Although the total amounts of most proteins were not significantly depleted for the first few hours after an ischemic episode, there were several notable exceptions. The levels of HSC73, a constitutively expressed member of the 70 kDa stress protein family, were reduced after in vitro ischemia. In addition, MAP-2 (microtubule-associated protein-2) and α-tubulin were depleted in the early hours after the insult, with MAP-2 exhibiting a detectable depletion earlier than tubulin. In contrast, the levels and distribution of a 68 kDa neurofilament protein localized to CA3 pyramidal neurons in the slice, apparently distinct from the band whose new synthesis was stimulated, were not affected by the 5 min in vitro ischemia insult. Thus, the responses of individual proteins to ischemia varied considerably. These individual responses could play an important role in the damage mechanism that is initiated in response to in vitro ischemia.     

Modulation of high-threshold Ca current and spontaneous postsynaptic transient currents by phorbol 12, 13-diacetate, 1-(5-isoquinolinesulfonyl)-2-methyl piperazine (H-7), and monosialoganglioside (GM1) in CA1 pyramidal neurons of rat hippocampus in vitro

Phorbol esters, which activate protein kinase C (PKC), enhance synaptic transmission in the CA1 subfield of hippocampus, both in situ and in vitro. The increase in synaptic transmission could be the consequence of enhanced Ca influx into nerve terminals, and perhaps a more general increase in voltage-dependent Ca currents. The effects of phorbol 12, 13-diacetate (PDAc) on the high-voltage activated (HVA) Ca currents, as well as spontaneous transient currents were therefore investigated by intracellular recording in hippocampal slices. PDAc selectively augmented, by 45% ± 10%, the early peak of the HVA Ca current (but not its sustained component), and also spontaneous inhibitory postsynaptic currents. The inactive phorbol ester, 4 α-PDAc, had no comparable effects. The actions of PDAc were reversible on prolonged washing, and they were antagonized by the PKC inhibitors (1-(5-isoquinolinesulfonyl)-2-methyl) piperazine (H-7) and monosialoganglioside (GM1). In addition, GM1, which also activates the Ca/calmodulin-dependent kinase, enhanced spontaneous excitatory postsynaptic currents, while inhibiting the IPSCs. It is concluded that activation of PKC increases HVA (probably N-type) Ca current and facilitates ongoing GABAergic IPSCs.     

The translocation and involvement of protein kinase C in mossy fiber-CA3 long-term potentiation in hippocampus of the rat brain

The detailed mechanisms underlying long-term potentiation (LTP) are not known. In hippocampal CA1, translocation of protein kinase C (PKC) activity from cytosol to membrane and subsequent phosphorylation of growth associated protein (GAP)-43 have been demonstrated to be critical events for the maintenance phase of LTP. LTP in mossy fiber (MF)-CA3 pathway and the Schaffer collateral/commissural (SC)-CA1 pathway differ in a number of ways: SC-CA1 LTP depends on NMDA receptors while MF-CA3 LTP does not, and SC-CA1 LTP is primarily postsynaptic while MF-CA3 LTP is primarily presynaptic. The role of PKC in MF-CA3 LTP has not been studied. We investigated the role of PKC in CA3 and show that PKC inhibitors prevent LTP, but that PKC activators produce a reversible synaptic potentiation, indicating that PKC activation is an essential but not sufficient component of LTP in CA3. Then using antibodies against specific PKC isozymes we have determined the membrane vs. cytosolic distribution of various PKC isozymes in slices subjected to low or tetanic stimulation, or perfused with phorbol esters (PDAc). Compared with control, LTP and PDAc slices show greater PKC-α and -ε immunoreactivity in the membrane fraction, indicating that both LTP and phorbol ester treatment induce translocation of PKC-α and -ε from cytosol to membrane. However, with PKC-β and PKC-γ the only detectable translocation from cytosol to membrane was in the phorbol ester-treated slices. Thus, while phorbol ester treatment causes translocation of PKC-α, -β, -γ and -ε, the only detectable translocation associated with CA3 LTP is that of PKC-α and -ε.