Facilitation of cholinergic transmission by substance P methyl ester in the mouse hippocampal slice preparation

Using sharp microelectrode recording from CA1 pyramidal neurons of the adult mouse hippocampal slice preparation, we studied the modulatory action of the selective neurokinin 1 (NK1) receptor agonist substance P methyl ester (SPME), a peptidase-resistant analogue of the peptide substance P (SP), on cholinergic responses. While SPME (0.1-1 microM) had only slight effects on membrane potential and input resistance of CA1 neurons, it largely and reversibly enhanced the membrane depolarization and oscillatory activity induced by the cholinergic agonist carbachol (CCh; 0.1-100 microM). This effect of SPME was prevented by the selective NK1 receptor antagonist SR 140333 (4 microM). In about half of the tested neurons the action of SPME was preserved in tetrodotoxin (TTX) solution, suggesting that it partly occurred at the level of pyramidal cells. Cholinergic slow excitatory postsynaptic potentials (sEPSPs) were reversibly enhanced by SPME which increased their amplitude and prolonged any associated bursting activity. This action was also blocked by SR 140333. The present results suggest that SPME largely enhances cholinergic activity in the mouse hippocampus, an effect which can help to explain, in this brain area, the recently reported facilitation of seizures by SP.     

Immunohistochemical characterization of substance P receptor (NK(1)R)-expressing interneurons in the entorhinal cortex

It has been reported that application of substance P (SP) to the medial portion of the entorhinal cortex (EC) induces a powerful antiepileptic effect (Maubach et al. [1998] Neuroscience 83:1047-1062). This effect is presumably mediated via inhibitory interneurons expressing the neurokinin-1 receptor (NK(1)R), but the existence of NK(1)R-expressing inhibitory interneurons in the EC has not yet been reported. The present immunohistochemical study was performed in the rat to examine the existence and distribution of NK(1)R-expressing neurons in the EC as well as any co-expression of other neurotransmitters/neuromodulators known to be associated with inhibitory interneurons: gamma-aminobutyric acid (GABA), parvalbumin (PARV), calretinin (CT), calbindin (CB), somatostatin (SST), and neuropeptide Y (NPY). Our results indicated that NK(1)R-positive neurons were distributed rather sparsely (especially in the medial EC), primarily in layers II, V, and VI. The results of our double-immunohistochemical staining indicated that the vast majority of NK(1)R-expressing neurons also expressed GABA, SST, and NPY. In addition, CT was co-expressed in a weakly stained subgroup of NK(1)R-expressing neurons, and CB was co-expressed very rarely in the lateral EC, but not in the medial EC. In contrast, SP-immunopositive axons with fine varicosities were distributed diffusely throughout all layers of the EC, appearing to radiate from the angular bundle. SP may be released in a paracrine manner to activate a group of NK(1)R-expressing entorhinal neurons that co-express GABA, SST, and NPY, exerting a profound inhibitory influence on synchronized network activity in the EC.     

Selective impairment of GABAergic synaptic transmission in the flurothyl model of neonatal seizures

Neonatal seizures can result in long-term adverse consequences including alteration of seizure susceptibility and impairment in spatial memory. However, little is known about the effects of neonatal seizures on developmental changes occurring in synaptic transmission during the first postnatal weeks. The purpose of the present study was to examine the effect of neonatal seizures on several aspects of gamma-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission in the developing rat hippocampus. Flurothyl was used to induce multiple recurrent seizures in rat pups during the first postnatal days. Whole-cell patch-clamp recordings from the hippocampal CA3 pyramidal cell and extracellular recordings from the CA3 pyramidal cell layer were made in slice preparations. In rats that experienced neonatal seizures the amplitude of spontaneous inhibitory postsynaptic currents at P15-17 was decreased by 27% compared with controls, whereas neither frequency nor the kinetic properties were altered. Neonatal seizures did not affect the timing of the developmental switch in the GABAA signaling from excitatory to inhibitory. None of the studied parameters of glutamatergic postsynaptic currents was different between the flurothyl and control groups, including the amplitude and frequency of the spontaneous excitatory postsynaptic currents, the ratio of the amplitudes and frequencies of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-mediated spontaneous postsynaptic currents, and the kinetics of AMPA and NMDA mediated postsynaptic currents in the age groups P8-10 and P15-17. We suggest that the selective depression of the amplitude of GABAergic synaptic responses may contribute to the adverse neurological and behavioral consequences that occur following neonatal seizures.     

Hyperthermia decreases GABAergic synaptic transmission in hippocampal neurons of immature rats

The mechanisms underlying the generation of febrile seizures are poorly understood. We suggest that high temperature contributes to febrile seizures and specifically tested the hypothesis that hyperthermia suppressed GABAA-receptor-mediated inhibition in hippocampal neurons using whole-cell patch clamp recordings. We found that heating from a baseline temperature of 32 degrees C to 40 degrees C suppressed the peak amplitude of GABAA-receptor-mediated inhibitory postsynaptic currents (IPSCs) by 50+/-4.7% and decreased the decay time constant of IPSCs by 60.6+/-6.7% in immature CA1 neurons in the rat hippocampus. This inhibitory effect partly results from reduced IPSC conductance and increased GABA uptake, as demonstrated by the fact that GABA uptake blocker N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SKF89976A) significantly reduced the peak suppression and decay time decrease of the IPSC during hyperthermia. In addition, hyperthermia (40 degrees C) produced a significantly larger depression of the IPSC peak than the slope or peak of the excitatory postsynaptic current (EPSC), and IPSCs recovered slower than EPSCs after hyperthermia. The larger decrease in GABAA-receptor-mediated inhibition during and after hyperthermia, as compared with excitation, may shift the excitation/inhibition balance and contribute to the generation of febrile seizures.     

Excitatory actions of substance P in the rat lateral posterior nucleus

The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher‐level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)‐containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole‐cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro‐caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5–2000 nm ) depolarized nearly all relay neurons tested (> 98%) in a concentration‐dependent manner. Voltage‐clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)₁ tachykinin receptors, although significantly smaller inward currents were produced by specific NK₂ and NK₃ receptor agonists. The selective NK₁ receptor antagonist RP67580 attenuated the SP‐mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK₂ and NK₃ receptor antagonists, GR159897 and SB222200, respectively. The SP‐mediated response showed voltage characteristics consistent with a K⁺ conductance, and was attenuated by Cs⁺, a K⁺ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.     

Fear conditioning enhances spontaneous AMPA receptor-mediated synaptic transmission in mouse hippocampal CA1 area

AMPA receptor-mediated synaptic modifications in the amygdala have been reported to sustain cued fear conditioning. However, the hippocampal formation is also critically involved in fear learning. Therefore, we examined whether fear conditioning is also accompanied by changes in AMPA receptor-mediated synaptic transmission in the hippocampus. We focused on spontaneous miniature excitatory post-synaptic currents (mEPSCs). Young adult mice were trained using tone/footshock pairings and contextual/cued memories were tested 3–4 h and 1 day later. We found that the mEPSC frequency was significantly enhanced when recorded 3 h, but not 24 h, after fear conditioning training. Fear training induced a slight enhancement in the mEPSC amplitude at 3 h after training. The increased mEPSC frequency and amplitude were absent in animals that were only exposed to footshock or novelty or unpaired tone/footshock training. This implies that learning the association between context, tone and footshock transiently enhances hippocampal CA1 spontaneous synaptic transmission, which may contribute to the encoding of the fearful event.     

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.     

Neuronal glutamate transporters regulate synaptic transmission in single synapses on CA1 hippocampal neurons

Glutamate is the major excitatory transmitter in CNS although it causes severe brain damage by pathologic excitotoxicity. Efficient neurotransmission is controlled by powerful protection and support afforded by specific high-affinity glutamate transporters in neurons and glia, clearing synaptic glutamate. While the role of glial cells in glutamate uptake is well defined, the role of neuronal transporters remains poorly understood. The evaluation of impact of neuronal transporters on spontaneous and evoked EPSC in hippocampal CA1 neurons within a model ‘single bouton preparation’ by pre- and postsynaptic uptake was addressed.In whole-cell patch clamp experiments the influence of blocking, pre- or both pre- and postsynaptic glutamate transporters (GluT) on spontaneous and evoked postsynaptic currents (sEPSC and eEPSC), was examined by manipulating the content of intracellular solution. Suppressing GluT by non-transportable inhibitor TBOA (10 μM) led to remarkable alteration of glutamate uptake process and was reflected in measurable changes of general properties of synaptic currents.Elimination of intracellular K⁺ concentration required for glutamate transporter operation by using Cs⁺-based internal solution (postsynaptic GluTs are non-functional apriori), causes the deficient of presynaptic glutamate transporters. Applied in such conditions glutamate transporter inhibitor TBOA (10 μM) affected the occurrence of synaptic event and thus unregulated the transmitter release. eEPSCs were generally suppressed both in amplitude (to 48.73 ± 7.03% vs. control) and in success rate (R_suc) by TBOA (from 91.1 ± 7.5% in control to 79.57 ± 13.2%). In contrast, with K⁺-based solution in patch pipette (pre- and postsynaptic GluT are intact), amplitude of eEPSC was substantially potentiated by pre-treatment with TBOA (152.1 ± 11%), whereas (R_suc) was reduced to 79.8 ± 8.3% in average. The identical reduction of event success rate as well as increased pair-pulse ratios (PPF ratio) for eEPSC in both cases indicates the effect of TBOA on presynaptic uptake. sEPSCs simultaneously recorded from neurons, showed the same pattern of regulation but with less potency, indicating the similar processes in most of excitatory synapses.In conclusion, presynaptic transporters are suggested to act mainly as negative feedback signal on presynaptic release and/or referred to vesicle refilling processes.     

Spermine depresses NMDA, K/AMPA and GABAA-mediated synaptic transmission in the rat hippocampal slice preparation

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.     

Enhanced synaptic excitation-inhibition ratio in hippocampal interneurons of rats with temporal lobe epilepsy

A common feature of all epileptic syndromes is the repetitive occurrence of pathological patterns of synchronous neuronal activity, usually combined with increased neuronal discharge rates. Inhibitory interneurons of the hippocampal formation control both neuronal synchronization as well as the global level of activity and are therefore of crucial importance for epilepsy. Recent evidence suggests that changes in synaptic inhibition during temporal lobe epilepsy are rather specific, resulting from selective death or alteration of interneurons in specific hippocampal layers. Hence, epilepsy-induced changes have to be analysed separately for different types of interneurons. Here, we focused on GABAergic neurons located at the border between stratum radiatum and stratum lacunosum-moleculare of hippocampal area CA1 (SRL interneurons), which are included in feedforward inhibitory circuits. In chronically epileptic rats at 6-8 months after pilocarpine-induced status epilepticus, frequencies of spontaneous and miniature inhibitory postsynaptic currents were reduced, yielding an almost three-fold increase in excitation-inhibition ratio. Consistently, action potential frequency of SRL interneurons was about two-fold enhanced. Morphological alterations of the interneurons indicate that these functional changes were accompanied by remodelling of the local network, probably resulting in a loss of functional inhibitory synapses without conceivable cell death. Our data indicate a strong increase in activity of interneurons in dendritic layers of the chronically epileptic CA1 region. This alteration may enhance feedforward inhibition and rhythmogenesis and--together with specific changes in other interneurons--contribute to seizure susceptibility and pathological synchronization.     

Interneurons targeting similar layers receive synaptic inputs with similar kinetics

GABAergic interneurons play diverse and important roles in controlling neuronal network dynamics. They are characterized by an extreme heterogeneity morphologically, neurochemically, and physiologically, but a functionally relevant classification is still lacking. Present taxonomy is essentially based on their postsynaptic targets, but a physiological counterpart to this classification has not yet been determined. Using a quantitative analysis based on multidimensional clustering of morphological and physiological variables, we now demonstrate a strong correlation between the kinetics of glutamate and GABA miniature synaptic currents received by CA1 hippocampal interneurons and the laminar distribution of their axons: neurons that project to the same layer(s) receive synaptic inputs with similar kinetics distributions. In contrast, the kinetics distributions of GABAergic and glutamatergic synaptic events received by a given interneuron do not depend upon its somatic location or dendritic arborization. Although the mechanisms responsible for this unexpected observation are still unclear, our results suggest that interneurons may be programmed to receive synaptic currents with specific temporal dynamics depending on their targets and the local networks in which they operate.     

Modulation of excitatory synaptic transmission by glycine and zinc in cultures of mouse hippocampal neurons

The monosynaptic EPSP between cultured hippocampal neurons is mediated by activation of 2 classes of excitatory amino acid receptors. Kainate or quisqualate receptors generate a fast conventional EPSP, while NMDA receptors mediate a slow, voltage-sensitive EPSP. Recently, 2 substances have been shown to modulate the activity of the NMDA receptor-channel complex: glycine increases the probability of channel opening, while zinc acts as a noncompetitive antagonist. Since these substances are present in the CNS and thus may function as neuromodulators, we have examined their role in excitatory synaptic transmission in hippocampal cultures using the whole-cell-patch-recording technique. The slow, NMDA-receptor-mediated EPSP was strikingly dependent on the presence of a conditioning substance that gradually accumulated in the extracellular fluid during a 30 min incubation in physiological saline. Washout of the conditioned medium eliminated the slow EPSP, and perfusion with physiological saline containing 1 microM glycine restored the slow EPSP to control levels. Furthermore, conditioned medium collected from astroglial-only cultures also potentiated the response to NMDA. Zinc (20-50 microM) overcame the potentiation of the response by glycine and resulted in a reversible block of the slow EPSP, providing the first evidence for a direct action of zinc on excitatory synaptic transmission. Our results show that the expression of the slow EPSP may be subject to regulation by several endogenous substances: positive modulation by glycine (or a glycine-like substance), which can be released from astroglial cells, and negative modulation by physiological levels of zinc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine can enhance synaptic transmission in hippocampal slices

Activity of 40 single antidromically identified supraoptic neurons was recorded and evaluated in response to a combination of tactile, vulvar massage, vaginal distension, and slow intrajugular 1.2 M sodium chloride infusion in unanesthetized, randomly hydrated ewes. Estradiol-implanted Southdown ewes were prepared according to techniques described by Jennings et al. Only 4 spontaneous firing patterns were observed in the supraoptic nuclei. Analysis of evoked activity indicated that each stimulus evoked alterations in mean firing rates or increased numbers of short interspike intervals in some cells. The resultant activity of units to sequential vulvar massage and 1.2 M sodium chloride infusion suggests a possibility of separate mechanisms of release of oxytocin and vasopressin.     

MPTP causes a non-reversible depression of synaptic transmission in mouse neostriatal brain slice

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinson's disease-like syndrome. The mechanism of MPTP's neurotoxicity is unknown; however, one hypothesis is that MPP+ (1-methyl-4-phenylpyridinium), a product of MPTP's oxidation, is the neurotoxic agent. Using a mouse brain slice preparation we studied the effects of MPTP and MPP+ on synaptic transmission. We found MPTP caused a decrease in amplitude of an excitatory synaptic response not reversed by washing. This non-reversible action of MPTP was prevented by GBR-32 and pargyline. MPP+s caused a decrease in synaptic transmission, but this decrease was reversed by washing. The results suggest that the toxic effect of MPTP on synaptic transmission is not accounted for by the action of MPP+.     

Effects of melatonin on synaptic transmission and long-term potentiation in two areas of mouse hippocampus

We have examined the effects of melatonin on synaptic transmission and long-term potentiation (LTP) in the Schaffer Collateral-CA1 and the mossy fiber-CA3 pathways in mouse hippocampus brain slices. Melatonin (0.1-1 mM) application had different actions on both the field excitatory postsynaptic potentials (fEPSPS) and LTP in the CA1 as compared to the CA3. In CA1, 0.1 mM melatonin blocked LTP, while 1 mM melatonin also depressed the fEPSP. In CA3, neither 0.1 nor 1 mM melatonin altered the fEPSP, whereas both concentrations only slightly reduced LTP. These results demonstrate that melatonin significantly alters synaptic transmission and LTP in the CA1 but has only modest actions in CA3.     

Increased density of substance P binding sites in specific brainstem nuclei of spontaneously hypertensive rats

Substance P binding sites were quantitated in 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats by incubation of brain sections with¹²⁵I-Bolton-Hunter substance P, autodiography and microdensitometry. The maximum binding capacity (B_max) was significantly increased in the dorsal motor nucleus of the vagus, in the hypoglossal, ambiguus and inferior olivary nuclei, and in lobes 9 and 10 of the vermis cerebelli of SHR.     

Perfusion with lithium modifies neurophysiological responses in the CA1 region of the hippocampal slice preparation

The effects of acute lithium exposure on extracellular electrophysiological responses in the CA1 region of the in vitro hippocampus were investigated. Field potentials were assessed while perfusing slices with normal media or media in which LiCl was substituted for NaCl in 30, 20, 10 and 2 mM amounts. Lithium concentration in the slice following 20 min perfusion with 20 mM lithium was determined to be about 14 mM. At the higher concentrations, lithium exposure depressed the presynaptic fiber volley and antidromic population spike. On the other hand, the population EPSP and orthodromic population spike were enhanced. No significant changes were found at 2 mM. The findings are compatible with one action of lithium being on the excitability of axons and synaptic terminals. Comparisons were drawn between previous studies involving chronic lithium exposure and the present results. In this acute preparation lithium effects, as reflected in the population EPSP, were in opposition to those found with chronic lithium exposure. Changes demonstrated in this preparation in fiber volley and antidromic population spike paralleled those found with chronic lithium exposure.     

MPDP causes a non-reversible decrease in neostriatal synaptic transmission in mouse brain slice

MPTP causes a Parkinson's disease-like syndrome in man and certain other animals. The toxic effect occurs if monoamine oxidase B is available, indicating that an MPTP metabolite may cause the toxic effect. We tested the effect of MPDP+, the first product of MPTP oxidation, and found that it, like MPTP, caused a non-reversible decrease in synaptic transmission in the mouse brain slice preparation. As the second oxidation product, MPP+ had been shown not to cause a similar, non-reversible decrease in synaptic transmission, MPDP+ is a better candidate for the role of toxic substance.     

Intracellular correlates of morphine excitation in the hippocampal slice preparation

The influences of morphine and opioid peptides on hippocampal CA1 pyramidal cells were investigated using intracellular recordings from the in vitro slice preparation. Morphine applied to somal and basal dendritic areas of CA1 cells via a pressure ejection system confirmed a number of excitatory actions of opiates and opioid peptides in this brain region. These included an increase in the amplitude and duration of orthodromically (radiatum) elicited EPSPs and a decrease in amplitude of the following IPSP. The increase in EPSP amplitude was accompanied by a reduction in stimulus intensity necessary for eliciting the action potential. Morphine delivered to the slice in this manner induced synaptically elicited and spontaneous multiple spike burst discharges. In slice maintained in 10⁻⁴ M pentobarbital¹, morphine reversed the presumably GABA mediated long-duration depolarization following orthodromic stimulation. Finally, depending on the specific site of application (apical or basal dendritic region) of the opiate, morphine produced two different effects on the resting membrane potential and input resistance of CA1 pyramidal cells. These findings are discussed as to whether opiates act directly excitatory influences in the hippocampus, or via blockade of GABA mediated inhibitory mechanisms.     

A hypoxic injury potential in the hippocampal slice

In rat hippocampal slices, neurons in the stratum pyramidale of the CA1 were stimulated orthodromically and antidromically while the resultant extracellular population spikes were monitored. Hypoxic conditions were then induced. After disappearance of the orthodromic population spike, a second orthodromic population spike appeared. We have titled this the hypoxic injury potential since it reflects the onset of permanent injury to neurons in area CA1 of the hippocampus.