Terminals Of Interneurons Research Articles

Neuronal activity and stress differentially regulate hippocampal and hypothalamic corticotropin-releasing hormone expression in the immature rat

Corticotropin-releasing hormone, a major neuromodulator of the neuroendocrine stress response, is expressed in the immature hippocampus, where it enhances glutamate receptor-mediated excitation of principal cells. Since the peptide influences hippocampal synaptic efficacy, its secretion from peptidergic interneuronal terminals may augment hippocampal-mediated functions such as learning and memory. However, whereas information regarding the regulation of corticotropin-releasing hormone’s abundance in CNS regions involved with the neuroendocrine responses to stress has been forthcoming, the mechanisms regulating the peptide’s levels in the hippocampus have not yet been determined. Here we tested the hypothesis that, in the immature rat hippocampus, neuronal stimulation, rather than neuroendocrine challenge, influences the peptide’s expression. Messenger RNA levels of corticotropin-releasing hormone in hippocampal CA1, CA3 and the dentate gyrus, as well as in the hypothalamic paraventricular nucleus, were determined after cold, a physiological challenge that activates the hypothalamic pituitary adrenal system in immature rats, and after activation of hippocampal neurons by hyperthermia. These studies demonstrated that, while cold challenge enhanced corticotropin-releasing hormone messenger RNA levels in the hypothalamus, hippocampal expression of this neuropeptide was unchanged. Secondly, hyperthermia stimulated expression of hippocampal immediate-early genes, as well as of corticotropin-releasing hormone. Finally, the mechanism of hippocampal corticotropin-releasing hormone induction required neuronal stimulation and was abolished by barbiturate administration. Taken together, these results indicate that neuronal stimulation may regulate hippocampal corticotropin-releasing hormone expression in the immature rat, whereas the peptide’s expression in the hypothalamus is influenced by neuroendocrine challenges.

Synaptic relationship of the neurons containing a metabotropic glutamate receptor, MGluR5, with nociceptive primary afferent and GABAergic terminals in rat spinal superficial laminae

Recent pharmacological evidence showed that metabotropic glutamate receptors (mGluRs), particularly mGluRs1/5, had a potential role in spinal nociceptive processing. However, previous morphological studies on mGluRs have been limited mainly to their distribution in the spinal cord. In the present study, electron microscopic immunocytochemistry was employed to identify the synaptic relationship of the neurons containing mGluR5, with nociceptive primary afferent and γ-aminobutyric acid-ergic (GABAergic) terminals in the superficial dorsal horn of the spinal cord. Nociceptive C- and A δ-primary afferent terminals selectively labeled with horseradish peroxidase conjugated to wheat-germ agglutinin were in asymmetric synaptic contacts with or in direct apposition to mGluR5 positive dendritic profiles. The double-labeling studies revealed that mGluR5 immunoreactive dendrites also received symmetric synaptic contacts from axon terminals labeled with immunogold particles indicating GABA. The present demonstration of mGluR5 neurons receiving inputs from both nociceptive primary afferents and GABAergic terminals of presumed interneurons further supports the involvement of mGluR5 in the transmission and modulation of nociceptive information in the spinal cord.

Presynaptic control of transmission along the pathway mediating disynaptic reciprocal inhibition in the cat.

In cat lumbar motoneurones, disynaptic inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of antagonist motor nerves were depressed for at least 150 ms following conditioning stimulation of flexor (1.7-2 times threshold (T)) and ankle extensor (5T) nerves. The aim of the present study was to investigate the possibility that this depression is caused by presynaptic inhibitory mechanisms acting at the terminals of group I afferent fibres projecting to the Ia inhibitory interneurones and/or the terminals of these interneurones to the target motoneurones. Conditioning stimulation of flexor, but not ankle extensor, nerves evoked a depression of the monosynaptic Ia excitatory postsynaptic potentials (EPSPs) recorded intracellularly in Ia inhibitory interneurones. This depression lasted between 200 and 700 ms and was not accompanied by a depression of the monosynaptic EPSPs evoked by stimulation of descending pathways. These results suggest that flexor, but not ankle extensor, group I afferent fibres can modulate sensory transmission at the synapse between Ia afferent fibres and Ia inhibitory interneurones. Conditioning stimulation of flexor muscle nerves, extensor muscle nerves and cutaneous nerves produced a long-lasting increase in excitability of the terminals of the Ia inhibitory interneurones. The increase in the excitability of the terminals was not secondary to an electrotonic spread of synaptic excitation at the soma. Indeed, concomitant with the excitability increase of the terminals there were signs of synaptic inhibition in the soma. The unitary IPSPs induced in target motoneurones following the spike activity of single Ia inhibitory interneurones were depressed by conditioning stimulation of muscle and cutaneous nerves. Since the conditioning stimulation also evoked compound IPSPs in those motoneurones, a firm conclusion as to whether unitary IPSP depression involved presynaptic inhibitory mechanism of the terminals of the interneurones could not be reached. The possibility that the changes in excitability of the Ia interneuronal terminals reflect the presence of a presynaptic inhibitory mechanism similar to that operating at the terminals of the afferent fibres (presynaptic inhibition) is discussed.1. In cat lumbar motoneurones, disynaptic inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of antagonist motor nerves were depressed for at least 150 ms following conditioning stimulation of flexor (1.7-2 times threshold (T)) and ankle extensor (5T) nerves. The aim of the present study was to investigate the possibility that this depression is caused by presynaptic inhibitory mechanisms acting at the terminals of group I afferent fibres projecting to the Ia inhibitory interneurones and/or the terminals of these interneurones to the target motoneurones. Conditioning stimulation of flexor, but not ankle extensor, nerves evoked a depression of the monosynaptic Ia excitatory postsynaptic potentials (EPSPs) recorded intracellularly in Ia inhibitory interneurones. This depression lasted between 200 and 700 ms and was not accompanied by a depression of the monosynaptic EPSPs evoked by stimulation of descending pathways. These results suggest that flexor, but not ankle extensor, group I afferent fibres can modulate sensory transmission at the synapse between Ia afferent fibres and Ia inhibitory interneurones. Conditioning stimulation of flexor muscle nerves, extensor muscle nerves and cutaneous nerves produced a long-lasting increase in excitability of the terminals of the Ia inhibitory interneurones. The increase in the excitability of the terminals was not secondary to an electrotonic spread of synaptic excitation at the soma. Indeed, concomitant with the excitability increase of the terminals there were signs of synaptic inhibition in the soma. The unitary IPSPs induced in target motoneurones following the spike activity of single Ia inhibitory interneurones were depressed by conditioning stimulation

Presynaptic impairment of cerebellar inhibitory synapses by an autoantibody to glutamate decarboxylase.

Glutamic acid decarboxylase (GAD), the enzyme responsible for converting glutamate to gamma-aminobutyric acid (GABA), is a target of humoral autoimmunity in stiff-man syndrome and subacute cerebellar ataxia. Recently, we found that an anti-GAD autoantibody in the CSF of an ataxic patient selectively suppressed GABA-mediated transmission on cerebellar Purkinje cells without affecting glutamate-mediated transmission. Here, we examine the mechanism by which the autoantibody impaired the inhibitory transmission, using immunohistochemistry and whole-cell recording in rat cerebellar slices. The present results indicate that CSF immunoglobulins prepared from an ataxic patient acted on the presynaptic terminals of GABAergic interneurons and decreased GABA release onto Purkinje cells.

A hippocampal interneuron associated with the mossy fiber system

Network properties of the hippocampus emerge from the interaction of principal cells and a heterogeneous population of interneurons expressing gamma-aminobutyric acid (GABA). To understand these interactions, the synaptic connections of different types of interneurons need to be elucidated. Here we describe a type of inhibitory interneuron of the hippocampal CA3 region that has an axon coaligned with the mossy fibers. Whole-cell patch-clamp recordings, in combination with intracellular biocytin filling, were made from nonpyramidal cells of the stratum lucidum under visual control. Mossy fiber-associated (MFA) interneurons generated brief action potentials followed by a prominent after-hyperpolarization. Subsequent visualization revealed an extensive axonal arbor which was preferentially located in the stratum lucidum of CA3 and often invaded the hilus. The dendrites of MFA interneurons were mainly located in the strata radiatum and oriens, suggesting that these cells are primarily activated by associational and commissural fibers. Electron microscopic analysis showed that axon terminals of MFA interneurons established symmetric synaptic contacts predominantly on proximal apical dendritic shafts, and to a lesser degree, on somata of pyramidal cells. Synaptic contacts were also found on GABAergic interneurons of the CA3 region and putative mossy cells of the hilus. Inhibitory postsynaptic currents (IPSCs) elicited by MFA interneurons in simultaneously recorded pyramidal cells had fast kinetics (half duration, 3.6 ms) and were blocked by the GABA(A) receptor antagonist bicuculline. Thus, MFA interneurons are GABAergic cells in a position to selectively suppress the mossy fiber input, an important requirement for the recall of memory traces from the CA3 network.

Differential innervation of parvalbumin-immunoreactive interneurons of the basolateral amygdaloid complex by cortical and intrinsic inputs

In the basolateral (BL) amygdaloid complex, the excitability of projection cells is regulated by intrinsic inhibitory interneurons using gamma-aminobutyric acid (GABA) as a transmitter. A subset of these cells are labeled in a Golgi-like manner by Parvalbumin (PV) immunohistochemistry. Recently, we have shown that the overwhelming majority of axon terminals contacting these PV-immunoreactive neurons form asymmetric synapses. The present study was undertaken to identify the source(s) of these inputs. Since previous work had revealed that thalamic axons form very few synapses on BL interneurons (< 1%), we focused on cortical and intra-amygdaloid inputs. Iontophoretic injections of the anterograde tracers Phaseolus vulgaris-leucoagglutinin or biotinylated dextran amine were performed in various cortical fields in cats (perirhinal, entorhinal, pre/infralimbic cortices) and monkeys (orbitofrontal region) or in the BL amygdaloid nucleus in cats. These injections resulted in a large number of anterogradely labeled terminals forming asymmetric synapses in the BL complex. Following cortical injections, numerous anterogradely labeled terminals were found in the vicinity of PV-immunoreactive interneurons in the BL amygdala. However, only approximately 1% of these terminals formed synaptic contacts with PV-immunoreactive profiles. In contrast, as many as 11% of the terminals contributed by the intranuclear axon collaterals of BL projection cells established synapses with PV-immunoreactive elements. Since the axon terminals of PV-immunoreactive interneurons are enriched in GABA and they exclusively form symmetric synapses, these results suggest that PV-immunoreactive interneurons are predominantly involved in feedback inhibition in the BL amygdaloid complex.

Effects of corticosteroids on synaptic transmission in rat dorsolateral septal nucleus.

The effects of corticosteroids on synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined, in vitro, by using intracellular and voltage-clamp recording methods. Prednisolone (100 microM) increased the amplitude of excitatory postsynaptic potential (EPSP) and depressed both fast and slow inhibitory postsynaptic potentials (IPSP). Under voltage-clamp conditions, prednisolone (100 microM) increased the amplitude of excitatory postsynaptic current (EPSC) and depressed the fast and slow inhibitory postsynaptic currents (IPSCs). Corticosterone (100 microM) mimicked the effects of prednisolone on the postsynaptic currents (PSCs). To examine the direct effects of prednisolone on the EPSC and slow IPSC, the fast IPSC was blocked by bicuculline (20 microM). Under these experimental conditions, prednisolone (100 microM) did not alter the isolated EPSC but depressed slow IPSC by 22 +/- 3% (n = 10). The fast IPSC was isolated by pretreatment with kynurenic acid and CGP55845A, where the EPSC and slow IPSC were blocked. Prednisolone (100 microM) depressed the isolated fast IPSC in DLSN neurons. Prednisolone (100 microM) did not change either the inward current produced by glutamate or the outward current produced by gamma-aminobutyric acid (GABA). The results suggest that corticosteroids facilitate excitatory synaptic transmission in the DLSN by reducing the release of GABA from the presynaptic nerve terminals of interneurons.

Nicotinic Receptor Activation in Human Cerebral Cortical Interneurons: a Mechanism for Inhibition and Disinhibition of Neuronal Networks

Cholinergic control of the activity of human cerebral cortical circuits has long been thought to be accounted for by the interaction of acetylcholine (ACh) with muscarinic receptors. Here we report the discovery of functional nicotinic receptors (nAChRs) in interneurons of the human cerebral cortex and discuss the physiological and clinical implications of these findings. The whole-cell mode of the patch-clamp technique was used to record responses triggered by U-tube application of the nonselective agonist ACh and of the alpha7-nAChR-selective agonist choline to interneurons visualized by means of infrared-assisted videomicroscopy in slices of the human cerebral cortex. Choline induced rapidly desensitizing whole-cell currents that, being sensitive to blockade by methyllycaconitine (MLA; 50 nM), were most likely subserved by an alpha7-like nAChR. In contrast, ACh evoked slowly decaying whole-cell currents that, being sensitive to blockade by dihydro-beta-erythroidine (DHbetaE; 10 microM), were most likely subserved by an alpha4beta2-like nAChR. Application of ACh (but not choline) to the slices also triggered GABAergic postsynaptic currents (PSCs). Evidence is provided that ACh-evoked PSCs are the result of activation of alpha4beta2-like nAChRs present in preterminal axon segments and/or in presynaptic terminals of interneurons. Thus, nAChRs can relay inhibitory and/or disinhibitory signals to pyramidal neurons and thereby modulate the activity of neuronal circuits in the human cerebral cortex. These mechanisms, which appear to be retained across species, can account for the involvement of nAChRs in cognitive functions and in certain neuropathological conditions.

The location of muscarinic type 2 receptors within the synaptic circuitry of the cat lateral posterior nucleus

The ultrastructural distribution of the muscarinic type 2 acetylcholine receptor (M2) was examined in the lateral division of the lateral posterior (LP) nucleus of the cat thalamus, using immunocytochemistry. Postembedding immunocytochemical staining for γ-aminobutyric acid (GABA) further characterized M2 stained profiles. M2 receptors were predominately found on small caliber (presumably distal) dendritic arbors of thalamocortical cells and interneurons in the lateral LP nucleus. While glomeruli were not abundant in the lateral LP nucleus, occasionally they contained dendritic terminals of interneurons (F2 profiles) stained for M2 receptors. Some GABAergic terminals throughout the neuropil also stained for M2 receptors. The location of M2 receptors correlates well with the cholinergic innervation of the lateral LP nucleus and suggests that muscarinic modulation of visual signals differs in the lateral LP nucleus as compared with the lateral geniculate and pulvinar nuclei.

K+Channel Expression Distinguishes Subpopulations of Parvalbumin- and Somatostatin-Containing Neocortical Interneurons

Kv3.1 and Kv3.2 K(+) channel proteins form similar voltage-gated K(+) channels with unusual properties, including fast activation at voltages positive to -10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3. 2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1-Kv3.2 current type can have functions other than facilitating high-frequency firing.

Metabotropic glutamate receptors in superficial laminae of the rat dorsal horn

Light and electron microscopic immunocytochemistry were employed here to show the distribution of metabotropic glutamate receptors (mGluRs) mGluR2/3 and mGluR5 in laminae I and II of the dorsal horn, to identify their pre- and postsynaptic location, and to test colocalization with gamma-aminobutyric acid (GABA). mGluR2/3 was mainly in the inner part of lamina II; mGluR5 was mainly in laminae I and II. Electron microscopy showed that both mGluR2/3 and mGluR5 were in perikarya, dendrites, and vesicle-containing profiles. Two main morphological types of primary afferent terminals can be distinguished in the superficial laminae: C1, likely to be endings of unmyelinated fibers, and C2, of small myelinated fibers. Quantitative data show that only a small fraction of C2s stained for either receptor; more common were immunopositive dendrites postsynaptic to these terminals, and most common were appositions between C2s and mGluR5 immunopositive dendrites. Vesicle-containing profiles were characteristically apposed to primary afferent terminals, mainly C2s. Immunopositivity for mGluRs, especially mGluR2/3, was present in vesicle-containing profiles apposed to C2, none to C1, and about half of the profiles immunostained for either receptor were also stained for GABA. The presence of presynaptic and postsynaptic mGluRs in both inhibitory and excitatory interneurons may contribute to complex processing of fast and slow responses to peripheral input in superficial laminae. As selective agonists of mGluRs may modulate GABA release, the present demonstration of mGluRs in GABAergic terminals of presumed interneurons suggests that facilitatory effects may involve a mechanism of disinhibition.

A Critical Role of the Nitric Oxide/cGMP Pathway in Corticostriatal Long-Term Depression

High-frequency stimulation (HFS) of corticostriatal glutamatergic fibers induces long-term depression (LTD) of excitatory synaptic potentials recorded from striatal spiny neurons. This form of LTD can be mimicked by zaprinast, a selective inhibitor of cGMP phosphodiesterases (PDEs). Biochemical analysis shows that most of the striatal cGMP PDE activity is calmodulin-dependent and inhibited by zaprinast. The zaprinast-induced LTD occludes further depression by tetanic stimulation and vice versa. Both forms of synaptic plasticity are blocked by intracellular 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, indicating that an increased cGMP production in the spiny neuron is a key step. Accordingly, intracellular cGMP, activating protein kinase G (PKG), also induces LTD. Nitric oxide synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and 7-nitroindazole monosodium salt (7-NINA) block LTD induced by either HFS or zaprinast, but not that induced by cGMP. LTD is also induced by the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine. SNAP-induced LTD occludes further depression by HFS or zaprinast, and it is blocked by intracellular ODQ but not by L-NAME. Intracellular application of PKG inhibitors blocks LTD induced by HFS, zaprinast, and SNAP. Electron microscopy immunocytochemistry shows the presence of NOS-positive terminals of striatal interneurons forming synaptic contacts with dendrites of spiny neurons. These findings represent the first demonstration that the NO/cGMP pathway exerts a feed-forward control on the corticostriatal synaptic plasticity.

Glutamate locally activates dendritic outputs of thalamic interneurons.

The relay of information through thalamus to cortex is dynamically gated, as illustrated by the retinogeniculocortical pathway. Important to this is the inhibitory interneuron in the lateral geniculate nucleus (LGN). For the typical neuron, synaptic information arrives through postsynaptic dendrites and is transmitted by axon terminals. However, the typical thalamic interneuron, in addition to conventional axonal outputs, has distal dendrites that serve both pre- and postsynaptic roles. These dendritic terminals participate in curious and enigmatic triadic arrangements, in which each contacts a relay cell dendrite and is contacted by a glutamatergic retinal terminal that innervates the same relay cell dendrite. Here we show that agonists of the metabotropic glutamate receptor (mGluR) activate dendritic terminals of interneurons in the absence of action potentials, thereby inhibiting the postsynaptic relay neuron. Somatic recordings from LGN interneurons reveal that there is no response to mGluR agonists, suggesting that their dendritic terminals are electrically isolated from their somata and axons, consistent with anatomical modelling of these cells. Our results offer insight into the functioning of triadic circuitry and indicate that thalamic interneurons can perform independent computations expressed through axonal as opposed to dendritic outputs.

Corticotropin-releasing hormone (CRH)-containing neurons in the immature rat hippocampal formation: Light and electron microscopic features and colocalization with glutamate decarboxylase and parvalbumin

Corticotropin-releasing hormone (CRH) excites hippocampal neurons and induces death of selected CA3 pyramidal cells in immature rats. These actions of CRH require activation of specific receptors that are abundant in CA3 during early postnatal development. Given the dramatic effects of CRH on hippocampal neurons and the absence of CRH-containing afferents to this region, we hypothesized that a significant population of CRHergic neurons exists in developing rat hippocampus. This study defined and characterized hippocampal CRH-containing cells by using immunocytochemistry, ultrastructural examination, and colocalization with gamma-aminobutyric acid (GABA)-synthesizing enzyme and calcium-binding proteins. Numerous, large CRH-immunoreactive (ir) neurons were demonstrated in CA3 strata pyramidale and oriens, fewer were observed in the corresponding layers of CA1, and smaller CRH-ir cells were found in stratum lacunosum-moleculare of Ammon's horn. In the dentate gyrus, CRH-ir somata resided in the granule cell layer and hilus. Ultrastructurally, CRH-ir neurons had aspiny dendrites and were postsynaptic to both asymmetric and symmetric synapses. CRH-ir axon terminals formed axosomatic and axodendritic symmetric synapses with pyramidal and granule cells. Other CRH-ir terminals synapsed on axon initial segments of principal neurons. Most CRH-ir neurons were coimmunolabeled for glutamate decarboxylase (GAD)-65 and GAD-67 and the majority also contained parvalbumin, but none were labeled for calbindin. These results confirm the identity of hippocampal CRH-ir cells as GABAergic interneurons. Further, a subpopulation of neurons immunoreactive for both CRH and parvalbumin and located within and adjacent to the principal cell layers consists of basket and chandelier cells. Thus, axon terminals of CRH-ir interneurons are strategically positioned to influence the excitability of the principal hippocampal neurons via release of both CRH and GABA.

Endogenous GABA activates small-conductance K+ channels underlying slow IPSCs in rat hippocampal neurons.

The objective of this study was to determine the properties of K+ channels activated by endogenously released trasmitter under synaptic conditions. First, the levels of gamma-aminobutyric acid (GABA) were depleted in hippocampal nerve endings to establish the relative contribution of endogenously released GABA to the activation of GABA(B) receptors mediating slow inhibitory postsynaptic currents (IPSCs). Inhibition of glutamic acid decarboxylase and GABA reuptake effectively depleted >85% of the releasable GABA pool, producing parallel reductions of GABA(A) and GABA(B) receptor-mediated IPSCs, indicating that both classes of receptors are activated synaptically by endogenously released GABA. Whole cell patch-clamp recordings of stimulus-evoked slow IPSCs at potentials hyperpolarized from the potassium reversal potential were consistent with the activation of a nonrectifying (n = 3) or slightly outwardly rectifying (n = 4) K+ conductance by the endogenously released GABA. Spectral analysis of the decay phase of GABA(B) IPSCs revealed several time constants indicating complex underlying channel kinetics. Nonstationary variance analysis yielded a small unitary conductance in the range of 5-13 pS, consistent with a large number of channels activated during evoked currents. These results indicate that in granule cells of the dentate gyrus, GABA released synaptically from interneuron terminals activates an unusually small K+ conductance, with no or slight outward rectification. This conductance is therefore unlike those typically reported for neuronal G protein-coupled K+ channels or those activated by exogenously applied baclofen with larger, inwardly rectifying conductances.

Evoked Excitability Changes at the Terminals of Midlumbar Premotor Interneurons in the Cat Spinal Cord

We present evidence that the electrical excitability of the terminals of a group of spinal premotor interneurons can be increased after stimulation of sensory afferents. The interneurons were located in the midlumbar segments of the spinal cord and had projections to the lower lumbar motor nuclei. Thresholds for antidromic activation of a substantial number of interneurons were reduced after electrical stimulation of group II muscle afferents. Several observations suggest that the excitability changes are unlikely to have arisen from electrotonic spread of depolarization from the interneuron soma to its terminals or by environmental changes in the vicinity of the terminals related to neuronal activity. A particularly interesting possibility is that the excitability of the central terminals of the interneurons is increased because they are depolarized by a mechanism similar to that acting at the terminals of primary sensory afferents (primary afferent depolarization, PAD), which accompanies one type of presynaptic inhibition. This type of presynaptic action has been shown in premotor interneurons in the lamprey but not in the mammalian spinal cord. From our observations the organization of the systems generating excitability changes at the interneuron terminals seem in general to parallel the organization of the systems generating PAD at afferent terminals, raising the possibility that common principles might underlie the operation of this form of presynaptic control.

High-threshold Ca2+ currents in rat hippocampal interneurones and their selective inhibition by activation of GABA(B) receptors.

1. Whole-cell calcium currents were recorded from visually identified inhibitory interneurones located in stratum radiatum (near the border with stratum lacunosum-moleculare) of area. CA1 in rat hippocampal slices. Current-voltage (I-V) relationships in relatively well-clamped neurones showed that inward current activated between -50 and -40 mV (holding potential, -80 mV) and was maximal near -10 mV. Currents showed little inactivation over the course of 85 ms steps, and were completely blocked by removal of Ca2+ or addition of Cd2+. Prominent low-threshold currents were not observed under these conditions. 2. The calcium channels contributing to whole-cell currents in interneurones were examined using selective channel antagonists. The selective N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTX-GVIA; 10 microM) irreversibly blocked 23.2 +/- 2.8% of whole-cell currents. The P/Q-type antagonist omega-agatoxin IVA (omega-Aga-IVA; 1-5 microM) blocked 10.4 +/- 3.3% of whole-cell currents. Block by omega-Aga-IVA was highly variable, ranging from 0 to 30%. The less selective conotoxin, omega-conotoxin MVIIC (omega-CTX-MVIIC; 5 microM) blocked 31.0 +/- 2.7% of whole-cell currents. The selective L-type channel antagonist nifedipine (20 microM) blocked 27.5 +/- 3.5% of whole-cell currents. 3. Whole-cell calcium currents were reversibly inhibited by the selective GABA(B) receptor agonists (+/-)-baclofen or CGP 27492 (1-3 microM; 18.9 +/- 1.4%). This inhibition was reversed or prevented by the selective GABAB receptor antagonist CGP 55845A (1 microM). Inhibition of inward current activated by voltage ramps was voltage dependent, being greatest near -10 mV, and less pronounced at more positive or negative potentials. Inhibition of calcium currents by GABAB receptor agonists was accompanied by an apparent change in the kinetics of whole-cell currents consistent with a slowing of the rate of activation. CGP 27492 depressed calcium currents by 16.1 +/- 1.9% before application of omega-CgTX-GVIA, and by 3.9 +/- 2.0% after application of omega-CgTX-GVIA in the same cells (P < 0.005), consistent with preferential block of N-type calcium channels. 4. Neither adenosine (200 microM) nor the selective mu-opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO; 2 microM) inhibited calcium currents. Similarly, CGP 27492, but not adenosine or DAMGO, induced an outward current (at - 70 mV) consistent with activation of inwardly rectifying potassium channels. 5. These results indicate that hippocampal inhibitory neurones located in stratum radiatum possess multiple calcium channel subtypes, including N-type, L-type, and at least two other types of high-threshold channels. Activation of GABAB receptors (but not adenosine or mu-opioid receptors) preferentially inhibits N-type channels in these neurones. Similar inhibition occurring in the terminals of interneurones could contribute to depression of inhibitory synaptic transmission by activation of GABAB autoreceptors.

A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5'-nucleotidase.

No attempt has been made so far to classify the subtypes of presynaptic inhibitory adenosine receptors located in the myenteric plexus and to localize ecto-ATPase and 5'-nucleotidase in the intestine. The release of [3H]acetylcholine and smooth muscle responses to acetylcholine were measured and the effect of selective adenosine receptor ligands was studied using field-stimulated isolated longitudinal muscle strips of guinea-pig ileum. Release of ATP and its hydrolysis rate were also measured using the luciferin-luciferase technique. A histochemical method combined with electron microscopy was used for localization of ecto-ATPase and 5'-nucleotidase, enzymes responsible for destruction of extracellular ATP, ADP and AMP. Subtype-selective A1-receptor agonists and antagonists inhibited and enhanced, respectively, the release of acetylcholine associated with neuronal activity. A significant amount of ATP was released in response to electrical stimulation and administration of carbamylcholine. The release of ATP was inhibited by atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3-receptor antagonist. Hydrolysis of ATP was rapid and resulted in an accumulation of extracellular adenosine involved in presynaptic A1-receptor-mediated inhibition of acetylcholine release. While the inhibitory effect of adenosine and ATP was significantly potentiated by dipyridamol, an adenosine uptake blocker, that of 2-ms ATP was not. The effect of ATP was not competitively antagonized by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1-receptor antagonist. In conclusion, axon terminals of cholinergic interneurons are equipped with inhibitory A1- and P2 gamma-receptors. Therefore, both adenosine and ATP control the release of acetylcholine through these receptors. ATP is mainly released from the smooth muscle in response to stimulation of M3-muscarinic receptors by endogenous acetylcholine (cascade transmission [Vizi E. S. et al. (1992) Neuroscience 50, 455-465]) and is rapidly hydrolysed by ecto-ATPase localized on the surface of the smooth muscle and axon terminals producing ADP and AMP, and by 5'-nucleotidase present only on the surface of smooth muscle cells producing adenosine.

Paradoxical enhancement by bicuculline of dentate granule cell IPSPs evoked by fimbria stimulation in rat hippocampal slices

Stimulation of the fimbria in rat hippocampal slices evoked an extracellular negativity in the granule cell layer and a small depolarization in granule cells at their resting potentials. The intracellular potentials appeared to be GABA A receptor-mediated IPSPs because they reversed at −69.1 ± 1.0 mV (mean ± S.E.M., n = 14) and were blocked by the GABA A receptor antagonist bicuculline (10–50 μM, n = 14). However, during the first few minutes of perfusion with bicuculline, IPSPs transiently and paradoxically increased in amplitude. As IPSPs increased, the reversal potential and latency to onset remained the same. These effects were reversible, and during the wash period IPSPs first increased and then stabilized at a smaller amplitude, similar to IPSPs evoked in control conditions. As the GABA A receptor-mediated IPSP decreased, it was followed by a second hyperpolarization. This late hyperpolarization appeared to be a GABA B receptor-mediated IPSP, because it reversed near the equilibrium potential for potassium (mean −81.8 ± 2.3 mV, n = 12, [K +] o = 5 mM) and was blocked by the GABA B receptor antagonist 2-hydroxy saclofen (250–500 μM, n = 5). The results suggest that GABA A and GABA B receptor-mediated IPSPs evoked in granule cells by fimbria stimulation are normally inhibited by activation of GABA A receptors. The inhibition by GABA A receptors is strong enough that, in control conditions, the GABA A IPSPs are barely detectable and the GABA B IPSPs are undetectable. The relevant GABA A receptors could be located presynaptically, on the nerve terminals of inhibitory interneurons that innervate granule cells, or on the dendrites and somata of the interneurons, where they may be affected by GABAergic inputs activated by fimbria stimulation. These data demonstrate the strength and complexity of pathways utilizing GABA A receptors and GABA B receptors to inhibit dentate granule cells.

Combined intracellular injection of Neurobiotin and pre-embedding immunocytochemistry using silver-intensified gold probes in myenteric neurons

We have developed methods to examine the neurochemistry of synaptic inputs to individual myenteric neurons labeled by dye injection through intracellular recording electrodes. Myenteric neurons of the guinea-pig ileum were filled with Neurobiotin, fixed, washed in 50% ethanol, exposed to sodium cyanoborohydride, incubated in avidin-biotin-horseradish peroxidase and incubated in antisera to calretinin or calbindin. The Neurobiotin-filled cells were revealed using the diaminobenzidine (DAB) reaction. The tissue was examined at the light microscope level to determine the morphology and projections of the Neurobiotin-filled neurons, and then incubated in 1 nm gold-labeled secondary antibodies. Following osmication, the gold probes were silver-intensified and the tissue embedded flat in resin. The tissue was re-examined at the light microscope level. Neurons containing a DAB reaction product could be distinguished from neurons containing a silver-intensified gold reaction product using oblique or epipolarized illumination. Ultrathin sections were taken through the injected neurons and examined. At the ultrastructural level, Neurobiotin-filled cell bodies and their processes (labeled with DAB) were easily distinguished from the structures labeled by silver-intensified gold. Gold-labeled terminals of enteric interneurons made synapses and close contacts with Neurobiotin-filled nerve cell bodies and their processes. This technique is valuable for the neurochemical identification of synaptic inputs to morphologically and/or functionally characterized myenteric neurons and could be easily applied to other preparations, such as brain slices.