GABAergic input of cholecystokinin-immunoreactive neurons in the hilar region of the rat hippocampus. An electron microscopic double immunostaining study

Double immunostaining was performed for electron microscopy to analyze the synaptic connections between glutamate decarboxylase (GAD)-immunoreactive axons and cholecystokinin (CCK)-immunoreactive neurons in the hilar region of the rat hippocampal formation. Following immunostaining for CCK, the diaminobenzidine (DAB) reaction product was silver-intensified and gold-substituted. In a subsequent second immunostaining for GAD, the immunoreactive elements were labeled using a single DAB reaction. Electron microscopic analysis of the double-stained Vibratome sections demonstrated that the single DAB-labeled GAD-immunoreactive boutons form symmetrical synaptic connections on the soma and primary dendrites of the DAB-gold-labeled CCK-immunoreactive neurons.     

The use of gold-substituted silver-intensified diaminobenzidine (DAB) and non-intensified DAB for simultaneous electron microscopic immunoperoxidase labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area

An improved gold-substituted silver intensification procedure for the peroxidase-diaminobenzidine (DAB) reaction product was developed. The method was applied in the rat medial preoptic area to label tyrosine hydroxylase (TH)-immunoreactive profiles. Following the gold toning, the same sections were immunostained for glutamic acid decarboxylase (GAD) immunoreactivity with non-intensified peroxidase-DAB. Single DAB-labeled GAD axons were found in symmetric synaptic connection with unlabeled dendrites as well as with gold-toned immunoperoxidase-containing TH neurons.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus. Dual electron microscopic immunolabeling

Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diaminobenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Catecholaminergic innervation of pyramidal and GABAergic nonpyramidal neurons in the rat hippocampus. Double label immunostaining with antibodies against tyrosine hydroxylase and glutamate decarboxylase

This study describes the catecholaminergic innervation of rat hippocampal neurons at the electron microscopic level by using an antibody against tyrosine hydroxylase (TH) and immunocytochemical techniques. In a first series of experiments, the course and distribution as well as the synaptic contacts of TH-immunoreactive fibers were analyzed with the peroxidase-antiperoxidase (PAP) method. Next, peroxidase immunostaining of TH fibers was combined with glutamate decarboxylase (GAD) immunostaining, using avidinated ferritin as a second electrondense marker. Our results demonstrate that TH-immunostained terminals establish asymmetric synaptic contacts with spines of pyramidal neurons, and symmetric synaptic contacts with cell bodies and dendritic shafts of ferritin-labeled GAD-immunoreactive nonpyramidal cells.     

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.     

Catecholaminergic innervation of pyramidal and GABAergic nonpyramidal neurons in the rat hippocampus

Summary This study describes the catecholaminergic innervation of rat hippocampal neurons at the electron microscopic level by using an antibody against tyrosine hydroxylase (TH) and immunocytochemical techniques. In a first series of experiments, the course and distribution as well as the synaptic contacts of TH-immunoreactive fibers were analyzed with the peroxidase-antiperoxidase (PAP) method. Next, peroxidase immunostaining of TH fibers was combined with glutamate decarboxylase (GAD) immunostaining, using avidinated ferritin as a second electrondense marker. Our results demonstrate that TH-immunostained terminals establish asymmetric synaptic contacts with spines of pyramidal neurons, and symmetric synaptic contacts with cell bodies and dendritic shafts of ferritin-labeled GAD-immunoreactive nonpyramidal cells.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Detection of elastin by immunoelectronmicroscopy

Summary By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptie membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.     

Immunocytochemical identification of GABAergic neurons in the main olfactory bulb of the rat

With the aid of a sheep antiserum against rat brain glutamate decarboxylase (GAD), the endogenous marker for GABAergic neurons, we have labeled immunocytochemically various types of nerve cells in the main olfactory bulb of rats, with and without topic injections of colchicine. The peroxidase-antiperoxidase procedure was applied to floating Vibratome and frozen sections. A large part of the periglomerular cell population and practically all granule cells in the deep layers contain GAD-like immunoreactivity in untreated rats, while tufted and mitral cells (the projection neurons) are unstained. This observation confirms a previous study with a rabbit antiserum against mouse brain GAD, which suggested that GABAergic neurons with presynaptic dendrites contain high somatal concentrations of GAD. We show, however, that immunostaining of granule cell bodies decreases progressively from the internal plexiform layer to the deep portion of the granule cell layer. Many cell processes in the glomeruli are densely stained. They presumably represent synaptic gemmules of the numerous GAD-positive periglomerular cells, which thus could provide initial, inhibitory modulation of the afferent input. In the external plexiform layer immunostaining of the neuropil is substantially denser in the superficial half than in the deep half. This may reflect a corresponding gradient of inhibition related to unequal frequency of occurrence of synaptic gemmules of granule cell dendrites. Alternatively such a graded immunostaining of cell processes could be related to the corresponding gradient in the density of immunostaining of granule cell bodies in the deep layers, in accordance with recent data indicating that superficial and deep granule cells project their ascending dendrites respectively to superficial and deep portions of the external plexiform layer. Furthermore, we have demonstrated the presence of additional classes of GAD-positive neurons, microneurons in the external plexiform layer, small neurons in the periglomerular region, the external plexiform layer, the mitral cell layer, the internal plexiform layer, and medium-size neurons in the granule layer and the white matter. The small- and medium-size GAD-positive neurons appear weakly immunoreactive in untreated rats, but become densely stained after topic colchicine injection. Such cells presumably lack presynaptic dendrites and may correspond to different types of short axon cells demonstrated by the Golgi method.(ABSTRACT TRUNCATED AT 400 WORDS)     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.     

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.     

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.     

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity and its protein levels in the gerbil hippocampus during normal aging

Phosphorylated extracellular signal-regulated kinase (pERK) mediates neuronal synaptic plasticity, long-term potentiation, and learning and memory in the hippocampus. In this study, we examined pERK1/2 immunoreactivity and its protein level in the gerbil hippocampus at various ages. In the postnatal month 1 (PM 1) group, very weak pERK1/2 immunoreactivity was detected in the hippocampus. In the CA1 region, pERK1/2 immunoreactivity was considerably increased in the stratum pyramidale in the PM 6 group. Thereafter, pERK1/2 immunoreactivity was decreased. In the CA2/3 region, pERK1/2 immunoreactivity increased in an age-dependent manner until PM 12. Thereafter, numbers of pERK1/2-immunoreactive neurons were decreased. However, in the mossy fiber zone, pERK1/2 immunostaining became stronger with age. In the dentate gyrus, a few pERK1/2-immunoreactive cells were observed until PM 12. In the PM 18 and 24 groups, numbers of pERK1/2-immunoreactive cells were increased, especially in the polymorphic layer. In Western blot analysis, pERK1/2 level in the gerbil hippocampus was increased with age. These results indicate that total pERK1/2 levels are increased in the hippocampus with age. However pERK1/2 immunoreactivity in subregions of the gerbil hippocampus was changed with different pattern during normal aging.     

Gastrin-cholecystokinin immunoreactivity in the central nervous system of Helix aspersa during rest and activity

The immunostaining pattern for the peptide gastrin/cholecystokinin 8 (gastrin/CCK8) in the molluscan central nervous system has been considered. The changes in the distribution of gastrin/CCK8 immunoreactivity were analyzed in the neurons of different areas of the cerebral ganglia (mesocerebrum and metacerebrum) and in the buccal ganglia of the terrestrial snail Helix aspersa, during rest and active phases. During the period of inactivity and after one day of activity, there were several immunoreactive neurons in the mesocerebrum and metacerebrum of the snails and in the buccal ganglia, whereas after 7 days of activity the number of labeled neurons decreased. Data suggested a storage of gastrin/CCK8 in the neurons when behavioral activities in which the peptide is involved (such as feeding-related behavior) are suppressed or reduced. The different percentage of gastrin/CCK8 immunoreactive neurons in the left and right mesocerebrum provides information about the activities controlled by these neurons, which could be related to the adaptive evolution and plasticity of the brain in terrestrial pulmonates.     

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.     

In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCK8s, 30 pM-300 nM, EC50 approximately 4 nM) induced a concentration-dependent inward current in 35 and 41% of corpus- and antrum/pylorus-projecting DMV neurons, respectively. Conversely, none of the fundus-projecting DMV neurons responded to perfusion with CCK8s. The CCK8s-induced inward current was accompanied by a 65 +/- 17% increase in membrane input resistance and reversed at 90 +/- 4 mV, indicating that the excitatory effects of CCK8s were mediated by the closure of a potassium conductance. Pretreatment with the synaptic blocker TTX (0.3-1 microM) reduced the CCK8s-induced current, suggesting that a portion of the CCK8s-induced current was mediated indirectly via an action on presynaptic neurons apposing the DMV membrane. Pretreatment with the selective CCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCK8s-induced inward current in a concentration-dependent manner, with a maximum inhibition of 69 +/- 12% obtained with 3 microM lorglumide. Conversely, pretreatment with the selective CCK-B antagonist triglumide did not attenuate the CCK8s-induced inward current; pretreatment with triglumide (3 microM) and lorglumide (1 microM) attenuated the CCK8s-induced current to the same extent as pretreatment with lorglumide alone. Immunohistochemical experiments showed that CCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.     

Role of GABAA-Mediated Inhibition and Functional Assortment of Synapses onto Individual Layer 4 Neurons in Regulating Plasticity Expression in Visual Cortex

Layer 4 (L4) of primary visual cortex (V1) is the main recipient of thalamocortical fibers from the dorsal lateral geniculate nucleus (LGN_d). Thus, it is considered the main entry point of visual information into the neocortex and the first anatomical opportunity for intracortical visual processing before information leaves L4 and reaches supra- and infragranular cortical layers. The strength of monosynaptic connections from individual L4 excitatory cells onto adjacent L4 cells (unitary connections) is highly malleable, demonstrating that the initial stage of intracortical synaptic transmission of thalamocortical information can be altered by previous activity. However, the inhibitory network within L4 of V1 may act as an internal gate for induction of excitatory synaptic plasticity, thus providing either high fidelity throughput to supragranular layers or transmittal of a modified signal subject to recent activity-dependent plasticity. To evaluate this possibility, we compared the induction of synaptic plasticity using classical extracellular stimulation protocols that recruit a combination of excitatory and inhibitory synapses with stimulation of a single excitatory neuron onto a L4 cell. In order to induce plasticity, we paired pre- and postsynaptic activity (with the onset of postsynaptic spiking leading the presynaptic activation by 10ms) using extracellular stimulation (ECS) in acute slices of primary visual cortex and comparing the outcomes with our previously published results in which an identical protocol was used to induce synaptic plasticity between individual pre- and postsynaptic L4 excitatory neurons. Our results indicate that pairing of ECS with spiking in a L4 neuron fails to induce plasticity in L4-L4 connections if synaptic inhibition is intact. However, application of a similar pairing protocol under GABA_ARs inhibition by bath application of 2μM bicuculline does induce robust synaptic plasticity, long term potentiation (LTP) or long term depression (LTD), similar to our results with pairing of pre- and postsynaptic activation between individual excitatory L4 neurons in which inhibitory connections are not activated. These results are consistent with the well-established observation that inhibition limits the capacity for induction of plasticity at excitatory synapses and that pre- and postsynaptic activation at a fixed time interval can result in a variable range of plasticity outcomes. However, in the current study by virtue of having two sets of experimental data, we have provided a new insight into these processes. By randomly mixing the assorting of individual L4 neurons according to the frequency distribution of the experimentally determined plasticity outcome distribution based on the calculated convergence of multiple individual L4 neurons onto a single postsynaptic L4 neuron, we were able to compare then actual ECS plasticity outcomes to those predicted by randomly mixing individual pairs of neurons. Interestingly, the observed plasticity profiles with ECS cannot account for the random assortment of plasticity behaviors of synaptic connections between individual cell pairs. These results suggest that connections impinging onto a single postsynaptic cell may be grouped according to plasticity states.