Application of a flexible polymer microECoG array to map functional coherence in schizophrenia model.

Anatomically, connections form the fundamental brain network, functionally the different types of oscillatory electric activities are creating a temporarily connected fraction of the anatomical connectome generating an output to the motor system. Schizophrenia can be considered as a connectome disease, in which the sensory input generates a schizophrenia specific temporary connectome and the signal processing becomes diseased showing hallucinations and adverse behavioral reactions. In this work, flexible, 32-channel polymer microelectrode arrays fabricated by the authors are used to map the functional coherence on large cortical areas during physiological activities in a schizophrenia model in rats.-Fabrication of a flexible microECoG array is shown.-Protocol to use a flexible microECoG is demonstrated to characterize connectome diseases in rats.-Customized method to analyze the functional coherence between different cortical areas during visually evoked potential is detailed.-R-based implementation of the analysis method is presented.

Electrophysiological and behavioral properties of 4-aminopyridine-induced epileptic activity in mice.

4-aminopyridine (4-AP) is a widely used drug that induces seizure activity in rodents, especially in rats, although there is no consensus in the literature on the dose to be used in mice. The aim of the present study was to investigate the effect of the intraperitoneal administration of 4-AP in two doses (4 and 10 mg/kg) in vivo. EEG, movement, and video recordings were made simultaneously in male B6 mice to specify the details of the seizures and to determine whether there is a suitable non-lethal dose for seizure induction and for further molecular studies. Seizure behavior in mice differs from that seen in rats, with no characteristic stages of epileptic seizures, but with spiking and seizure activity. Seizure activity, although produced at both doses without being lethal, induced different changes of the EEG pattern. Smaller dose induced a lower amplitude seizure activity, decreased spiking activity and later onset of seizures, while higher dose induced a much more intense brain seizure activity and severe trembling. It is concluded that the intraperitoneal administration of 4-AP at a dose of 10 mg/kg induces explicit seizure activity in mice which is repeatable and can be suitable for further molecular research.

Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus. We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the axon loses parvalbumin or immunoreactivity for parvalbumin. The hyperinnervation of axon initial segments might be a compensatory change in the inhibitory network, but at the same time may lead to a more effective synchronization of granule cell firing that could contribute to the generation or amplification of epileptic seizures.

Intracellular features predicted by extracellular recordings in the hippocampus in vivo.

Multichannel tetrode array recording in awake behaving animals provides a powerful method to record the activity of large numbers of neurons. The power of this method could be extended if further information concerning the intracellular state of the neurons could be extracted from the extracellularly recorded signals. Toward this end, we have simultaneously recorded intracellular and extracellular signals from hippocampal CA1 pyramidal cells and interneurons in the anesthetized rat. We found that several intracellular parameters can be deduced from extracellular spike waveforms. The width of the intracellular action potential is defined precisely by distinct points on the extracellular spike. Amplitude changes of the intracellular action potential are reflected by changes in the amplitude of the initial negative phase of the extracellular spike, and these amplitude changes are dependent on the state of the network. In addition, intracellular recordings from dendrites with simultaneous extracellular recordings from the soma indicate that, on average, action potentials are initiated in the perisomatic region and propagate to the dendrites at 1.68 m/s. Finally we determined that a tetrode in hippocampal area CA1 theoretically should be able to record electrical signals from approximately 1, 000 neurons. Of these, 60-100 neurons should generate spikes of sufficient amplitude to be detectable from the noise and to allow for their separation using current spatial clustering methods. This theoretical maximum is in contrast to the approximately six units that are usually detected per tetrode. From this, we conclude that a large percentage of hippocampal CA1 pyramidal cells are silent in any given behavioral condition.

Changes in the distribution and connectivity of interneurons in the epileptic human dentate gyrus.

The distribution, size, dendritic morphology and synaptic connections of calbindin-, calretinin- and substance P receptor-positive interneurons and pathways have been examined in control and epileptic human dentate gyrus. In the epileptic dentate gyrus, calbindin-containing interneurons are preserved, but their dendrites become elongated and spiny, and several cell bodies appear hypertrophic. The relative laminar distribution of calretinin-containing cells did not change, but their number was considerably reduced. The calretinin-positive axonal bundle at the top of the granule cell layer originating from the supramammillary nucleus expanded, forming a dense network in the entire width of the stratum moleculare. Substance P receptor-immunopositive cells were partially lost in epileptic samples, and in addition, the laminar distribution and dendritic morphology of the surviving cells differed considerably from the controls. In the control human dentate gyrus, the majority of substance P receptor-positive cells can be seen in the hilus, while most are present in the stratum moleculare in the epileptic tissue. Their synaptic input is also changed. The extent of individual pathological abnormalities correlates with each other in most cases. Our data suggest, that although a large proportion of inhibitory interneurons are preserved in the epileptic human dentate gyrus, their distribution, morphology and synaptic connections differ from controls. These functional alterations of inhibitory circuits in the dentate gyrus are likely to be compensatory changes with a role to balance the enhanced excitatory input in the region.

Dual projection from the medial septum to the supramammillary nucleus in the rat.

The supramammillary nucleus, collecting information about the physiological state of the animal, innervates medial septal neurons that are involved in the generation of hippocampal theta activity. Here we demonstrate that septal neurons located in an area bordering the medial and lateral septal nucleus project back to the supramammillary nucleus, and most of these cells contain calretinin, calbindin or both. GABA-immunoreactive boutons of these neurons (60%) form symmetrical synapses, whereas the remaining GABA-negative terminals form asymmetrical synapses (40%) with their supramammillary targets. We hypothesize that the septosupramammillary feedback, because of the specific location of its parent cells, carries information about the activity of theta generator cells in the medial septum and supramammillary nucleus, as well as about the resulting theta activity in the hippocampus.

Dual (excitatory and inhibitory) calretinin innervation of AMPA receptor-containing neurons in the rat lateral septum.

A recent study demonstrated both an extrinsic and an intrinsic calretinin (CR) innervation of the rat septal complex and that a population of the extrinsic calretinin fibers is aspartate/glutamate-containing. The aim of this study was to determine which types (GluR1, GluR2/3, or both) of AMPA receptor-containing lateral septal area neurons are innervated by extrinsic and intrinsic CR neurons and whether the intrinsic CR cells are GABAergic. Light- and electron-microscopic single immunostaining for CR, GluR1, and GluR2/3, as well as light- and electron-microscopic-double immunostaining experiments for CR plus GluR1 and CR plus GluR2/3 were performed in the lateral septal area. Furthermore, the "mirror" colocalization technique was employed on consecutive vibratome sections of the septal complex to investigate whether the intrinsic septal CR neurons are GABAergic. The results are summarized as follows: (1) both GluR1- and GluR2/3-immunoreactive neurons are innervated by CR-containing fibers; (2) the majority of these synapses, observed mainly on the soma and, to a lesser extent, on proximal dendrites of AMPA receptor-containing neurons, represent asymmetric synaptic membrane specializations; (3) a minority of CR-containing axon terminals associated with both GluR1- and GluR2/3-immunoreactive neurons form symmetric contacts, predominantly on their soma; and (4) 93% of the lateral septal area CR cells are GABAergic. These observations indicate that both GluR1- and GluR2/3-containing lateral septal area neurons receive a dual intrinsic and extrinsic CR innervation. The former (intrinsic) CR boutons are GABAergic, while the latter form asymmetric synaptic contacts, are excitatory, and probably originate in the supramammillary area, since previous work has demonstrated that a population of supramammillo-septal fibers contain aspartate and/or glutamate.

The supramammillary nucleus innervates cholinergic and GABAergic neurons in the medial septum-diagonal band of Broca complex.

In the present study, the connectivity between two subcortical nuclei involved in hippocampal theta activity, the supramammillary nucleus and the medial septum-diagonal band of Broca complex, was examined. Targets of the supramammillary afferents in the medial septum-diagonal band of Broca complex were identified by combining anterograde transport of Phaseolus vulgaris leucoagglutinin with immunostaining for putative postsynaptic neurons, i.e. for parvalbumin and choline acetyltransferase that are known to label the GABAergic and cholinergic neurons, respectively, of the medial septum-diagonal band of Broca complex. Double retrograde transport experiments using the tracers horseradish peroxidase and wheat germ agglutinin-conjugated colloidal gold were employed to identify supramammillary neurons that project both to the hippocampus and the medial septum-diagonal band of Broca complex. Phaseolus vulgaris leucoagglutinin injections into the supramammillary nucleus of the rat resulted in dense fibre and terminal labelling in the medial septum-diagonal band of Broca complex. Labelled terminals formed asymmetric synapses mainly on distal dendrites of medial septal neurons. Proximal dendrites and somata were rarely contacted. The supramammillary afferents showed no target selectivity for a particular cell type; they innervated both cholinergic and GABAergic cells. Occasionally, perisomatic, basket-like terminals of supramammillary origin were found around parvalbumin-containing neurons. Double-retrograde experiments revealed that at least 25% of the supramammillo-hippocampal cells also projected to the medial septum-diagonal band of Broca. These data suggest that the nucleus, known to modulate the hippocampal electrical activity directly by the supramammillo-hippocampal pathway, also has the potential for an indirect action via the innervation of both the GABAergic and cholinergic septohippocampal neurons. This dual modulation may originate, at least in part, from the same population of supramammillary neurons.

Substance P innervation of the rat hippocampal formation.

Light and electron microscopic substance P (SP) immunostaining was performed on hippocampal sections of colchicine-pretreated, control, untreated fimbria-fornix-transected (5 days), as well as perforant path-stimulated Sprague-Dawley rats fixed in 5% acrolein. Numerous SP-immunoreactive neurons could be observed in the stratum oriens of the Ammon's horn and subiculum, fewer were seen in the dentate hilar area and stratum radiatum of CA2 and CA3, and even fewer were seen at the border between the CA1 strata radiatum and the lacunosum moleculare of CA1 subfield. A higher dose of colchicine resulted in SP immunoreactivity in a large population of granule cells and mossy axon terminals. The entire CA2 region, the stratum oriens of CA1, CA3, and the subiculum were densely innervated by SP-containing axon terminals. A homogeneous SP innervation was found in the stratum radiatum of CA1. Only a few SP fibers were seen adjacent to the granule cell layer. Symmetric axosomatic contacts were seen between SP-containing boutons and somata in the stratum oriens of the Ammon's horn. However, throughout the hippocampal formation, the majority of SP-containing axons formed axodendritic symmetric synapses. A dense population of SP-immunoreactive boutons that formed axodendritic asymmetric synapses was observed in the strata oriens and radiatum of the CA3a and CA2 regions, and a few were found in the supragranular and subgranular layers of the dentate gyrus. Fimbria-fornix transection resulted in a marked loss of SP fibers in the strata oriens, pyramidale, and radiatum of the CA3a and CA2 subfields. In perforant pathway-stimulated animals, a population of granule cells and a large number of mossy axon terminals were immunoreactive for SP. These observations suggest two sources of SP innervation to the hippocampal formation: one arising from intrinsic sources (interneurons and granule cells) and one arising from extrinsic sources, most likely the supramammillary region.

Distinct substance P- and calretinin-containing projections from the supramammillary area to the hippocampus in rats; a species difference between rats and monkeys.

Our recent studies showed the co-existence of substance P and calretinin in the supramammillo-hippocampal pathway of monkeys, as well as species differences in the synaptic targets of extrinsic substance P fibers in the hippocampi of monkeys and rats. Experiments used: (1) single and multiple stereotaxic injection of wheat germ agglutinin-conjugated HRP into the hippocampus and immunostaining for substance P in the supramammillary area; (2) colocalization of substance P and calretinin in supramammillary area cells; and (3) colocalization of these two neurochemicals in retrogradely labeled supramammillary projective cells of both male and female rats. These demonstrated: (a) many calretinin- and fewer substance P-immunoreactive neurons retrogradely labeled in the ipsilateral supramammillary area; (b) approximately 74% of all substance P cells contain calretinin and 9% of the calretinin neurons co-contain substance P; and, most importantly (c) none of the retrogradely labeled supramammillary cells colocalize calretinin and substance P. These results indicate the presence of two distinct supramammillo-hippocampal projections in the rat, one that contains substance P and the other calretinin. The latter innervates the same areas as those in the monkey, and the former terminates only in the CA2 hippocampal subfield.

Parvalbumin-containing cells of the angular portion of the vertical limb terminate on calbindin-immunoreactive neurons located at the border between the lateral and medial septum of the rat.

In the septal complex, both parvalbumin and calbindin neurons cocontain GABA. In the same area, a large number of GABA-GABA synaptic connections can be observed. In order to further characterize their neurochemical nature, as well as the extrinsic and/or intrinsic origin of these GABA terminals, the following experiments were performed: (1) correlated light- and electron-microscopic double immunostaining for calbindin and parvalbumin on septal sections of control rats: (2) light microscopic parvalbumin immunostaining of septal sections after surgical isolation (5 days) of the septum from its telencephalic or (3) hypothalamic afferents; and (4) parvalbumin immunostaining of sections prepared from the entire brain 2 days following horseradish peroxidase injection into the border between the lateral and medial septum. The results demonstrated that: (1) in a well-circumscribed, vertically longitudinal area located between the lateral and medial septum, 0.1-0.6 mm anterior to the bregma, a group of calbindin-containing, nonsomatospiny neurons are surrounded by parvalbumin-immunoreactive baskets; (2) these basket-forming axon terminals establish symmetric synaptic contacts with their targets; and (3) their cells of origin are not in the medial septum, but in the angular portion of the vertical limb. These observations indicate that a portion of the septal complex GABA-GABA synaptic connections represent functional interaction between two different types of GABAergic neurons. The presynaptic GABAergic neurons contain parvalbumin, and the postsynaptic GABAergic cells are immunoreactive for calbindin. Furthermore, a population of the medial septum/diagonal band parvalbumin neurons project only to the hippocampus, while others, which may also send axons to the hippocampus, terminate on lateral septum calbindin cells as well.

Postsynaptic targets of GABAergic hippocampal neurons in the medial septum-diagonal band of broca complex.

The termination pattern of hippocamposeptal nonpyramidal cells was investigated by injecting Phaseolus vulgaris leucoagglutinin (PHAL) into stratum oriens of the CA1 region. Electron microscopic analysis showed that the majority of the anterogradely labeled boutons formed symmetric synapses with dendrites and occasionally with cell bodies located in the medial septum-diagonal band of Broca complex. We have demonstrated with postembedding GABA immunocytochemistry that the majority of PHAL-labeled axon terminals were GABAergic. The neurochemical character of the postsynaptic target cells was also investigated using immunocytochemical double staining. Our data showed that the majority of the labeled hippocamposeptal axons innervated parvalbumin-immunoreactive cells representing GABAergic projection neurons, and a smaller number of contacts were found on ChAT-positive neurons. Septohippocampal neurons identified by retrograde HRP transport were also shown to reactive direct hippocamposeptal input. According to recent results, the lateral septum is unlikely to relay the hippocampal feedback to the medial septum; therefore, the direct hippocampal projection to the medial septum, arising from GABAergic nonpyramidal cells, seems to be the only feedback pathway to the area containing septohippocampal neurons. A novel circuit diagram, based on our recent morphological-immunocytochemical findings, is shown for the synaptic organization of the septo-hippocampo-septal loop. We suggest that the GABAergic hippocamposeptal feedback controls the activity of septal (mostly GABAergic) projection neurons as a function of hippocampal synchrony. The newly discovered reciprocal interactions may give a better insight into septohippocampal physiology.