Katalin Halasy

A parvalbumin-containing, axosomatic synaptic network in the rat medial septum: relevance to rhythmogenesis*

The medial septal diagonal band complex (MS/DB), made up of cholinergic and GABAergic neurons, plays an important role in the generation of the hippocampal theta rhythm. A GABAergic neuron type in the MS/DB that has fast spiking properties was shown previously to contain parvalbumin immunoreactivity and to form axosomatic connections with unidentified somata. The aim in the current study was to determine the neurochemical identities of these target neurons. In slices and in perfused‐fixed brain, staining for parvalbumin immunoreactivity first of all revealed the presence of two types of parvalbumin‐positive somata in the MS/DB: medially located neurons with parvalbumin‐positive basket‐like terminals on them, and more laterally located neurons with fewer parvalbumin‐positive contacts on them. In MS/DB slices, the terminals of fast spiking neurons filled with biocytin correspondingly made either numerous contacts that surrounded the parvalbumin‐positive cell body in basket‐like formation, or 1–5 contacts on a localized patch of the soma. These contacts were shown by electron microscopy to form synaptic junctions. No terminals of biocytin‐filled fast spiking neurons were observed on cholinergic neurons, and dual staining in perfused‐fixed brain did not reveal the presence of parvalbumin‐containing terminals on cholinergic somata. Our results suggest therefore that there are two subtypes of parvalbumin‐containing neuron in the MS/DB, and that these are interconnected via axosomatic synapses. The contrasting topographical organization of the two types of parvalbumin‐containing neuron suggests that they may receive different types of afferent input, but this will require substantiation in future studies. We propose that generation of rhythmic activity in the MS/DB is controlled by contrasting contributions from two types of parvalbumin‐positive neuron, and that the role of the cholinergic neuron is modulatory.

Distribution and role of Kv3.1b in neurons in the medial septum diagonal band complex

The medial septum diagonal band complex (MS/DB) projects via cholinergic and GABAergic pathways to the hippocampus and plays a key role in the hippocampal theta rhythm. In the MS/DB we have previously described a population of fast spiking GABAergic neurons that contain parvalbumin and mediate theta frequency activity in vitro. The Kv3.1 potassium channel is a delayed rectifier channel that plays a major role in fast spiking neurons in the CNS, and has previously been localized in the MS/DB. To determine which cell types in the MS/DB express the Kv3.1b ion channel subunit, transgenic mice in which the expression of GABAergic and glutamate markers are associated with the expression of green fluorescent protein (GFP; GAD67-GFP and VGluT2-GFP mice, respectively) were used for immunofluorescence and axonal tract tracing. Electrophysiological studies were also carried out on rat MS/DB slices to examine the role of the Kv3.1 channel in theta frequency oscillations. The results for the MS/DB were as follows: (1) cholinergic cells did not express GFP in either GAD67-GFP or VGluT2-GFP mice, and there was GAD67 immunoreactivity in GFP-positive neurons in GAD67-GFP mice and in a small proportion (6%) of GFP-positive neurons in VGluT2-GFP mice. (2) Kv3.1b immunofluorescence was associated with the somata of GABAergic neurons, especially those that contained parvalbumin, and with a minority of glutamatergic neurons, but not with cholinergic neurons, and with GABAergic axonal terminal-like processes around certain GABAergic neurons. (3) Both Kv3.1b-positive and -negative GABAergic neurons were septo-hippocampal, and there was a minor projection to hippocampus from VGluT2-GFP neurons. (4) Kainate-induced theta oscillations in the MS/DB slice were potentiated rather than inhibited by the Kv3.1 blocker 4-aminopyridine, and this agent on its own produced theta frequency oscillations in MS/DB slices that were reduced by ionotropic glutamate and GABA receptor antagonists and abolished by low extracellular calcium. These studies confirm the presence of heterogeneous populations of septo-hippocampal neurons in the MS/DB, and suggest that presence of Kv3.1 in the GABAergic neurons does not contribute to theta activity through fast spiking properties, but possibly by the regulation of transmitter release from axonal terminals.

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent

The medial septal diagonal band area (MS/DB), made up of GABAergic and cholinergic neurones, plays an essential role in the generation and modulation of the hippocampal theta rhythm. To understand the part that the cholinergic neurones might play in this activity, we sought to determine whether postsynaptic nicotinic receptor responses can be detected in slices of the rodent MS/DB by puffing on acetylcholine (ACh). Neurones were characterized electrophysiologically into GABAergic and cholinergic neurones according to previous criteria. Responses of the MS/SB neurones to ACh were various combinations of fast depolarizations (1.5–2.5 s), fast hyperpolarizations (3–4 s) and slow depolarizations (20–30 s), the latter two being blocked by atropine. The fast depolarizations were partially or not blocked with cadmium and low calcium, tetrodotoxin, and antagonists of other ionotropic receptors, and were antagonized with 25 μm mecamylamine. Pharmacological investigation of the responses showed that the α7* nicotinic receptor type is associated with cholinergic neurones and 10% of the GABAergic neurones, and that nonα7* nicotinic receptor subtypes are associated with 50% of the GABAergic neurones. Pharmacological dissection of evoked and spontaneous postsynaptic responses, however, did not provide evidence for synaptic nicotinic receptor transmission in the MS/DB. It was concluded that nicotinic receptors, although prevalent on the somatic and/or dendritic membrane compartments of neurones in the MS/DB, are on extrasynaptic sites where they presumably play a neuromodulatory role. The presence of α7* nicotinic receptors on cholinergic neurones may also render these cells specifically vulnerable to degeneration in Alzheimers disease.

Kappa opioid receptor is expressed by somatostatin- and neuropeptide Y-containing interneurons in the rat hippocampus

In our previous studies (J. Chem. Neuroanat. 2000;19:233-241), kappa opioid receptors were immunocytochemically identified in inhibitory interneurons of the dentate hilus and CA1 area of the rat hippocampus. From among the known interneuron subtypes, somatostatin- (SOM) and neuropeptide Y- (NPY) immunoreactive (IR) hippocampal interneurons show morphology and distribution similar to the kappa opioid receptor (KOR) immunopositive cells. In the present study, with the help of double immunocytochemical labelling, we provide direct evidence that the majority of the interneurons immunoreactive for SOM and/or NPY also express the kappa opioid receptor. The receptor was localized on the perikaryal and proximal dendritic region of the SOM- and NPY-immunopositive neurons in the dentate hilus and the CA1 region. From among the SOM-immunoreactive cells, 77% in the dentate hilus and 51% in the CA1 stratum oriens was double labelled. In the case of NPY-immunoreactive neurons this proportion was 56 and 65%, respectively. The co-expression of KOR and SOM/NPY suggests that hippocampal interneurons can selectively be activated by the different opioids under different physiological circumstances.

Kappa opioid receptors are expressed by interneurons in the CA1 area of the rat hippocampus: a correlated light and electron microscopic immunocytochemical study

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szûcs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897-1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa(2) subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested

Laminar analysis of initiation and spread of epileptiform discharges in three in vitro models

Overexcitation of neuronal networks in some forebrain structures and pathological synchronization of neuronal activity play crucial role in epileptic seizures. Seizure activity can be elicited experimentally by different convulsants. Because of various distribution of excitatory and inhibitory connections in the neocortex there might be laminar differences in seizure sensitivity. Current source density (CSD) analysis or immunocytochemical c-Fos localization offer suitable tools to localize increased activation of neurons during seizure. In the present experiments, interictal epileptiform activity elicited by 4-aminopiridine, bicuculline or Mg(2+)-free solution was recorded with a 16-channel multielectrode assembly in different layers of the somatosensory cortex, and CSDs were calculated. Parallel c-Fos immunocytochemistry was applied. Each convulsant elicited characteristic activation pattern. 4-aminopiridine induced relatively short discharges, which were associated with a huge sink in layer V, the sink and source pattern was relatively simple. Mg(2+)-free solution elicited the longest discharges, sinks appeared typically in the supragranular layers II and III than quickly distributed toward layers V and VI. Bicuculline induced rather similar seizure pattern as Mg(2+)-free solution, but the amplitudes of field potentials were larger, while the durations shorter. The peak of c-Fos activation, however, was not parallel with the largest electrical activation. Larger amount of stained cells appeared in layers II and III in 4-aminopiridine and bicuculline, respectively. In Mg(2+)-free solution the highest c-Fos activity was detected in upper layer VI. Long-lasting cellular effects do not always correspond to the largest electrical responses, which are primarily determined by the activation of asymmetrical pyramidal neurons. Interneurons, which possess more symmetric process arborisation, play less important role in the generation of field potentials, although they may be intensively activated during seizure.

Chronic fasting-induced changes of neuropeptide Y immunoreactivity in the lateral septum of intact and ovariectomized female rats.

The effect of 40% food deprivation for 1 week on the immunohistochemically detectable amount of neuropeptide Y (NPY) was studied in the lateral septum (LS) of intact and ovariectomized (OVX) female rats. Animals were either fed ad libitum or 40% food-deprived. Densitometric analysis of immunostained material showed a significant decrease in NPY-immunoreactivity (NPY-IR) in OVX rats compared to the control group. Food deprivation increased the density of punctate NPY-IR profiles in both intact and OVX animals, however, the density in food-deprived OVX animals was increased compared to baseline but remained reduced compared to intact rats. Our study indicates that the lack of gonadal hormones - most likely estrogen - results in a decrease in the density of NPY-IR axonal fibers within the LS, while food deprivation induced considerable elevation in NPY density. Food restriction-induced changes in the density of NPY-containing neural elements suggest that the LS may play a crucial role in the regulation of food intake and energy balance, in concert with the relevant hypothalamic areas.

Cocaine- and amphetamine- regulated transcript (CART) peptide- immunopositive neuronal elements in the lateral septum: Rostrocaudal distribution in the male rat

The morphological features and distribution of cocaine- and amphetamine-regulated transcript peptide immunoreactivity (CART-IR) were studied in the lateral septum (LS) of male rats using light and electron microscopic immunocytochemistry and computer-aided densitometry. CART-IR was detected along the rostrocaudal axis of the LS in varicose axonal fibers only, although immunoreactive cell bodies and dendrites were not detected. Pericellular basket-like arrangements around immunonegative cell bodies were present. From among the targets of such pericellular baskets, glutamic acid decarboxylase (GAD)-immunopositive and NPY-immunoreactive somata were identified. Thin varicose axons were present in each section, whereas thick varicose axons were restricted to the sections of rostral position only. CART-IR was observed in varicose fibers forming a dense subependymal plexus, from which solitary varicose fibers entered the ependymal layer. The fine structure of varicosities was similar to that of other neuropeptide-containing fibers. Small varicosities established asymmetrical synaptic contacts mainly with dendrites and dendritic spines, and larger varicosities established symmetrical synapses with somata and dendritic shafts. CART-to-CART connections were not revealed. The density curve of the CART-IR along the rostrocaudal axis of LS was found to be paraboloid. CART is known as one of the most anorexigenic peptides. These results serve as basis for further physiological studies concerning the biological significance of lateral septal CART peptide in the regulation of food intake.

Differential distribution of calpain small subunit 1 and 2 in rat brain

Calpains, the Ca2+‐dependent thiol proteases, are abundant in the nervous tissue. The ubiquitous enzyme forms in mammals are heterodimers consisting of a specific, µ or m, large (catalytic) subunit and, apparently, a common small (regulatory) subunit (CSS1). Recently, however, we described a second form of small subunit (CSS2), which is of restricted occurrence [Schád, E., Farkas, A., Jékely, G., Tompa, P. & Friedrich, P. (2002) Biochem. J., 362, 383–388]. Here we analysed the distribution of immunoreactivity in various parts of rat brain against two anti‐CSS1 and two anti‐CSS2 antibodies by correlated light and electron microscopy. Remarkably, the antibodies showed differential distribution in various parts of rat cortex: anti‐CSS1 reacted mainly with perikarya and dendrites, whereas anti‐CSS2 was more prominent in axons. In serial sections CSS2 and synaptophysin gave very similar patterns, i.e. these epitopes seem to colocalize. Electron microscopy confirmed that CSS1 was mainly localized postsynaptically in dendrites and somata, whereas CSS2 was found presynaptically. The hypothesis is advanced that these distinct distributions of calpain subunits may be related to the transport of these enzymes in nerve cells.

Enteric neuromuscular junctions: Comparison of ultrastructural features in different phylogenetic groups

The enteric neuromuscular junctions of snail (Helix pomatia), locust (Locusta migratoria migratorioides), cockroach (Periplaneta americana), carp (Cyprinus carpio) and tench (Tinca tinca) were studied by means of different light and electron microscopic methods. The nitroblue tetrazolium staining revealed that the myenteric plexuses of the above species are composed of nerve cells, a network of varicose nerves and nerve bundles. Instead of highly organized ganglia, single neurons or small groups of 2-4 cells are characteristic of the invertebrates and fish studied. Catecholaminergic fluorescence induced by glyoxylic acid was detected in the muscular layer of the entire alimentary tract in snail and the hindgut of tench. Fluorescent nerves and perikarya were frequent in the snail gut, while only nerves and no perikarya were found in tench. A close contact between enteric muscles and nerves is the most common form of enteric neuromuscular junction in both the smooth (i.e. the molluscan and fish gut) and the striated (i.e. the insect gut) musculature. The striated musculature (i.e. the insect gut, the oesophagus of carp, and the oesophagus, stomach and the midgut of tench) also receives a synaptic input. Cytochemical evidence is provided of the cholinergic character of fish motor endplates. The ultrastructural appearance and vesicle population of certain nerve terminals suggest a universal role of aminergic and peptidergic control in gut motility.