Loss of GABAergic neurons in medial septum after fimbria-fornix transection

Neurons in the medial septum of the rat brain undergo retrograde degeneration after transection of their projection to the hippocampal formation, the fimbria-fornix. This cell death has been characterized for both Nissl-stained neurons and acetylcholinesterase-stained neurons. The major cell type in the medial septum is GABAergic, and many of these GABAergic neurons project to the hippocampal formation. Because the fimbria-fornix transection causes more neuronal death than can be accounted for by the loss of cholinergic neurons, we have sought to determine if the GABAergic neurons undergo a cell death similar to that reported for the cholinergic neurons. We report here that GABAergic neurons are indeed lost after the transection but the time course is considerably slower than that for the cholinergic neurons.     

Rhythmical bursting activity and GABAergic mechanisms in the medial septum of normal and pertussis toxin-pretreated rats

Summary The possible involvement of GABA in the control of the rhythmical bursting activity (RBA) of septo-hippocampal neurons (SHNs) has been studied in the rat in vivo. The discharge frequency of SHNs was modified by the iontophoretic application of a GABA agonist and antagonist as well as by the application of the GABA uptake blocker, nipecotic acid. The GABA_B agonist baclofen inhibited the SHNs' activity, this effect being antagonized by the GABA_B antagonist phaclofen. However, these different pharmacological manipulations did not modify the RBA frequency. Pretreatment of the rats with pertussis toxin, a substance which is known to block the events mediated by G-proteins (Gi or Go), decreased the RBA frequency. Neither agonists nor antagonists of GABA_A or GABA_B types had significant effects on the rhythmical bursting activity of SHNs. The effect of pertussis toxin suggests that other neurotransmitters or intrinsic mechanisms involving a G-protein influence this rhythm.     

Excitatory effects induced by carbachol on bursting neurons of the rat subiculum

Conventional intracellular recordings were made from neurons of the rat subiculum in an in vitro slice preparation. Intracellular pulses of depolarizing current (duration, 10–120 ms) delivered at a resting membrane potential of −62.2 ± 7.7 mV (mean ± SD, n = 14) imduced bursts of 3–5 fast, action potentials riding on a slow depolarization. The burst was terminated by an afterhyperpolarization (burst AHP) that lasted 117 ± 26 ms and reached peak amplitude of 5.1 ± 1.8 mV (n = 8). Bath application of the cholinergic agonist carbachol (CCh; 30–100 μM; n = 20) in the presence of ionotropic excitatory amino acid receptor antagonists induced a steady depolarization (4.6 ± 2.7 mV) of the membrane potential, and a small increase in input resistance. Action potential bursts continued to occur in response to intracellular depolarizing pulses during CCh application. However, this cholinergic agonist reduced and eventually blocked the burst AHP, which was replaced by action potentials firing. In the presence of CCh (> 70 μM; n = 9) the burst response, was followed by a depolarizing plateau potential (PP) that outlasted the intracellular depolarizing pulse by 731 ± 386 ms (range 160–1900 ms), and could trigger repetitive action potential firing at 35–116 Hz. The effects induced by CCh were reversed by bath application of the muscarinic antagonist atropine (0.5–1 μM; n = 4). Our findings demonstrate that CCh exerts in the rat subiculum an excitatory action that is dependent upon muscarinic receptor stimulation. This cholinergic mechanism may play a physiological role in the subicular processing of signals arising from the hippocampus proper, and may also contribute to the generation of sustained epileptiform discharges induced in the limbic system by cholinergic agents.     

A distinct group of non-cholinergic neurons along the mid-line of the septum and within the rat medial septal nucleus

The septum is a critical and integral component of the limbic brain that serves as a link between diverse brain structures while being necessary for human cognition and emotionality. A major anatomical component of the septum is designated as the medial septum/diagonal band of Broca complex (MS/DB). A primary focus of much research has been to investigate cholinergic neurons within the MS/DB, as these are the rodent brain's main source of acetylcholine to the cortex and hippocampus. On the other hand, we have chosen to investigate a specific group of neurons that lie on the midline of the MS/DB in an area distinguished anatomically as the medial septal nucleus (MSN). Based on somatic morphology and electrophysiological characteristics we conclude that these neurons, characterized into three different types, are non-cholinergic.     

Parvalbumin-immunoreactive, fast-spiking neurons in the medial septum/diagonal band complex of the rat: intracellular recordings in vitro.

The medial septum/diagonal band complex is composed predominantly of cholinergic and GABAergic neurons, and it projects to the hippocampal formation. A proportion of the GABAergic neurons contain parvalbumin, a calcium-binding protein that has previously been localized in fast-spiking, non-accommodating GABAergic neurons in the cerebral cortex and neostriatum. The aim of the present study was to determine whether parvalbumin is localized preferentially in a similar electrophysiological class of neuron in the medial septum/diagonal band complex. The study was carried out using in vitro intracellular recording, intracellular biocytin filling and parvalbumin immunocytochemistry. Three main classes of neurons were identified according to standard criteria: burst-firing, slow-firing and fast-firing neuronal populations. The fast-firing neurons were subdivided into two subpopulations based on whether or not they displayed accommodation. The fast-spiking, non-accommodating cells were furthermore found to be spontaneously active at resting potentials, and to possess action potentials of significantly (P < 0.05) shorter duration (half width: 0.61 +/- 0.12 ms) than those of the regular-spiking, accommodating neurons (1.0 +/- 0.34 ms). Of the neurons that were successfully filled with biocytin and processed for parvalbumin immunoreactivity, 82% of the fast-spiking, non-accommodating cells possessed parvalbumin immunoreactivity, while none of the regular-spiking, accommodating neurons were found to be immunoreactive for parvalbumin. The slow-firing neurons, shown previously to be cholinergic, did not stain for parvalbumin immunoreactivity, in agreement with studies showing parvalbumin to be localized solely in GABAergic neurons in the medial septum/diagonal band complex. In conclusion, these findings suggest the presence of a previously uncharacterized population of neurons in the medial septum/diagonal band complex that generate high-frequency, non-adaptive discharge. This property correlates with the localization of parvalbumin in these neurons, which suggests that parvalbumin fulfils the same role in the medial septum/diagonal band complex that it does in other parts of the brain. The fast-spiking neurons in the medial septum/diagonal band complex may play an essential role in the GABAergic influence of the septum on the hippocampal formation.     

Ultrastructure of the anterior medial glands of the rat nasal septum

The anterior medial glands lying in the submucosa of the rat nasal septum were studied by light and electron microscopy. The glands consist of a single long duct, which is studded with numerous solitary acinar formations connected perpendicularly to the main duct by short intercalated ducts. Proximal acini (those furthest from the stoma of the main duct) consist of typical serous cells with many dense secretory granules and an extensive rough endoplasmic reticulum. The most distal acini consist of cells whose major feature is the enwrapment of each mitochondrion by a cisternal profile of rough endoplasmic reticulum. Myoepithelial cells are absent from proximal acini, but are abundant on distal acini. Intracellular nerve terminals are extremely common, particularly in distal acini. The main ducts resemble, to a degree, the striated ducts of salivary glands.     

Multiple connections of medial hypothalamic neurons in the rat

Summary The responses of 700 single neurons in the hypothalamus to electrical stimulation of the preoptic area, limbic structures, and midbrain were studied to determine the location of neurons with multiple inputs and to identify by antidromic activation the projection areas of those neurons.Converging excitatory inputs, observed in 134 responsive hypothalamic neurons, were principally derived from the preoptic, limbic, and midbrain areas. Inputs from separate nuclei of the amygdala were noted in the response of individual hypothalamic neurons. Two classes of short latency transsynaptic responses to amygdala stimulation were defined, indicating either separate pathways from the amygdala to the medial hypothalamus or two types of fibers conducting at different velocities. Stimulation of single or multiple sites in the preoptic and limbic areas, as well as in the arcuate nucleus and medial forebrain bundle produced inhibition of hypothalamic neuronal activity.Most antidromically identified medial hypothalamic neurons projected to the preoptic area, median eminence (tuberoinfundibular neurons), or midbrain. Evidence is presented for collateral projections of tuberoinfundibular neurons to the preoptic area and reticular formation. Medial hypothalamic neurons received inputs from the preoptic area, lateral septal nucleus, amygdala, ventral hippocampus (subiculum), and fornix. These findings illustrate a pattern of reciprocal connections between the medial hypothalamus and limbic and midbrain structures.It was concluded that the hypothalamus contains a type of neuron that is equipped to perform complex integrations and to coordinate directly the behavior of neurons in a diversity of anatomical regions.Abbreviations ABL basolateral nucleus of the amygdala - ACO cotical nucleus of the amygdala - AHA anterior area of the hypothalamus - ARH arcuate nucleus of the hypothalamus - DMH dorsomedial nucleus of the hypothalamus - FX fornix - HPC ventral hippocampus (subiculum) - LS lateral septal nucleus - ME median eminence - MH medial hypothalamus - MFB medial forebrain bundle - MP posterior mamillary nucleus - PH posterior nucleus of the hypothalamus - PMD dorsal premamillary nucleus - PMV ventral premamillary nucleus - POA preoptic area - PVG periventricular gray - PVH paraventricular nucleus of the hypothalamus - RF reticular formation of the mesencephalon - RT reticular nucleus of the thalamus - SUM supramamillary nucleus - VMH ventromedial nucleus of the hypothalamus Performed with financial support from the National Institutes of Health (Grants NS 09688 and RR 00165)     

Quisqualate receptor-mediated rhythmic bursting of rat hypothalamic neurons in dissociated cell culture

Dissociated hypothalamic neurons from embryonic rat brain exhibit a level of spontaneous synaptic activity after 21 days in culture. When GABA-mediated responses are blocked by picrotoxin or bicuculline (20 microM), the neurons burst rhythmically. Rhythmic burst activity is generated in most cells by postsynaptic excitatory currents (EPSCs) through non-specific cationic channels rather than by intrinsic pacemaker currents. We present evidence that EPSCs are mediated by an excitatory amino acid and a quisqualate receptor type.     

Activity-independent coregulation of IA and Ih in rhythmically active neurons

The fast transient potassium or A current (IA) plays an important role in determining the activity of central pattern generator neurons. We have previously shown that the shal K+ channel gene encodes IA in neurons of the pyloric network in the spiny lobster. To further study how IA shapes pyloric neuron and network activity, we microinjected RNA for a shal-GFP fusion protein into four identified pyloric neuron types. Neurons expressing shal-GFP had a constant increase in IA amplitude, regardless of cell type. This increase in IA was paralleled by a concomitant increase in the hyperpolarization-activated cation current Ih in all pyloric neurons. Despite significant increases in these currents, only modest changes in cell firing properties were observed. We used models to test two hypotheses to explain this failure to change firing properties. First, this may reflect the mislocalization of the expressed shal protein solely to the somata and initial neurites of injected neurons, rendering it electrically remote from the integrating region in the neuropil. To test this hypothesis, we generated a multicompartment model where increases in IA could be localized to the soma, initial neurite, or neuropil/axon compartments. Although spike activity was somewhat more sensitive to increases in neuropil/axon versus somatic/primary neurite IA, increases in IA limited to the soma and primary neurite still evoked much more dramatic changes than were seen in the shal-GFP-injected neurons. Second, the effect of the increased IA could be compensated by the endogenous increase in Ih. To test this, we modeled the compensatory increases of IA and Ih with a cycling two-cell model. We found that the increase in Ih was sufficient to compensate the effects of increased IA, provided that they increase in a constant ratio, as we observed experimentally in both shal-injected and noninjected neurons. Thus an activity-independent homeostatic mechanism maintains constant neuronal activity in the face of dramatic increases in IA.     

The presence of pacemaker HCN channels identifies theta rhythmic GABAergic neurons in the medial septum

The medial septum (MS) is an indispensable component of the subcortical network which synchronizes the hippocampus at theta frequency during specific stages of information processing. GABAergic neurons exhibiting highly regular firing coupled to the hippocampal theta rhythm are thought to form the core of the MS rhythm-generating network. In recent studies the hyperpolarization-activated, cyclic nucleotide-gated non-selective cation (HCN) channel was shown to participate in theta synchronization of the medial septum. Here, we tested the hypothesis that HCN channel expression correlates with theta modulated firing behaviour of MS neurons by a combined anatomical and electrophysiological approach. HCN-expressing neurons represented a subpopulation of GABAergic cells in the MS partly overlapping with parvalbumin (PV)-containing neurons. Rhythmic firing in the theta frequency range was characteristic of all HCN-expressing neurons. In contrast, only a minority of HCN-negative cells displayed theta related activity. All HCN cells had tight phase coupling to hippocampal theta waves. As a group, PV-expressing HCN neurons had a marked bimodal phase distribution, whereas PV-immunonegative HCN neurons did not show group-level phase preference despite significant individual phase coupling. Microiontophoretic blockade of HCN channels resulted in the reduction of discharge frequency, but theta rhythmic firing was perturbed only in a few cases. Our data imply that HCN-expressing GABAergic neurons provide rhythmic drive in all phases of the hippocampal theta activity. In most MS theta cells rhythm genesis is apparently determined by interactions at the level of the network rather than by the pacemaking property of HCN channels alone.     

Muscarinic agonists block a late-afterhyperpolarization in medial septum/diagonal band neurons in vitro.

Intracellular recordings were made from neurons located in the medial septum (MS), and nucleus of the diagonal band (nDB) from slices of guinea pig brain. These forebrain nuclei contain both cholinergic and noncholinergic neurons that project to the cortex and hippocampus and are involved in many cortical functions. Muscarinic agonists (bethanechol, 2-30 microM) had the specific action to reduce a long-duration afterhyperpolarization (long-AHP) while leaving other shorter duration AHPs intact. Since the long-AHP was observed in both cholinergic and non-cholinergic neurons, muscarinic agonists were not selective for any one cell type. Block of a long-AHP was not associated with a consistent increase in cell excitability and therefore can not fully explain the excitatory actions of acetylcholine (ACh) observed in vivo within the MS/nDB.     

Modulation of late phases of long-term potentiation in rat dentate gyrus by stimulation of the medial septum

The prolonged maintenance of hippocampal long-term potentiation (LTP) seems to require heterosynaptic events during its induction. We have previously shown that stimulation of the basolateral nucleus of the amygdala (BLA) within a distinct time window can reinforce a transient early-LTP into a long-lasting late-LTP in the dentate gyrus (DG) in freely moving rats. We have shown that this reinforcement was dependent on beta-adrenergic and/or muscarinergic receptor activation and protein synthesis. However, since the BLA does not directly stimulate the DG the question remained by which inputs such heterosynaptic processes are triggered. We have now directly stimulated the medial septal pathway 15 min after induction of early-LTP in the DG and show that this input is capable of reinforcing early into late-LTP in a frequency-dependent manner. This septal reinforcement of DG LTP was dependent on beta-adrenergic receptor activation and protein synthesis. We suggest that the reinforcing effect of the BLA stimulation can, potentially, be mediated via the septal input to the DG, though it differs in its ability to induce or modulate functional plasticity.     

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.     

The morphology of neurons in rat tectal transplants as revealed by Golgi-Cox impregnation

Summary The Golgi technique has been used to examine the morphology of neurons within tectal transplants. Embryonic tectal tissue was transplanted to the midbrain of newborn rats. Four to eight months later, host animals were decapitated under anaesthesia, the unfixed brains removed and processed by Golgi-Cox impregnation. In tectal grafts, different types of neuron were recognized on the basis of the size and shape of their somata and the morphology of their dendritic trees. Neuronal types found in transplants resembled cell classes found in normal rat superior colliculus (SC). Neurons characteristic of the superficial collicular layers such as marginal, ganglion type I, stellate and horizontal cells and multipolar cells typical of the deeper collicular layers were identified in the transplants. Compared with normal cells, grafted neurons often had smaller dendritic fields and fewer dendritic spines. No laminar organization was discernable in the grafts and there was commonly no preferential orientation of perikarya or dendrites. Small cells with similar dendritic morphology were sometimes found grouped together in patches within the graft neuropil. These patches resembled cytologically and histochemically distinct areas described in previous studies and may represent areas homologous to the superficial layers of normal SC.     

AMPA receptors are modulated by tachykinins in rat cerebellum neurons

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as neuromodulators, although their direct action on cerebellum granule cells have not yet been demonstrated. We have examined the effect of the best known members of the family, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from rat cerebellar granule cells in culture to assess the ability of these peptides to regulate the glutamatergic input. Both NKA and NKB, but not SP, produce a significant enhancement of ionic current through AMPA receptors activated by the agonist kainate in 53.5 and 46% of patched neurons, respectively. This effect was not observable in the presence of MEN 10,627 and Trp(7)betaAla(8), NKA and NKB competitive antagonist receptors, respectively, indicating that the current modulations were mediated by the respective receptors. NKB also produces a significant enhancement of ionic current through the AMPA receptors activated directly by its agonist AMPA and cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA receptors. The presence of NK3 receptors was demonstrated in these neurons by RT-PCR amplification of total RNA extracted from cerebellar granule cells, using NK3-specific primer pairs. Immunocytochemistry experiments, using a specific polyclonal antibody directed against NK3, also confirmed the presence of NK3 receptors and their co-localization with the GLUR2 AMPA subunit in about 54% of cerebellar granule neurons. This study adds the tachykinins to the list of neuromodulators capable of exerting a excitatory action on cerebellar granule cells.