Glenn I. Hatton

Anatomy of the brain neurogenic zones revisited : Fractones and the fibroblast/macrophage network

Cytogenesis in adult peripheral organs, and in all organs during development, occurs nearby basal laminae (BL) overlying connective tissue. Paradoxically, cytogenesis in the adult brain occurs primarily in the subependymal layer (SEL), a zone where no particular organization of BL and connective tissue has been described. We have reinvestigated the anatomy of the area considered the most neurogenic in the adult brain, the SEL of the lateral ventricle, in zones adjacent to the caudate putamen, corpus callosum, and lateral septal nucleus. Here, we report structural (confocal microscopy using laminin as a marker) and ultrastructural evidence for highly organized extravascular BL, unique to the SEL. The extravascular BL, termed fractones because of their fractal organization, were regularly arranged along the SEL and consisted of stems terminating in bulbs immediately underneath the ependyma. Fractones contacted local blood vessels by means of their stems. An individual fractone engulfed in its folds numerous processes of astrocytes, ependymocytes, microglial cells, and precursor cell types. The attachment site (base) of stems to blood vessels was extensively folded, overlying large perivascular macrophages that belong to a fibroblast/macrophage network coursing in the perivascular layer and through the meninges. In addition, collagen‐1, which is associated with BL and growth factors during developmental morphogenetic inductions, was immunodetected in the SEL and particularly regionalized within fractones. Because macrophages and fibroblasts produce cytokines and growth factors that may concentrate in and exert their effect from the BL, we suggest that the structure described is implicated in adult neurogenesis, gliogenesis, and angiogenesis. J. Comp. Neurol. 451:170–188, 2002.

CALBINDIN-D28K : ROLE IN DETERMINING INTRINSICALLY GENERATED FIRING PATTERNS IN RAT SUPRAOPTIC NEURONES

1. Physiological activation of rat supraoptic nucleus (SON) neurones leads to phasic firing in vasopressin neurones and fast, continuous firing in oxytocin neurones. Using whole‐cell patch clamp methods in brain slices, we investigated the role of endogenous calbindin‐D28k (calbindin) in determining these intrinsically generated patterns of firing. 2. Direct introduction of calbindin (0.1‐0.2 mM) into twelve of twelve phasically firing neurones suppressed Ca(2+)‐dependent depolarizing after‐potentials (DAPs) and changed activity from phasic to continuous firing. Bovine calcium binding protein (0.3 mM), an analogue of calbindin, had similar effects on both DAPs and firing patterns in five of five cells tested. 3. Introduction of anti‐calbindin antiserum (1:2000‐5000) into thirteen of thirteen continuously firing neurones unmasked DAPs and converted continuous into phasic firing. Such effects could not be mimicked either by diffusion of normal rabbit serum or antibodies directed against glial fibrillary acidic protein or against neurophysin. 4. Immunocytochemical staining with antisera directed against calbindin revealed more intense staining in the dorsal, oxytocin‐rich and less intense staining in the ventral, vasopressin‐rich areas of the SON. 5. Elevated intracellular Ca2+ concentration ([Ca2+]i; 0.1 mM) induced DAPs and phasic firing in all twenty‐nine SON cells recorded. During chelation of intracellular Ca2+ with (1.1‐11 mM) BAPTA, fifty‐eight of fifty‐eight neurones recorded displayed regular continuous activity and had no DAPs. 6. These data suggest that firing activities in SON cells are dependent on [Ca2+]i and that calbindin, acting as an endogenous Ca2+ buffer, is involved in regulation of intrinsic firing patterns. It is likely that calcium binding proteins have a similar influence on the firing patterns of many neuronal types throughout the nervous system.

Electrophysiology of excitatory and inhibitory afferents to rat histaminergic tuberomammillary nucleus neurons from hypothalamic and forebrain sites

Anatomical evidence exists for projections to the tuberomammillary nucleus (TM) from the nucleus of the diagonal band of Broca (DBB) and the lateral preoptic area (LPO). The physiological effects of activating these inputs were studied by recording postsynaptic responses intracellularly from TM cells during both electrical stimulation and local nanodrop application of glutamate in horizontally cut brain slices. Electrical stimulation of the DBB, LPO and anterior lateral hypothalamic area (LH) usually evoked fast IPSPs (approximately 75% of responses) which were blocked by bicuculline or picrotoxin, suggesting GABA(A) mediation. The remaining excitatory responses evoked by stimulation of the LPO and LH were blocked by non-NMDA receptor antagonists (CNQX or NBQX) and the NMDA receptor antagonist, AP-5. Glutamate applied to the above areas induced postsynaptic responses in TM cells similar to those seen with electrical stimulation. Spontaneous firing in TM cells was suppressed by glutamate applied in the DBB. Glutamate applied in the LPO or LH evoked both inhibitory and excitatory responses. Changes in PSPs and firing rates were interpreted to result from glutamate activation of the neurons in the DBB, LPO and LH areas with inhibitory or excitatory connections to recorded TM neurons. These results support previous anatomical findings and suggest that inhibitory and excitatory synaptic control of TM activity is exerted by the DBB, LPO and LH areas.

Reduced outward K+ conductances generate depolarizing after–potentials in rat supraoptic nucleus neurones

1 Whole‐cell patch clamp recordings were obtained from sixty‐five rat supraoptic nucleus (SON) neurones in brain slices to investigate ionic mechanisms underlying depolarizing after‐potentials (DAPs). When cells were voltage clamped around ‐58 mV, slow inward currents mediating DAPs (Idap), evoked by three brief depolarizing pulses, had a peak of 17 ± 1 pA (mean ± s.e.m.) and lasted for 2.8 ± 0.1 s. 2 No significant differences in the amplitude and duration were observed when one to three preceding depolarizing pulses were applied, although there was a tendency for twin pulses to evoke larger Idap than a single pulse. The Idap was absent when membrane potentials were more negative than ‐70 mV. In the range ‐70 to ‐50 mV, Idap amplitudes and durations increased as the membrane became more depolarized, with an activation threshold of ‐65.7 ± 0.7 mV. 3 I dap with normal amplitude and duration could be evoked during the decay of a preceding Idap. As frequencies of depolarizing pulses rose from 2 to 20 Hz, the times to peak Idap amplitude were reduced but the amplitudes and durations did not change. 4 A consistent reduction in membrane conductance during the Idap was observed in all SON neurones tested, and averaged 34.6 ± 3.3%. Small hyperpolarizing pulses used to measure membrane conductances appeared not to disturb major ionic mechanisms underlying Idap, since the slope and duration of Idap with and without test pulses were similar. 5 The Idap had an averaged reversal potential of ‐87.4 ± 1.6 mV, which was close to the K+ equilibrium potential. An elevation in [K+]O reduced or abolished the Idap, and shifted its reversal potential toward more positive levels. Perifusion of slices with 7.5–10 mm TEA, a K+ channel blocker, reversibly suppressed the Idap. 6 Both Na+ and Ca2+ currents failed to induce an Idap‐like current during perifusion of slices with media containing high [K+]o or TEA. However, the Idap was abolished by replacing external Ca2+ with Co2+, or replacing 82% of external Na+ with choline or Li+. Perifusion of slices with media containing 1–2 μm TTX also reduced Idap by 55.5 ± 9.0 %. 7 These results suggest that the generation of DAPs in SON neurones mainly involves a reduction in outward K+ current(s), which probably has little or no inactivation and can be inhibited by [Ca2+]i transients, due to Ca2+ influx during action potentials and Ca2+ release from internal stores. Na+ influx might provide a permissive influence for Ca2+‐induced reduction of K+ conductances and/or help to raise [Ca2+]i via reverse‐mode Ca2+‐Na+ exchange. Other conductances, making minor contributions to the Idap, may also be involved.

Ca2+ release from internal stores: role in generating depolarizing after‐potentials in rat supraoptic neurones.

1. Influences of Ca2+ release from internal stores on the generation of depolarizing after‐potentials (DAPs) were investigated in magnocellular neurones of rat supraoptic nucleus (SON) using whole‐cell patch recording techniques in brain slices. 2. DAPs were recorded from more than half of the cells encountered, and following evoked single spikes had an amplitude of 3.00 +/‐ 0.19 mV (mean +/‐ S.E.M.) and lasted for 1.02 +/‐ 0.06 s. Their sizes usually increased with the number of preceding spikes, but could be reduced or eliminated when intervals between consecutive current pulses evoking tens of spikes were short. 3. DAPs were eliminated by removal of external Ca2+, and significantly reduced by bath application of nifedipine or omega‐conotoxin. 4. Blockade of Ca2+ release from internal stores by perifusion with ryanodine or dantrolene, or direct diffusion of Ruthenium Red into cells suppressed DAP amplitudes by approximately 50% and shortened their durations. 5. Depletion of internal Ca2+ stores by perifusion with thapsigargin or cyclopiazonic acid also reduced DAP amplitudes by approximately 50% and eliminated phasic patterns of firing. 6. Caffeine, an agent known to enhance intracellular Ca2+ release, amplified DAPs and promoted phasic firing. 7. These results suggest that Ca2+ influx via high‐voltage‐activated Ca2+ channels in SON cells triggers ryanodine receptor‐mediated Ca2+ release from internal stores. This process enhances DAPs and promotes phasic firing in SON cells, and would thus contribute to vasopressin release.

Oscillatory bursting of phasically firing rat supraoptic neurones in low‐Ca2+ medium: Na+ influx, cytosolic Ca2+ and gap junctions.

1. Whole‐cell patch recordings were obtained from supraoptic nucleus (SON) neurones in horizontal brain slices of adult male rats. Low‐Ca2+ or Ca(2+)‐free perifusion medium induced oscillatory bursting activity in all sixty‐nine cells displaying both phasic firing and depolarizing after‐potentials (DAPs). In fifteen non‐phasic cells without DAPs, Ca(2+)‐free medium produced little or no oscillatory bursting. 2. Typical bursts started with rapid membrane depolarization, resulting in a plateau with superimposed action potentials, and ended several hundred milliseconds later in swift repolarization. Prominent bursting was observed at membrane potentials from ‐50 to ‐70 mV, with maximum amplitudes of 12.2 +/‐ 0.7 mV (mean +/‐ S.E.M.) around ‐70 mV. Development of oscillatory bursting was dependent on reduction of [Ca2+]o, with a threshold for the bursting < or = 1.2 mM Ca2+. 3. Bursting was abolished by addition of TTX, Co2+, Ni2+ or Mg2+ into the Ca(2+)‐free medium, or by replacement of external Na+ with choline or Li+. Low concentrations of TEA or increased [K+]o prolonged burst durations and enlarged oscillation amplitudes. 4. Voltage‐clamp techniques were used to examine the persistent Na+ current (INaP), and revealed that low [Ca2+]o shifted the threshold for INaP activation in a negative direction and enhanced the amplitude of this current. These changes in INaP were abolished by adding Co2+ or Mg2+ to Ca(2+)‐free medium. 5. Direct diffusion of BAPTA or heparin into neurones or bath application of ryanodine suppressed bursting. Oscillations were also eliminated by the uncoupling agents heptanol, halothane or acidification. 6. CNQX, APV, bicuculline, CGP35348 (GABAB receptor antagonist), promethazine, atropine, d‐tubocurarine and suramin had no obvious effects on oscillatory bursting. Blockers of transient Ca2+, or hyperpolarization‐activating cation currents also did not alter bursting activity. 7. These results suggest that intrinsic burst activity in SON neurons perifused with low‐Ca2+ or Ca(2+)‐free medium involves enhanced Na+ influx through persistent Na+ channels, and requires the presence of rapid intracellular Ca2+ mobilization that might also explain the selective existence of oscillatory bursting in phasically firing cells. Intercellular communication through gap junctions appears to be important in determining neuronal activity of the neuroendocrine cells in low‐Ca2+ medium.

Connexin 26 and basic fibroblast growth factor are expressed primarily in the subpial and subependymal layers in adult brain parenchyma: roles in stem cell proliferation and morphological plasticity?

The gap junction protein connexin 26 (Cx26) has been detected previously in the parenchyma of the developing brain and in the developing and adult meninges, but there is no clear evidence for the presence of this connexin in adult brain parenchyma. Confocal mapping of Cx26 through serial sections of the meningeal‐intact rat brain with four antibodies revealed an intense Cx26 immunoreactivity in both parenchyma and extraparenchyma. In the extraparenchyma, a continuum of Cx26‐immunoreactive puncta was observed throughout the three meningeal layers, the perineurium of cranial nerves, and meningeal projections into the brain, including sheaths of blood vessels and stroma of the choroid plexus. In the parenchyma, Cx26‐immunoreactive puncta were located primarily in subependymal, subpial, and perivascular zones and were associated primarily with glial fibrillary acidic protein‐positive (GFAP+) astrocytes, the nuclei of which are strongly immunoreactive for basic fibroblast growth factor (bFGF). Although it was found to a lesser extent than in astrocytes, bFGF immunoreactivity also was intense in the nuclei of meningeal fibroblasts. In addition, we have found a close correlation between the distribution of Cx26 and vimentin immunoreactivities in the meninges and their projections into the brain. We previously showed vimentin and S100β immunoreactivities through a network of meningeal fibroblasts in the three layers of meninges, perivascular cells, and ependymocytes and in a population of astrocytes. The related topography of this network with GFAP+ astrocytes has also been demonstrated. Considering that connexin immunoreactivity may reflect the presence of functional gap junctions, the present results are consistent with our hypothesis that all of these various cell types may communicate in a cooperative network. J. Comp. Neurol. 431:88–104, 2001.

Astroglial modulation of neurotransmitter/peptide release from the neurohypophysis: present status.

Reviewed in this article are those studies that have contributed heavily to our current conceptualizations of glial participation in the functioning of the magnocellular hypothalamo-neurohypophysial system. This system undergoes remarkable morphological and functional reorganization induced by increased demand for peptide synthesis and release, and this reorganization involves the astrocytic elements in primary roles. Under basal conditions, these glia appear to be vested with the responsibility of controlling the neuronal microenvironment in ways that reduce neuronal excitability, restrict access to neuronal membranes by neuroactive substances and deter neuron neuron interactions within the system. With physiological activation, the glial elements, via receptor-mediated mechanisms, take up new positions. This permissively facilitates neuron neuron interactions such as the exposure of neuronal membranes to released peptides and the formation of gap junctions and new synapses, enhances and prolongs the actions of those excitatory neurotransmitters for which there are glial uptake mechanisms, and facilitates the entry of peptides into the blood. In addition, subpopulations of these glia either newly synthesize or increase synthesis of neuroactive peptides for which their neuronal neighbors have receptors. Release of these peptides by the glia or their functional roles in the system have not yet been demonstrated.

Immunocytochemical basis for a meningeo-glial network

Evidence is presented here for a cellular network that courses through all layers of meninges, the vasculature of both the brain and meninges, and extends into the brain parenchyma. Confocal mapping of calcium‐binding protein S100β immunoreactivity (S100β‐ir) and of the intermediate filament vimentin‐ir through serial sections of the meningeal‐intact adult rat brain revealed this network. In all tissues examined, S100β‐ir and vimentin‐ir were primarily colocalized, and were found in cells with elongated processes through which these cells contacted one another to form a network. The location of labeling and the morphology of the cells labeled were consistent with the possibility that this network consists of fibroblasts in the meninges and the walls of large blood vessels, of pericytes at the level of capillaries, and of ependymocytes and a population of astrocytes in the brain parenchyma. At many sites along the borders of the brain parenchyma itself and of the brain blood vessels, it was possible to detect S100β‐ir and vimentin‐ir cell processes that cross the basal laminae. This suggested the probable means by which the S100β‐ir cells of the extraparenchymal tissues anatomically contact the cells that express the same markers in the brain. Privileged anatomical relationships of the S100β/vimentin network with the glial fibrillary acidic protein (GFAP) astrocytes further suggested that, together, they form the structural basis for a general meningeo‐glial network. This organization challenges the current model of brain architecture, calls for a reconsideration of the role of meninges and vascular tissues, and appears to reflect the existence of hitherto unsuspected systems of communication. J. Comp. Neurol. 420:445–465, 2000.

Histamine mediates fast synaptic inhibition of rat supraoptic oxytocin neurons via chloride conductance activation

Axons from the histaminergic neurons of the tuberomammillary nucleus project to both the anterior and tuberal portions of the supraoptic nucleus. Histamine is known to activate vasopressin neurons via a histamine receptor subtype 1 and to increase release of vasopressin, but effects on oxytocin neurons have been previously unexplored. Here we investigated the effects of tuberomammillary nucleus electrical stimulation as well as of histamine antagonists on supraoptic nucleus oxytocin and vasopressin neurons in slices of rat hypothalamus. Electrical stimulation evoked short constant latency (approximately 5 ms), fast (4-6 ms onset to peak) inhibitory postsynaptic potentials in oxytocin neurons and, as shown previously, fast excitatory postsynaptic potentials in vasopressin neurons. These synaptic responses followed paired-pulse stimulus frequencies up to 100 Hz and were, thus, probably reflecting monosynaptic connections. Inhibitory postsynaptic potentials were selectively blocked by histamine receptor subtype 2 antagonists (either cimetidine or famotidine) and by picrotoxin but not by histamine receptor subtype 1 antagonists or bicuculline. Similar synaptic responses to tuberomammillary nucleus stimulation were found in 16 of 16 neurons immunocytochemically identified as oxytocinergic and in seven putative oxytocin neurons. Perifusion of the slice with low chloride medium (4.8 mM) reversed stimulus-evoked inhibitory postsynaptic potentials. We conclude that histaminergic neurons monosynaptically contact both oxytocin and vasopressin cells of the supraoptic nucleus and inhibit the former via activation of chloride channels which can be blocked by the histamine receptor subtype 2 antagonists, famotidine and cimetidine.