J.J. Miller

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.

Calcium-binding protein distribution in the rat brain

The distribution in the rat brain of a protein with properties similar to vitamin D-dependent calcium binding protein (CaBP) was measured using a radioimmunoassay developed for mammalian brain CaBP. The cerebellum contained very high levels of CaBP in agreement with reports of CaBP distribution in the chick brain. The protein was distributed in large and unequal concentrations in different regions of the brain. Microdissection of the hippocampus revealed that, within a particular region, CaBP can have a marked differential distribution, suggesting that it is found in specific cell types. The significance of this protein is discussed in relation to the important functions of calcium in the CNS with particular emphasis being placed upon the possible role of CaBP as an intraneuronal calcium ion buffering system.

Hippocampal calcium-binding protein during commissural kindling-induced epileptogenesis: progressive decline and effects of anticonvulsants.

Changes in hippocampal calcium-binding protein (CaBP) were examined in rats given kindling stimuli via electrodes chronically implanted in the midline commissural pathway. CaBP levels decreased progressively and were significantly lower (16.6%) than controls after only 10 kindling trials. The maximum fall (33%) was achieved prior to the production of stage 5 motor seizures and additional kindling-induced seizures produced no further decline. Induction of motor seizures with pentylenetetrazol had no effect upon hippocampal CaBP levels. Diazepam treatment during the course of kindling significantly increased the number of stimulation trials required to produce stage 5 motor seizures but did not inhibit the fall in CaBP. Diazepam treatment of fully kindled rats was effective in blocking generalized motor seizures without causing any restoration of the depleted levels of CaBP. Diphenylhydantoin was neither effective during the course of kindling nor in modifying the effect of further stimulations in fully kindled rats. These data indicate that the highly specific decrease in hippocampal CaBP, previously demonstrated to be localized to dentate granule cells and their processes following kindling-induced epilepsy, does not result from the expression of full tonic-clonic (stage 5) motor seizures. The loss of CaBP may be a biochemical factor contributing either to the predisposition of neuronal tissue to seizure activity or to a protective attempt to overcome the deleterious effect of repeated high-frequency stimulation.

Biochemical and immunohistochemical correlates of kindling-induced epilepsy: role of calcium binding protein

The levels and distribution of calcium binding protein (CaBP) were examined in the central nervous system of rats exhibiting kindling-induced epilepsy. Following commissural kindling, the concentration of CaBP in the hippocampal formation was significantly reduced but no change was observed in levels of total soluble protein or calmodulin, another calcium-related protein. Histochemical examination of kindled animals revealed a localized depletion of CaBP-like immunoreactivity restricted to the dentate granule cell-mossy fiber system of the hippocampal formation. These data demonstrate a correlation between the loss of CaBP from dentate granule cells and kindling-induced epilepsy, the underlying mechanism of which may involve an impairment of granule cells to regulate their intracellular calcium environment when challenged with high frequency electrical stimulation.

Pharmacological identification of acetylcholine and glutamate excitatory systems in the dentate gyrus of the rat

The responses of dentate granule cells to medial septum (MS) and perforant path (PP) stimulation were examined in urethane anaesthetized rats. MS and PP stimulation evoked an orthodromic activation of granule cells which was correlated with the negative transient of the characteristic field potential elicited from each site. The effects of electrophoretic application of acetylcholine (ACh) and glutamate (Glu) were examined on granule cells identified in this manner. The excitatory action of ACh but not that of Glu was antagonized by atropine. Glutamate diethylester (GDEE) blocked the excitation produced by Glu but not ACh. The synaptically evoked excitation elicited by MS was blocked by atropine but unaltered by GDEE whereas the PP excitatory response was blocked by GDEE and unaltered by atropine. The results of this study indicate that two discrete excitatory systems are present in the dentate gyrus of the rat: a cholinergic system originating in the medial septum and a glutamate mediated system originating in the entorhinal cortex.

Mechanisms of excitation and inhibition in the nigrostriatal system

The extracellular responses of neurones in the neostriatum following single pulse stimulation of the substantia nigra were investigated in urethane anaesthetized rats. Low intensity stimulation (less than 10 V) evoked single large amplitude spikes while higher intensities (10-20 V) elicit a high frequency burst of small amplitude spikes or waves. When spontaneous or glutamate-induced large spikes are recorded, nigral stimulation causes their inhibition coincidentally with the development of a burst. If the burst is prevented, the inhibitory response disappears. Both the nigral evoked inhibition and burst response are unaffected by iontophoretically or systemically administered antonists of dopamine or by chemical lesions of the dopamine-containing nigral neurones. The monosynaptic activation of large amplitude striatal neurones, which could also be identified antidromically by stimulation of the globus pallidus, was reversibly blocked by dopamine antagonists. It is concluded (a) that the burst responses are induced through the antidromic excitation of striatonigral axons within the striatum; (b) that the striatal neurones thus activated are inhibitory interneurones and (c) that the dopamine-containing neurones of the nigra make excitatory synaptic contact with a population of striatal output cells, some of which at least project to the globus pallidus.

Extracellular fields influence transmembrane potentials and synchronization of hippocampal neuronal activity

The influence of extracellular fields on the transmembrane potential (TMP) of CA1 pyramidal neurons was investigated following both ortho- and antidromic stimulation in the in vitro hippocampal slice preparation. A short latency negative deflection on the intracellular potential coincided with the falling phase of the extracellular population spike. Subtraction of extracellular field potentials from ground referenced intracellular records revealed a sharp depolarizing wave of the TMP superimposed upon the underlying synaptic potential. This graded depolarization was capable of discharging CA1 cells and displayed a parallel shift in latency and amplitude with the extracellular population spike. A similar depolarizing wave was associated with the antidromically evoked population spike which persisted following blockade of synaptic activity. Finally, multiple population spike activity similar to that observed during epileptiform discharge was associated with repetitive depolarizing waves of the TMP. These data suggest that extracellular field potentials can ephaptically discharge CA1 neurons and may play a role in recruitment and synchronization of neuronal activity in the hippocampus.

Excitatory action of the mesolimbic dopamine system on septal neurones

Fluorescence histochemical studies have demonstrated a topographical distribution of dopaminergic nerve terminals in limbic forebrain and cortical regions4, 9-11. These data, together with biochemical evidence indicating significant amounts of dopamine (DA) in these regionsl,XS, zl, suggest the existence of a major anatomical pathway projecting to forebrain areas which originate in mesencephalic tegmental regions known to be rich in DA cell bodies3,10, 29. Of particular interest in the present study is the catecholaminergic innervation of the septal area, which has previously been considered to consist almost exclusively of noradrenergic nerve terminals 14. More recently histochemical mapping experiments 9, have demonstrated a high density of DA terminals in the medial aspect of the lateral septal region which remain after bilateral transection of the ascending noradrenergic projection. Lindvall 9 has shown that transection of the medial forebrain bundle at the level of the rostral substantia nigra, or bilateral electrolytic lesions of the A10 mesencephalic cell group (according to Dahlstr6m and Fuxe 3) result in a complete loss of septal DA terminals. These data suggest a direct dopaminergic fibre system innervating the lateral septal area which originates in the Al0 region of the ventromedial tegmentum. The present experiments were undertaken to further delineate the cells of origin of this pathway using retrograde axonal transport of horseradish peroxidase (HRP). The possibility that DA may be involved as a synaptic transmitter in this projection system was determined by observing (1) the change in septal cell discharge elicited by A10 stimulation and (2) the effects of 6-hydroxydopamine (6-OHDA) and the DA receptor blocking agent haloperidol on the synaptically evoked response patterns. In order to demonstrate the cells of origin of afferent projections to the septal area, 0.1-0.3 #1 of a 33 ~ solution of horseradish peroxidase (HRP, Type VI, Sigma Chemical) in saline was injected stereotaxically into either the lateral or medial septal region of 11 male Wistar rats anaesthetized with Nembutal (50 mg/kg i.p.). Following survival periods of 24 h the rats were killed, perfused and frozen sections cut and treated to reveal peroxidase activity according to methods described previously 1~. The