David M. Finch

Circuit Mechanisms of Seizures in the Pilocarpine Model of Chronic Epilepsy: Cell Loss and Mossy Fiber Sprouting

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epilep tic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilo carpine‐induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo‐Timm stains showed supra‐and intragranular mossy fiber sprouting. Supragranular mossy fiber sprout ing and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CAS showed later onset of chronic epilepsy (r= 0.83, p < 0.0005), suggest ing that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the fea tures of human temporal lobe epilepsy (hippocampal cell loss, suprar and intragranular mossy fiber sprouting, den tate granule cell dispersion, spontaneous recurrent sei zures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.

Neurophysiology of converging synaptic inputs from the rat prefrontal cortex, amygdala, midline thalamus, and hippocampal formation onto single neurons of the caudate/putamen and nucleus accumbens.

Neurophysiological responses mediated by projections from five telencephalic and diencephalic regions (the infra‐ and prelimbic portions of the prefrontal cortex, amygdala, midline and intralaminar thalamic nuclei, entorhinal cortex and subiculum/CA1) to the caudate/putamen (CPu) and nucleus accumbens (Acb) of the dorsal and ventral striatum were studied in chloral‐hydrate‐anesthetized rats. Both extra‐ and intracellular in vivo recording techniques were used. A retrograde tracer (wheatgerm agglutinin‐apo‐horseradish peroxidase‐5 nm colloidal Gold) was deposited in some animals in the vicinity of recording sites to confirm that stimulating electrodes were located near cells that projected to the striatum. Electrical stimulation of these five regions, respectively, evoked excitatory responses in 60%, 22%, 51%, 25%, and 17% of striatal neurons. Some responses, particularly with thalamic stimulation, showed short‐term frequency potentiation in which 5/s stimulation increased the probability of spike firing. About half of responsive cells showed convergent excitation to more than one stimulating site. It was possible with convergent excitatory responses to show synaptic interactions: simultaneous activation of more than one site produced spatial and temporal summation to increase the probability of spike firing. Up to 5‐way convergence onto single striatal neurons and up to 3‐way interactions could be shown. These results indicate that functional influences from the hippocampal formation can converge with other excitatory input onto single striatal neurons to effect synaptic integration.

Demonstration of axonal projections of neurons in the rat hippocampus and subiculum by intracellular injection of HRP

Hippocampal formation neurons of rat were injected intracellularly with horseradish peroxidase in order to trace intrinsic and extrinsic axonal projections. CA3 pyramids (n = 9) projected axons rostrally toward the fimbria, one or more Schafer collaterals toward CA1, and in two cases fibers that crossed the hippocampal commissure. Pyramids of CA1 (n = 5) projected axons to the alveus where they proceeded caudally toward the subiculum. A subset (n = 3) also projected an axonal branch rostrally toward the fimbria. These findings confirm not only major target regions of Ammons horn pyramids, but also emphasize their divergent axonal projections that are not necessarily lamellar in organization. Axons from subicular pyramids (n = 12) projected rostrally, caudally, or in both directions. They could be traced to several other cortical regions, specifically Ammons horn, entorhinal cortex and cingulate cortex. The results further confirm that subicular neurons are the recipient of input from the hippocampus proper and are a principal source of efferents from the hippocampal formation. A multi-process neuron in CA1 with physiologic properties associated with inhibitory interneurons was filled and traced in detail. It most resembled the poligonal basket cells that Lorente de Nó described, having long radially oriented dendrites extending as far as stratum lacunosum-moleculare. The presence of putative inhibitory interneuron dendrites in stratum lacunosum-moleculare suggests some role other than traditional recurrent inhibition for these dendritic segments, and two possible circuits are described.

Afferent fibers to rat cingulate cortex

Afferent fibers to the rat cingulate cortex were studied by the retrograde labeling technique using horseradish peroxidase-wheat germ agglutinin conjugate as the tracer. The results showed that the posterior cingulate cortex, but not the anterior, received input from the anterior dorsal and anterior ventral nuclei of the anterior thalamic group of nuclei (part of the so-called limbic thalamus), and from the subicular complex. The anterior cingulate cortex, but not the posterior, received input from the mediodorsal and ventral thalamic nuclei. Both posterior and anterior cingulate cortex received input from the hippocampus pars anterior; claustrum; globus pallidus; nucleus of the diagonal band of Broca (a particularly reliable source of afferent fibers); anterior medial, lateral, rhomboid, and reuniens nuclei of the thalamus; region of the medial forebrain bundle; periventricular nucleus of the hypothalamus; the dorsal and median raphe; and the locus ceruleus. Corticocortical projections were seen anterior, posterior, and lateral to the injection site, and in the homologous contralateral cingulate cortex. The results demonstrate a prominent source of cingulate afferent fibers from the subicular complex, provide evidence for a functional division of anterior and posterior cingulate cortices in the rat, and provide information about the relative anatomic importance of cingulate afferent fibers from those different regions.

Demonstration of caudally directed hippocampal efferents in the rat by intracellular injection of horseradish peroxidase

Axonal projections of rat hippocampal neurons were demonstrated by intracellular injections of horseradish peroxidase. The data indicated a prominent caudally directed projection from pyramidal neurons of hippocampal field CA1, provided evidence that the subiculum is one of its targets, and suggested that the caudally directed efferents from CA1 are more numerous than rostrally directed ones.

Neurophysiology of limbic system pathways in the rat: Projections from the subicular complex and hippocampus to the entorhinal cortex

We studied the responses of rat entorhinal neurons to electrical stimulation of the dentate gyrus, hippocampus and subicular complex. Three main results were obtained. Excitatory postsynaptic potentials were recorded in entorhinal neurons in response to electrical stimulation. Cell in layers II, III and V of the entorhinal cortex were responsive. Frequency potentiation of excitatory responses was observed when 10/s stimulation was used. Excitatory responses were followed by inhibitory postsynaptic potentials. The results provide evidence for an excitatory projection from the hippocampus and subiculum to the entorhinal cortex, and are consistent with the existence of feed-forward inhibition of entorhinal principal neurons.

Convergence of projections from the rat hippocampal formation, medial geniculate and basal forebrain onto single amygdaloid neurons: an in vivo extra- and intracellular electrophysiological study

We recorded extra- and intracellular responses from rat amygdaloid neurons in vivo after electrical stimulation of the hippocampal formation (dentate gyrus, hippocampal fields CA3 and CA4, entorhinal cortex, subicular complex); medial geniculate; and basal forebrain (diagonal band, ventral pallidum, olfactory tubercle, nucleus accumbens, bed nucleus of stria terminalis, lateral preoptic area, substantia innominata). Stimulation of hippocampal formation structures evoked IPSPs or EPSP-IPSP sequences in which the IPSP had a lower threshold than the EPSP. Recordings from candidate inhibitory neurons in the amygdala indicated that excitatory afferents from the hippocampal formation contact both amygdaloid inhibitory and principal neurons (feedforward inhibition), and that the inhibitory neurons have a lower threshold of activation. Medial geniculate stimulation also evoked EPSP-IPSP sequences. In marked contrast to these results, stimulation of basal forebrain structures evoked short latency IPSPs in amygdaloid neurons. This provides the first physiological evidence for direct inhibition of the amygdala by the basal forebrain. Basal forebrain stimulation also evoked EPSP-IPSP sequences in amygdaloid neurons. Individual amygdaloid neurons could show responses to stimulation of the hippocampal formation, basal forebrain, and medial geniculate, indicating that synaptic input from these areas converges onto single amygdaloid cells. The findings provide further information about the synaptic organization of afferents to the amygdala, and indicate that single amygdaloid neurons play a role in the synaptic integration of input from these diverse sources.

Physiologic properties of human dentate granule cells in slices prepared from epileptic patients

The neurophysiological properties of human dentate granule cells were studied in hippocampal slices prepared from patients undergoing surgical treatment for medically intractable temporal lobe epilepsy. In 24 neurons which were morphologically identified as dentate granule cells by intracellular staining with biocytin, there were 2 types of synaptic responses to perforant path stimulation: one showed an EPSP-IPSP sequence (n = 10) and the other showed prolonged EPSPs without accompanying hyperpolarizing IPSPs (n = 14). The prolonged EPSPs were markedly retarded by the application of an NMDA receptor antagonist, APV. Membrane properties of neurons showing the different classes of synaptic responses were similar in resting membrane potential (pooled average: -56.2 mV +/- 0.94 SEM) and spike amplitude (pooled average: 65.2 mV +/- 1.69 SEM). However, membrane resistance tended to be lower in neurons with prolonged EPSPs (31.8 M omega +/- 2.63 SEM) than in neurons that showed EPSP-IPSP responses (40.2 +/- 4.33) (P less than 0.05, Fisher). No spontaneous and/or evoked burst firing was observed. These data provide fuller information on the neurophysiological properties of human dentate granule cells in surgically resected epileptogenic hippocampus, implicating a role of NMDA receptor activation in human temporal lobe epilepsy.

Neurophysiology of limbic system pathways in the rat: Projections from the amygdala to the entorhinal cortex

We studied the responses of rat entorhinal neurons to electrical stimulation of the amygdala. Four main results were obtained: (1) excitatory postsynaptic potentials were recorded in entorhinal neurons in response to electrical stimulation of the amygdala. Cells in layers II, III and V of the entorhinal cortex were responsive. (2) Excitatory responses were followed by inhibitory postsynaptic potentials. (3) Frequency potentiation of both excitatory and inhibitory responses was observed when 10/s stimulation was used. (4) Three amygdala neurons were antidromically activated by entorhinal stimulation; and two layer II entorhinal cells that were excited by amygdala stimulation were also antidromically activated by dentate gyrus stimulation. These results provide evidence for a monosynaptic, excitatory projection from the amygdala to the entorhinal cortex. In addition, the data indicate that amygdala neurons are only one synapse removed from the excitation of dentate gyrus granule cells.

Presynaptic enhancement of synaptic transmission in hippocampal cell cultures by phorbol esters

The calcium-diacyglycerol activated protein kinase (C kinase) plays an important role in synaptic plasticity, and possibly in long-term potentiation (LTP). We have found that phorbol esters, which activate this protein kinase, increase the frequency of miniature excitatory postsynaptic currents (mEPCs) in hippocampal cell cultures. An analysis of distributions of these miniature synaptic currents shows that amplitudes, and thus postsynaptic function, are not influenced by phorbol esters. Synaptic transmission is therefore enhanced by phorbol esters at a presynaptic locus. The method of analysis employed here is applicable to a broad range of situations involving modification of synaptic transmission in the mammalian central nervous system.