Aiko M. Tan

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.

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.

Neurophysiology and neuropharmacology of projections from entorhinal cortex to striatum in the rat.

We studied projections from the entorhinal cortex (Ent) to the striatum in anesthetized rats using extra- and intracellular recording and multibarrel iontophoresis. The majority of recording were from the caudate-putamen (CPu) and core of the nucleus accumbens (AcbC). Electrical stimulation of the Ent evoked synaptic responses in 77% of tests with AcbC neurons and 48% of tests with CPu neurons. In the case of AcbC neurons, 61% of these tests proved to be excitatory and were often followed by inhibitory phases. In contrast to this, only 18% of tests from CPu neurons were excitatory. Intracellular HRP labeling showed that responsive cells were medium spiny neurons. During iontophoretic experiments, application of the glutamatergic AMPA antagonist DNQX could selectively decrease or block excitatory responses. The GABAA antagonist bicuculline methiodide increased cellular firing rates and could reveal excitatory responses, suggesting block of a short-latency, short-duration inhibitory component. Ejection of the GABAB antagonist CGP-35348 could attenuate a later, longer-duration component of inhibition. The results indicate that the Ent excites striatal neurons at least in part by glutamatergic receptors and suggest that this excitation is followed by secondary prolonged GABAergic inhibition.

GABAergic synaptic transmission in projections from the basal forebrain and hippocampal formation to the amygdala : an in vivo iontophoretic study

We recorded extracellular responses from rat amygdaloid neurons in vivo after electrical stimulation of the basal forebrain and hippocampal formation. Iontophoretic application of the GABAA receptor antagonist, bicuculline, lead to the appearance of short latency evoked bursts after stimulation of either region. This occurred whether the baseline response was inhibitory or excitatory. Bicuculline only affected an early phase of inhibition, leaving a longer latency, longer duration phase unchanged or even increased. By contrast, the GABAB receptor antagonist, phaclofen, never produced such short latency evoked bursts. Both bicuculline and phaclofen increased the spontaneous rate of firing of amygdaloid neurons. The excitatory burst response to hippocampal formation stimulation of an amygdaloid candidate inhibitory neuron was blocked by CNQX (an antagonist of the AMPA subtype of glutamate receptor). Based on these and prior studies, it seems likely that the effects of hippocampal formation stimulation are mediated by feed-forward inhibition, in which GABAergic amygdaloid inhibitory neurons are excited by glutamatergic projections from the hippocampal formation. The effects of basal forebrain stimulation may be mediated by both feed-forward inhibition and direct, GABAergic inhibition.

Functional reciprocal connections of the rat entorhinal cortex and subicular complex with the medial frontal cortex: an in vivo intracellular study

We used in vivo intracellular recording techniques in the rat in order to determine the properties of projections from the medial frontal cortex to the entorhinal cortex and subicular complex. Three main results were obtained. (1) A high proportion (65%) of neurons within the medial frontal cortex were antidromically activated at short latency (0.4-1.9 ms) by electrical stimulation of the entorhinal cortex or subicular complex. This provided physiological evidence for fast direct projections from the medial frontal cortex to the entorhinal cortex and subicular complex. (2) Clear excitatory postsynaptic potentials (EPSPs) were evoked in 8% of the cells within the entorhinal cortex, subicular complex, or adjacent cortices after electrical stimulation of the medial frontal cortex. (3) The most salient synaptic response was inhibition, as shown by the presence of inhibitory postsynaptic potentials (IPSPs) in 50% of the cells sampled. Similar results were obtained for the reciprocal pathway: 56% of the sampled cells in the entorhinal cortex or subicular complex responded with antidromic spikes to stimulation of the medial cortex; 4% of medial frontal neurons responded to stimulation of the entorhinal cortex or subicular complex with clear EPSPs, and 48% with IPSPs. The latencies of most synaptic responses, 15-25 ms, were inconsistent with monosynaptic activation. This suggests that oligosynaptic relays amplified the signal within or en route to their targets, and/or that cells with more slowly propagating axons were also present but not sampled by the intracellular electrodes. Finally, responsive fast-spiking cells (candidate inhibitory neurons) were encountered within target structures. The results provide evidence that these distant cortical regions are functionally connected in a reciprocal manner, and that both principal and inhibitory neurons are excited by this projection system.

Lack of Fos-like immunoreactivity after spontaneous seizures or reinduction of status epilepticus by pilocarpine in rats.

Acute seizures and status epilepticus induced by pilocarpine lead to the expression of Fos-like immunoreactivity in several specific brain areas in a manner similar to that of other models of limbic seizures. Upon development of status epilepticus after systemic pilocarpine injection, animals develop a state where chronic spontaneous seizures recur. Assessment of Fos-like immunoreactivity after such spontaneous seizures or after status epilepticus reinduction reveals either lack of staining or a weak reaction in a few brain areas including the ventral tip of the dentate gyrus, prepiriform, lateral piriform and perirhinal cortices, and scattered locations throughout temporal neocortex. Our results suggest that status epilepticus induction may lead to a long-lasting state of Fos down-regulation.

Functional coupling, desensitization and internalization of virally expressed μ opioid receptors in cultured dorsal root ganglion neurons from μ opioid receptor knockout mice

Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.

Functional connections of the rat medial cortex and basal forebrain: an in vivo intracellular study.

Projections between the medial cortex and basal forebrain in the rat were demonstrated by intracellular recordings and the anterograde tracer Phaseolus vulgaris leucoagglutinin. Direct projections between these areas were indicated by antidromic action potentials, short latency (less than 5 ms) orthodromic potentials, and labeled axon terminals in the basal forebrain subsequent to iontophoresis of Phaseolus vulgaris leucoagglutinin into posterior cingulate cortex. High proportions of antidromic action potentials were encountered in responsive cortical neurons (66%) and basal forebrain neurons (97%). Antidromic latencies recorded in the basal forebrain (less than 1.0 ms) revealed fast ascending projections; cortical neurons showed both fast and slow descending projections (latencies of 0.3-3.7 ms). Relatively few synaptic potentials (none in the diagonal band of Broca) and sparse labeling of axon terminals observed in the basal forebrain indicated that the ascending projections may be the more physiologically important or, at least, densest pathway. Polysynaptic feedforward pathways were suggested through long latency (greater than 20 ms) inhibitory and excitatory postsynaptic potentials, the former being the more common response. Candidate inhibitory neurons were identified in both cortex and basal forebrain. Possible monosynaptic (less than 5 ms) inhibitory postsynaptic and antidromic responses in these cells provided evidence that candidate inhibitory neurons participate in the reciprocal pathways.