Fos induction and persistence, neurodegeneration, and interneuron activation in the hippocampus of epilepsy-resistant versus epilepsy-prone rats after pilocarpine-induced seizures

Previous studies demonstrated that the spiny rat Proechimys guyannensis exhibits resistance to experimental epilepsy. Neural activation was studied in the Proechimys hippocampus, using Fos induction, within 24 h after pilocarpine-induced seizures; neurodegenerative events were investigated in parallel, using FluoroJade B histochemistry. These parameters were selected since pilocarpine-induced limbic epilepsy is known to elicit immediate early gene expression and cell loss in the hippocampus of seizure-prone laboratory rodents. At variance with matched experiments in Wistar rats, pilocarpine injection resulted in Proechimys in seizure episodes that, as previously reported, did not develop into status epilepticus. At 3 h and 8 h after seizure onset, Fos immunoreactivity filled the dentate gyrus of both rat species, and was quite marked in pyramidal cells of the Proechimys Ammon's horn. At 24 h, Fos immunoreactivity dropped in the Wistar hippocampus and persisted in Proechimys. At 8 h and 24 h, FluoroJade-stained neurons were very few in the Proechimys hippocampus, whereas they were abundant in that of Wistar rats. Double immunohistochemistry for Fos and parvalbumin, the protein expressed by fast-spiking hippocampal interneurons, indicated that Fos was induced up to 24 h in the vast majority of parvalbumin-containing cells of the Proechimys hippocampus, and in a minority of these cells in the Wistar hippocampus. The findings demonstrate that early postepileptic neurodegeneration is very limited in the Proechimys hippocampus, in which sustained Fos induction persists for several hours. The findings also indicate that Fos induction and persistence may not correlate with seizure intensity and may not be associated with neuronal death. Finally, the data implicate differential mechanisms of interneuron activity in anti-convulsant and pro-convulsant phenomena.     

Selective changes in inhibition as determinants for limited hyperexcitability in the insular cortex of epileptic rats

The insular cortex (IC) is involved in the generalization of epileptic discharges in temporal lobe epilepsy (TLE), whereas seizures originating in the IC can mimic the epileptic phenotype seen in some patients with TLE. However, few studies have addressed the changes occurring in the IC in TLE animal models. Here, we analyzed the immunohistochemical and electrophysiological properties of IC networks in non‐epileptic control and pilocarpine‐treated epileptic rats. Neurons identified with a neuron‐specific nuclear protein antibody showed similar counts in the two types of tissue but parvalbumin‐ and neuropeptide Y‐positive interneurons were significantly decreased (parvalbumin, approximately ?35%; neuropeptide Y, approximately ?38%; P < 0.01) in the epileptic IC. Non‐adapting neurons were seen more frequently in the epileptic IC during intracellular injection of depolarizing current pulses. In addition, single‐shock electrical stimuli elicited network‐driven epileptiform responses in 87% of epileptic and 22% of non‐epileptic control neurons (P < 0.01) but spontaneous postsynaptic potentials had similar amplitude, duration and intervals of occurrence in the two groups. Finally, pharmacologically isolated, GABA_A receptor‐mediated inhibitory postsynaptic potentials had more negative reversal potential (P < 0.01) and higher peak conductance (P < 0.05) in epileptic tissue. These data reveal moderate increased network excitability in the IC of pilocarpine‐treated epileptic rats. We propose that this limited degree of hyperexcitability originates from the loss of parvalbumin‐ and neuropeptide Y‐positive interneurons that is compensated by an increased drive for GABA_A receptor‐mediated inhibition.     

Perirhinal cortex hyperexcitability in pilocarpine‐treated epileptic rats

The perirhinal cortex (PC), which is heavily connected with several epileptogenic regions of the limbic system such as the entorhinal cortex and amygdala, is involved in the generation and spread of seizures. However, the functional alterations occurring within an epileptic PC network are unknown. Here, we analyzed this issue by using in vitro electrophysiology and immunohistochemistry in brain tissue obtained from pilocarpine‐treated epileptic rats and age‐matched, nonepileptic controls (NECs). Neurons recorded intracellularly from the PC deep layers in the two experimental groups had similar intrinsic and firing properties and generated spontaneous depolarizing and hyperpolarizing postsynaptic potentials with comparable duration and amplitude. However, spontaneous and stimulus‐induced epileptiform discharges were seen with field potential recordings in over one‐fifth of pilocarpine‐treated slices but never in NEC tissue. These network events were reduced in duration by antagonizing NMDA receptors and abolished by NMDA + non‐NMDA glutamatergic receptor antagonists. Pharmacologically isolated isolated inhibitory postsynaptic potentials had reversal potentials for the early GABA_A receptor‐mediated component that were significantly more depolarized in pilocarpine‐treated cells. Experiments with a potassium‐chloride cotransporter 2 antibody identified, in pilocarpine‐treated PC, a significant immunostaining decrease that could not be explained by neuronal loss. However, interneurons expressing parvalbumin and neuropeptide Y were found to be decreased throughout the PC, whereas cholecystokinin‐positive cells were diminished in superficial layers. These findings demonstrate synaptic hyperexcitability that is contributed by attenuated inhibition in the PC of pilocarpine‐treated epileptic rats and underscore the role of PC networks in temporal lobe epilepsy. © 2010 Wiley‐Liss, Inc.     

Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures

In the pilocarpine model of chronic limbic seizures, vulnerability of GABAergic interneurons to excitotoxic damage has been reported in the hippocampal CA1 region. However, little is known about the specific types of interneurons that degenerate in this region. In order to characterize these interneurons, we performed quantitative analyses of the different populations of GABAergic neurons labeled for their peptide or calcium-binding protein content. Our data demonstrate that the decrease in the number of GAD mRNA-containing neurons in the stratum oriens of CA1 in pilocarpine-treated rats involved two subpopulations of GABAergic interneurons: interneurons labeled for somatostatin only (O-LM and bistratified cells) and interneurons labeled for parvalbumin only (basket and axo-axonic cells). Stratum oriens interneurons labeled for somatostatin/calbindin or somatostatin/parvalbumin were preserved. The decrease in number of somatostatin- and parvalbumin-containing neurons was observed as early as 72 hours after the sustained seizures induced by pilocarpine injection. Many degenerating cell bodies in the stratum oriens and degenerating axon terminals in the stratum lacunosum-moleculare were observed at 1 and 2 weeks after injection. In addition, the synaptic coverage of the axon initial segment of CA1 pyramidal cells was significantly decreased in pilocarpine-treated animals. These results indicate that the loss of somatostatin-containing neurons corresponds preferentially to the degeneration of interneurons with an axon projecting to stratum lacunosum-moleculare (O-LM cells) and suggest that the death of these neurons is mainly responsible for the deficit of dendritic inhibition reported in this region. We demonstrate that the loss of parvalbumin-containing neurons corresponds to the death of axo-axonic cells, suggesting that perisomatic inhibition and mechanisms controlling action potential generation are also impaired in this model.     

Fos induction in subtypes of cerebrocortical neurons following single picrotoxin-induced seizures

In adult rats single seizures of varying behavioural severities were caused by slow, systemic infusion of picrotoxin, an antagonist of the Cl⁻ channel at the GABA_A receptor. We used a double labelling immunohistochemical method to define the subclasses of neurons that contained Fos protein following seizures. In four cortical regions (piriform, entorhinal, motor and sensory) neuronal subclasses were defined with antibodies against the calcium-binding proteins calbindin D-28K, parvalbumin and calretinin (aspiny neurons), and neurofilament protein (spiny neurons). The remaining spiny neuron population was estimated by subtraction of defined subclasses from total neuronal numbers determined from Nissl stain. After seizures, most of the calbindin D-28K immunoreactive interneurons (> 80%) and many of the unlabelled spiny neurons (60–80%) were Fos positive. Co-localisation of Fos was found in about 30% of parvalbumin, calretinin and neurofilament protein immunoreactive neurons. Paradoxically, mild seizures were associated with induction of Fos in up to 80% of cortical cells and more severe seizures with 60%, the difference being due to different levels of Fos induction in spiny neurons. These results also demonstrate that seizures induce Fos predominantly in excitatory cortical neurons.     

Fos induction in cortical interneurons during spontaneous wakefulness of rats in a familiar or enriched environment

It has been repeatedly reported that Fos is spontaneously induced in several brain structures, including the cerebral cortex, during wakefulness. To ascertain whether cortical interneurons are involved in this state-dependent oscillation of gene regulation, we combined Fos immunocytochemistry with immunostaining of either parvalbumin or calbindin, known markers of cortical interneurons. Immunopositive neurons were examined in the sensorimotor and cingulate cortex. In rats perfused in basal conditions, a minor proportion (around 8%) of Fos-immunoreactive neurons in the parietal cortex were also parvalbumin- or calbindin-immunoreactive; these double immunostained cells accounted for 13% of the parvalbumin- and 34% of the calbindin-labeled neurons. Colocalization of Fos with either calcium-binding protein was instead not observed in the cingulate cortex. In rats stimulated by novel environmental cues during the period of wakefulness preceding perfusion, Fos-positive neurons increased markedly relative to unstimulated animals, and involved the majority of the calbindin- or parvalbumin-labeled cell populations (60-75% and over 95%, respectively). In the neuronal populations in which Fos was induced by exposure to the enriched environment, the proportion of calbindin- and parvalbumin-labeled cells was larger than in the unstimulated cases, and the increment was statistically significant in the cingulate cortex. The results demonstrate that Fos induction occurring in the cortex during undisturbed wakefulness in a familiar environment involves a minor proportion of interneurons. Furthermore, the findings indicate that the addition of novel environmental stimuli results in an increase of Fos-expressing neurons whose recruitment, at least in the cingulate cortex, involves a higher proportion of interneurons than of projection neurons.     

Interneuron activity leads to initiation of low‐voltage fast‐onset seizures

Seizures in temporal lobe epilepsy can be classified as hypersynchronous and low‐voltage fast according to their onset patterns. Experimental evidence suggests that low‐voltage fast‐onset seizures mainly result from the synchronous activity of γ‐aminobutyric acid–releasing cells. In this study, we tested this hypothesis using the optogenetic control of parvalbumin‐positive interneurons in the entorhinal cortex, in the in vitro 4‐aminopyridine model. We found that both spontaneous and optogenetically induced seizures had similar low‐voltage fast‐onset patterns. In addition, both types of seizures presented with higher ripple than fast ripple rates. Our data demonstrate the involvement of interneuronal networks in the initiation of low‐voltage fast‐onset seizures. Ann Neurol 2015;77:541–546     

Limbic seizures produced by pilocarpine in rats: behavioural, electroencephalographic and neuropathological study

Behavioural, electroencephalographic and neuropathological responses to increasing doses of pilocarpine (100-400 mg/kg) administered intraperitoneally to rats were studied. At the dose of 400 mg/kg pilocarpine produced a sequence of behavioural alterations including staring spells, olfactory and gustatory automatisms and motor limbic seizures that developed over 1-2 h and built up progressively into limbic status epilepticus. Smaller doses showed different threshold for these behavioural phenomena but a similar time course of development. The earliest electrographic alterations occurred in the hippocampus and then epileptiform activity propagated to amygdala and cortex. Subsequently electrographic seizures appeared in both limbic and cortical leads. The ictal periods recurred each 5-15 min and were followed by variable periods of depression of the electrographic activity. The sequence of electrographic changes correlated well with the development of behavioural phenomena. Histological examination of frontal forebrain sections revealed disseminated, apparently seizure-mediated pattern of brain damage. Neuropathological alterations were observed in the olfactory cortex, amygdaloid complex, thalamus, neocortex, hippocampal formation and substantia nigra. Pretreatment of animals with scopolamine (20 mg/kg) and diazepam (10 mg/kg) prevented the development of convulsive activity and brain damage. These results show that systemic pilocarpine in rats selectively elaborates epileptiform activity in the limbic structures accompanied by motor limbic seizures, limbic status epilepticus and widespread brain damage. It is suggested that a causative relationship between excessive stimulation of cholinergic receptors in the brain and epileptic brain damage may exist.     

Comparison of numbers of interneurons in three thalamic nuclei of normal and epileptic rats

The inhibitory sources in the thalamic nuclei are local interneurons and neurons of the thalamic reticular nucleus. Studies of models of absence epilepsy have shown that the seizures are associated with an excess of inhibitory neurotransmission in the thalamus. In the present study, we used light-microscopic gamma-aminobutyric acid (GABA) immunocytochemistry to quantify the interneurons in the lateral geniculate (LGN), ventral posteromedial (VPM), and ventral posterolateral (VPL) thalamic nuclei, and compared the values from normal Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS). We found that in both Wistar rats and GAERS, the proportion of interneurons was significantly higher in the LGN than in the VPM and VPL. In the LGN of Wistar rats, 16.4% of the neurons were interneurons and in the GAERS, the value was 15.1%. In the VPM, the proportion of interneurons was 4.2% in Wistar and 14.9% in GAERS; in the VPL the values were 3.7% for Wistar and 11.1% for the GAERS. There was no significant difference between Wistar rats and the GAERS regarding the counts of interneurons in the LGN, whereas the VPM and VPL showed significantly higher counts in GAERS. Comparison of the mean areas of both relay cells and interneuronal profiles showed no significant differences between Wistar rats and GAERS. These findings show that in the VPL and the VPM there are relatively more GABAergic interneurons in GAERS than in Wistar rats. This may represent a compensatory response of the thalamocortical circuitry to the absence seizures or may be related to the production of absence seizures.     

The role of the inherited genetic background on the consequences of lithium-pilocarpine status epilepticus: study in Genetic Absence Epilepsy Rats from Strasbourg and Wistar audiogenic rats

The susceptibility of rats with genetically inherited epilepsy to the genesis and consequences of secondary temporal lobe epilepsy is unknown. Here, we induced lithium-pilocarpine status epilepticus (SE) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) or in Wistar audiogenic sensitive (AS) rats. Wistar AS needed less pilocarpine than GAERS and Non-Epileptic Rats (NERs) to develop SE. Sixty six, 40 and 5% of Wistar AS, GAERS and NERs, respectively, died within 24 h after SE. In GAERS, SE prevented the occurrence of absence seizures for 5 days. Thereafter a limited number of absence seizures with low amplitude and short duration were recorded. Wistar AS developed limbic epilepsy within 9 days after SE while GAERS and NERs needed 36-39 days to develop spontaneous motor seizures. Neuronal loss consecutive to SE was similar in the three strains and particularly marked in limbic forebrain and parahippocampal cortices. In conclusion, the development of focal limbic epilepsy in GAERS largely impairs the expression of absence seizures. The genetic background underlying the expression of audiogenic seizures sensitizes strongly the rats to a further insult and compromises their survival.     

Long‐term changes in dopamine‐stimulated gene expression after single‐day methamphetamine exposure

Methamphetamine (mAMPH) is a highly addictive psychostimulant drug that injures monoaminergic neurons and results in behavioral impairments in humans and animals. Although evidence exists for changes in cortical volume, metabolism, and blood oxygenation levels in human mAMPH abusers, animal models have instead emphasized this drug's long‐lasting influence on ascending monoaminergic (dopamine, serotonin) projections. The aim of this study was to investigate cortical and subcortical function in rats long after administration of a single‐day mAMPH regimen known to damage monoaminergic systems, at a time point when behavioral impairments are still evident. Rats were given either saline or a neurotoxic (4 × 4 mg/kg, sc) mAMPH regimen. Five weeks later, they were given pharmacological treatments that stimulate cortical gene expression: either the dopaminergic agonist apomorphine (3 mg/kg, sc) or the muscarinic acetylcholine agonist pilocarpine (25 mg/kg, ip). Cortical and subcortical immediate early gene (IEG) responses were measured by immunocytochemical analysis of Fos or JunB, protein products of the IEGs, c‐fos and junB. Compared with saline‐pretreated controls, mAMPH‐pretreated animals had about 50–70% fewer Fos‐ and JunB‐immunoreactive cells in anterior cingulate, infralimbic, orbital, somatosensory, and rhinal cortices as well as caudate‐putamen and nucleus accumbens, 90 min after apomorphine challenge. By contrast, mAMPH‐pretreated rats had no reductions in the numbers of Fos or JunB‐positive cells following pilocarpine challenge. This study demonstrates the profound and enduring effects of mAMPH administration on dopamine‐stimulated cortical function in animals. Synapse 63:403–412, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanisms of status epilepticus: α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor hypothesis

Prolonged seizures of status epilepticus (SE) result from failure of mechanisms of seizure termination or activation of mechanisms that sustain seizures. Reduced γ‐aminobutyric acid type A receptor–mediated synaptic transmission contributes to impairment of seizure termination. However, mechanisms that sustain prolonged seizures are not known. We propose that insertion of GluA1 subunits at the glutamatergic synapses causes potentiation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptor (AMPAR)‐mediated neurotransmission, which helps to spread and sustain seizures. The AMPAR‐mediated neurotransmission of CA1 pyramidal neurons was increased in animals in SE induced by pilocarpine. The surface membrane expression of GluA1 subunit–containing AMPARs on CA1 pyramidal neurons was also increased. Blockade of N‐methyl‐d ‐aspartate receptors 10 minutes after the onset of continuous electrographic seizure activity prevented the increase in the surface expression of GluA1 subunits. N‐methyl‐d ‐aspartate receptor antagonist MK‐801 in conjunction with diazepam also terminated seizures that were refractory to MK‐801 or diazepam alone. Future studies using mice lacking the GluA1 subunit expression will provide further insights into the role of GluA1 subunit–containing AMPAR plasticity in sustaining seizures of SE.     

Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats

Mesiotemporal sclerosis (MTS), the most frequent form of drug‐resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)‐induced changes in expression of key components in the glutamate–glutamine cycle, known to be affected in MTS patients. SE was induced by Li⁺/pilocarpine injection in 21‐day‐old rats. At 2–19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15–18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin‐positive GABAergic interneurons. Hilar neuron loss (or end‐folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)‐immunoreactivity from 2 (?15%) to 19 weeks (?33.5%) after SE. Immunoreactivity for excitatory amino‐acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21‐day‐old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate–glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.     

Nuclear translocation of mitochondrial cytochrome c,lysosomal cathepsins B and D,and three other death‐promoting proteins within the first 60 minutes of generalized seizures

We have shown that generalized seizures produce necrotic neurons with caspase‐independent nuclear pyknosis and DNA fragmentation. In this study, we determined the time course of translocation of mitochondrial cytochrome c, apoptosis‐inducing factor, endonuclease G, lysosomal cathepsins B and D, and DNase II with respect to signs of irreversible neuronal damage. Adult male Wistar rats underwent lithium‐pilocarpine‐induced seizures lasting for 60 min, 3 hr, and 3 hr with 6‐ or 24‐hr survival periods, after which the brains were prepared for immunofluorescence microscopic examination of piriform cortex. Contrary to expectation, cytochrome c and cathepsins B and D translocated to neuronal nuclei with DNase II, endonuclease G, and apoptosis‐inducing factor within 60 min of seizure onset and persisted for 24 hr after 3‐hr seizures. After 60‐min seizures, some neurons showed translocation of the death‐promoting proteins in normal‐appearing neurons, prior to their appearance in irreversibly damaged neurons. Western blots of subcellular fractions of cytochrome c and cathepsins B and D confirmed their nuclear translocation. This is the first evidence of nuclear translocation of cathepsins B and D and the first in vivo evidence of nuclear translocation of cytochrome c. The appearance of these mitochondrial proteins and lysosomal enzymes before signs of irreversible neuronal death suggests that they could contribute to seizure‐induced nuclear pyknosis and DNA fragmentation. © 2010 Wiley‐Liss, Inc.     

Axonal sprouting in commissurally projecting parvalbumin‐expressing interneurons

Previous research has shown that in vivo on‐demand optogenetic stimulation of inhibitory interneurons expressing parvalbumin (PV) is sufficient to suppress seizures in a mouse model of temporal lobe epilepsy (TLE). Surprisingly, this intervention was capable of suppressing seizures when PV‐expressing interneurons were stimulated ipsilateral or contralateral to the presumed seizure focus, raising the possibility of commissural inhibition in TLE. There are mixed reports regarding commissural PV interneuron projections in the healthy hippocampus, and it was previously unknown whether these connections would be maintained or modified following the network reorganization associated with TLE. Using retrograde labeling and viral vector technology in both sexes and the intrahippocampal kainate mouse model of TLE, we therefore examined these issues. Our results reveal that healthy controls possess a population of commissurally projecting hippocampal PV interneurons. Two weeks post kainate injection, we observed a slight, but not statistically significant decrease in retrogradely labeled PV interneurons in the hippocampus contralateral to kainate and tracer injection. By 6 months post kainate, however, there was a significant increase in retrogradely labeled PV interneurons, suggesting commissural inhibitory axonal sprouting. Using viral green fluorescent protein expression selectively in PV neurons, we demonstrated sprouting of commissural PV projections in the dentate gyrus of the kainate‐injected hippocampus 6 months post kainate. These findings indicate that PV interneurons supply direct inhibition to the contralateral hippocampus and undergo sprouting in a mouse model of TLE. © 2017 Wiley Periodicals, Inc.     

Antiepileptic drugs combined with high‐frequency electrical stimulation in the ventral hippocampus modify pilocarpine‐induced status epilepticus in rats

Purpose: To evaluate the effects of high‐frequency electrical stimulation (HFS) in both ventral hippocampi, alone and combined with a subeffective dose of antiepileptic drugs, during the status epilepticus (SE) induced by lithium‐pilocarpine (LP). Methods: Male Wistar rats, stereotactically implanted in both ventral hippocampi, were injected with pilocarpine (30 mg/kg, i.p.) 24 h after lithium (3 mEq/kg) administration. One minute following pilocarpine injection, HFS (pulses of 60 μs width at 130 Hz at subthreshold intensities and applied during 3 h) was applied alone or combined with subeffective doses of antiepileptic drugs. Results: HFS alone reduced the incidence of severe generalized seizures. This effect was not evident when HFS was combined with phenytoin (33.3 mg/kg, i.p.). HFS combined with diazepam (0.41 mg/kg, i.p.) or phenobarbital (10 mg/kg, i.p.) reduced the incidence of severe generalized seizures and mortality rate, and augmented the latency to first forelimb clonus, generalized seizure, and status epilepticus (SE). When combined with gabapentin (46 mg/kg, i.p.), HFS reduced the incidence of severe generalized seizures, enhanced latency to SE, and decreased mortality rate. Discussion: Subeffective doses of antiepileptic drugs that increase the γ‐aminobutyric acid (GABA)ergic neurotransmission may represent a therapeutic tool to augment the HFS‐induced anticonvulsant effects.     

Disinhibition of the mediodorsal thalamus induces Fos-like immunoreactivity in both pyramidal and GABA-containing neurons in the medial prefrontal cortex of rats,but does not affect prefrontal extracellular GABA levels

Stimulation of the mediodorsal and midline thalamic nuclei excites cortical neurons and induces c-fos expression in the prefrontal cortex. Data in the literature data suggest that pyramidal neurons are the most likely cellular targets. In order to determine whether cortical interneurons are also impacted by activation of mediodorsal/midline thalamic nuclei, we studied the effects of thalamic stimulation on (1) Fos protein expression in γ-aminobutyric acid (GABA)-immunoreactive neurons and on (2) extracellular GABA levels in the prefrontal cortex of rats. Perfusion of the GABA-A receptor antagonist bicuculline for 20 minutes through a dialysis probe implanted into the mediodorsal thalamus induced Fos-like immunoreactivity (IR) approximately 1 hour later in the thalamus and in the medial prefrontal cortex of freely moving rats. Immunohistochemical double-labeling for Fos-like IR and GABA-like IR showed that about 8% of Fos-like IR nuclei in the prelimbic and infralimbic areas were located in GABA-like IR neurons. Fos-like IR was detected in three major subsets of GABAergic neurons defined by calbindin, parvalbumin, or vasoactive intestinal peptide (VIP)-like IR. Dual probe dialysis showed that the extracellular levels of GABA in the prefrontal cortex did not change in response to thalamic stimulation. These data indicate that activation of thalamocortical neurons indeed affects the activity of GABAergic neurons as shown by the induction of Fos-like IR but that these metabolic changes are not reflected in changes of extracellular GABA levels that are sampled by microdialysis. Synapse 30:156–165, 1998. © 1998 Wiley-Liss, Inc.     

Expression of m1‐type muscarinic acetylcholine receptors by parvalbumin‐immunoreactive neurons in the primary visual cortex: A comparative study of rat,guinea pig,ferret, macaque,and human

Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin‐immunoreactive (PV) neurons in macaque V1 express the m1‐type (pirenzepine‐sensitive, Gq‐coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine‐sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV‐ and mAChR‐immunoreactive somata by dual‐immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV‐ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ～25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq‐coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. J. Comp. Neurol. 522:986–1003, 2014. © 2013 Wiley Periodicals, Inc.     

Deletion of the citron kinase gene selectively affects the number and distribution of interneurons in barrelfield cortex

Citron kinase (CIT‐K), a ser/thr kinase, is required during neurogenesis for cytokinesis of neuronal precursors. Deletion of the cit‐k gene in mice (cit‐k^?/? mice) leads to a severe malformative central nervous system syndrome characterized by microencephaly, ataxia, and epileptic seizures; affected mice die by the third week of postnatal life. We have used NADPH‐diaphorase histochemistry, immunostaining for calbindin, calretinin, parvalbumin, and glutamic acid decarboxylase 67 (GAD67), and histological staining to undertake qualitative and quantitative analyses of the morphology and distribution of interneurons in the barrelfield cortex of cit‐k^?/? mice. By postnatal day 13, lack of CIT‐K results in profoundly altered cortical cell morphology: the infragranular layers are populated by large, binucleate interneurons bearing many more dendrites than in control mice, an anatomical profile that has also been reported for the cortex of humans with cortical dysplasias and epilepsy. Tessellation analyses reveal that a clustered distribution of interneurons is maintained in cit‐k^?/? mice, but that their nearest neighbor distance is significantly increased, and thus their density is reduced; the overall number of interneurons is more dramatically decreased in the absence of CIT‐K than would be predicted on the basis of the reduced brain size of affected mice. This reduction of inhibitory γ‐aminobutyric acid (GABA)ergic neurons likely underlies the occurrence of epileptic seizures in the cit‐k^?/? mice. Furthermore, the altered distribution of NADPH‐diaphorase‐positive interneurons could be responsible for an impaired coupling of cortical activity to blood flow, also affecting cortical growth and functioning. J. Comp. Neurol. 513:249–264, 2009. © 2009 Wiley‐Liss, Inc.     

Laminar Distribution of Fos/Calcium-Binding Protein and Fos/Neurofilament Protein-Labeled Neurons in Rat Motor and Sensory Cortex after Picrotoxin-Induced Seizures

Cerebrocortical Fos induction after picrotoxin-induced seizure occurs in spiny neurons and, to a lesser extent, in neurons defined by calcium-binding protein immunoreactivity. In motor and sensory cortex of rats we have defined the laminar distribution of Fos expression in these neurons. Initially we defined the laminar distributions of parvalbumin-, calbindin-D 28K-, and calretinin-immunoreactive aspiny neurons; these were unique for each class and similar across cortical regions. Spiny cells defined by SMI32 immunoreactivity were distributed with two peaks and there were differences between cortical regions. Parvalbumin-immunoreactive neurons exhibited peak numbers where numbers of SMI32-immunoreactive neurons were low. The distribution of Fos induction across laminae matched that of its class for calbindin-D 28K and calretinin neurons; however, Fos induction was less in infragranular compared with supragranular for parvalbumin in motor cortex and SMI32 containing neurons in both cortices. In both these latter cell classes Fos induction was inversely correlated with neuronal size. It is suggested that cell size within some cell classes is one factor that determines the extent of Fos induction within that class following seizures.