Sanjay Khanna

Immunolocalization of NMDA receptor subunit NR3B in selected structures in the rat forebrain, cerebellum, and lumbar spinal cord

N‐methyl‐D‐aspartate (NMDA) receptors have been implicated in many neurological disorders. Although NMDA receptors are best known for their high calcium permeability, the recently discovered NR3 subunits, NR3A and NR3B, have been shown to reduce the calcium permeability of the NMDA receptor. Thus, NR3 subunits may be important players in modulating synaptic plasticity in neurons. Although NR3B expression in the rodent and human brain has been studied, little is known about its distribution in different cell types. Here we used immunolabeling with a specific NR3B antibody together with antibodies against established neurochemical markers to determine the cellular and subcellular localization of NR3B. The nucleus was concurrently stained with NR3B immunolabeling to show that NR3B is widely expressed by many cells in each brain region. Our findings indicate that NR3B is widely expressed in the structures examined in the rat forebrain (hippocampus, cerebral cortex, caudoputamen, and nucleus accumbens), cerebellum, and lumbar sections of the spinal cord. Within these regions NR3B was found to be expressed in all the substructures of the hippocampus (CA1, CA3, dentate gyrus), the various layers of the cerebral cortex, projection neurons and interneurons of the striatum, different cell types of the cerebellum, and motor neurons of the spinal cord. Furthermore, when stained with NR1—the obligatory subunit responsible for forming functional NMDA receptors—the distribution of NR3B appears to be as ubiquitous as NR1. Taken together, our data suggest that there may be a population of NR3B‐containing NMDA receptors conferring new functional roles in the mammalian central nervous system. J. Comp. Neurol. 509:118–135, 2008.

A nociceptive-intensity-dependent role for hydrogen sulphide in the formalin model of persistent inflammatory pain

The present study investigated the hypothesis that hydrogen sulfide (H2S) is pro-nociceptive in the formalin model of persistent inflammatory pain in the adult rat. Hind paw injection of formalin evoked a concentration-dependent increase in the hind paw concentration of H2S. Increased concentration of H2S was found in homogenates prepared from hind paws injected with 5% (but not 1.25%) formalin. Correspondingly, animal nociceptive flinching and hind paw edema were maximal with 5% formalin. Both nociceptive flinching and hind paw edema induced by injection of 5% formalin were attenuated by pretreatment with DL-propargylglycine (PPG; 50 mg/kg, i.p.) which is an inhibitor of the H2S synthesizing enzyme cystathionine-gamma-lyase (CSE). The effect of pretreatment with PPG was selective and the drug did not influence animal behavior or hind-paw edema with injection of 1.25% formalin. Furthermore, PPG pretreatment attenuated the induction of c-Fos in spinal laminae I-II following injection of 5% formalin. In contrast, co-injection of 1.25% formalin with sodium hydrogen sulfide (NaHS; 1 nmol/0.1 ml), a H2S donor, into the hind paw increased animal nociceptive behavior. Collectively, these findings show that the effect of peripheral H2S in the pathogenesis of inflammatory pain depends, at least in part, on the nociceptive intensity level.

Nociception-driven decreased induction of Fos protein in ventral hippocampus field CA1 of the rat

To test the hypothesis that the hippocampus field CA1 is recruited in nociceptive intensity-dependent fashion in the formalin model of inflammatory pain, we determined the effect of injection of formalin (0.625-2.5%) on the induction of Fos protein along the length of the hippocampus. Compared to injection of saline, injection of formalin (0.625-2.5%) evoked a concentration-dependent increase in nociceptive behavior and a significant linear increase in the number of Fos-positive cells in the spinal cord, especially in the deeper laminae. Injection of saline also increased induction of Fos along the length of hippocampus. On the other hand, injection of formalin decreased the number of Fos-positive cells in whole CA1, CA3 and dentate gyrus, with a greater significant effect in the posterior-ventral regions of the hippocampus. Indeed, a formalin concentration-dependent decrease was observed in the ventral CA1. A systematic pattern of change in Fos induction was not observed in the medial septum region. Of the regions examined, only the formalin-induced changes in Fos cell counts in the posterior and ventral CA1 were tightly correlated with the changes observed in the spinal cord. The foregoing findings suggest that nociceptive information is processed in distributed fashion by the hippocampus, and at least the ventral CA1 is implicated in nociceptive intensity-dependent integrative functions.

Glutamate receptor 1‐immunopositive neurons in the gliotic CA1 area of the mouse hippocampus after pilocarpine‐induced status epilepticus

Significant reduction in glutamate receptor 1 (GluR1)‐ and GluR2/3‐immunopositive neurons was demonstrated in the hilus of the dentate gyrus in mice killed on days 1, 7 and 60 after pilocarpine‐induced status epilepticus (PISE). In addition, GluR1 and GluR2/3 immunostaining in the strata oriens, radiatum and lacunosum moleculare of areas CA1–3 decreased drastically on days 7 and 60 after PISE. Neuronal loss observed in the above regions may account, at least in part, for a decrease in GluR immunoreactivity. By contrast, many GluR1‐immunopositive neurons were observed in the gliotic area of CA1. Of these, about 42.8% were immunopositive for markers for hippocampal interneurons, namely calretinin (7.6%), calbindin (12.8%) and parvalbumin (22.4%). GluR1 or GluR2/3 and BrdU double‐labelling showed that the GluR1‐ and GluR2/3‐immunopositive neurons at 60 days after PISE were neurons that had survived rather than newly generated neurons. Furthermore, anterograde tracer and double‐labelling studies performed on animals at 60 days after PISE indicated a projection from the hilus of the dentate gyrus to gliotic areas in both CA3 and CA1, where the projecting fibres apparently established connections with GluR1‐immunopositive neurons. The projection to CA1 was unexpected. These novel findings suggest that the intrinsic hippocampal neuronal network is altered after PISE. We speculate that GluR1‐immunopositive neurons in gliotic CA1 act as a bridge between dentate gyrus and subiculum contributing towards epileptogenesis.

Selective destruction of medial septal cholinergic neurons attenuates pyramidal cell suppression, but not excitation in dorsal hippocampus field CA1 induced by subcutaneous injection of formalin.

Using extracellular recording techniques in urethane- (1g/kg, i.p.) anaesthetized rats, we investigated the influence exercised by medial septal cholinergic neurons on dorsal hippocampus field CA1 neural responses to a hind paw injection of formalin (5%, 0.05 ml, s.c.). Cholinergic neurons of the medial septal region were destroyed by local microinjection of the immunotoxin 192 IgG-saporin. Compared to control vehicle microinjected animals, immunotoxin-treatment attenuated the amplitude, but not frequency, of CA1 theta induced by intraseptal injection of carbachol. This suggested a selective destruction of medial septal cholinergic neurons by the immunotoxin. Such destruction also abolished; (i) intraseptal carbachol-induced suppression of CA1 population spike, and (ii) stimulation-intensity dependent increase in amplitude, but not frequency, of theta evoked on electrical stimulation in the region of oral part of pontine reticular nucleus. Further, in comparison to vehicle-treated animals, selective cholinergic destruction attenuated formalin-induced; (i) theta activation, (ii) suppression of CA1 pyramidal cell population spike and dendritic field excitatory post-synaptic potential, (iii) inhibition of complex spike cell extracellular activity, and (iv) excitation and theta-rhythmicity of local putative GABAergic interneurons. However, pretreatment with the immunotoxin did not alter the strength and proportion of complex spike cells excited following injection of formalin. From these findings we suggest that medial septal cholinergic neurons mediate, at least partly, the amplitude of theta and pyramidal cell suppression via an inhibitory network involving CA1 interneurons. The data also indicates that during formalin theta, the cholinergic-mediated inhibitory processing does not modulate the strength and selectivity of complex spike cell excitation. This points to formalin-induced, non-overlapping inhibitory and excitatory processes that might have different functional relevance.

Hippocampal field CA1 interneuronal nociceptive responses: modulation by medial septal region and morphine

A majority (24/32) of the extracellularly recorded dorsal hippocampus field CA1 putative GABAergic interneurons were excited in conjunction with theta activation on formalin injection (5%, 0.05 ml, s.c. into right hind-paw) in urethane (1.0 g/kg, i.p.)-anaesthetized rats. An increase in activity was observed to the 10th minute (n=24) and also at later time-periods at which a few of the neurons were recorded following injection of formalin. The mean peak increase in activity within 5 min of formalin injection was 6.43+/-0.81 Hz over the average background activity for these neurons (6.46+/-1.04 Hz). Of 24 neurons, 14 exhibited an increase in activity which was rhythmically modulated with theta. With a concurrent administration of formalin and morphine (5 mg/kg, i.p.), the presumed interneurons recorded displayed an initial increase in discharge rate (mean peak increase within 5 min of 6.95+/-1.10 Hz) which then declined with a decrease in theta activity. The effect of concurrent morphine was naloxone reversible. Morphine administration alone resulted in an immediate decrease in the interneuronal firing rate. In presence of the medial septal region lesions, formalin did not evoke an excitation of intemeurons or theta activation. Further, such lesions prevented the decrease in intemeuron activity to morphine administration. The above data are consistent with the notion that (i) the field CA1 interneurons participate in a noxious stimulus-induced and medial septal region mediated pyramidal cell suppression, and (ii) morphine affects CA1 nociceptive responses partly in a fashion consistent with the effect of the drug on septohippocampal neural network processing.

Hippocampal theta state in relation to formalin nociception.

Abstract In the present study using extracellular electrophysiological recording techniques, we explored the temporal characteristics of hippocampal theta activation in relation to formalin nociception. Results indicate that, compared to hind paw injection of saline, formalin injection in behaving rat evoked biphasic increase in duration of dorsal CA1 theta. Such an increase broadly paralleled animal biphasic behavioral activation, especially lick and moment‐to‐moment agitated behaviors. Correspondingly, theta‐modulated cell firing was observed following formalin injection in anesthetized rat. The formalin‐induced theta activation in behaving rat was most marked during peak of theta activation in the 2nd theta state (11–40 min post‐injection) comprising 73% of the time in the 5 min block. An increase in theta peak frequency was also observed with respect to pre‐injection control. However, the peak of theta in the 2nd theta state mostly preceded the peak of lick and flinch of the affected paw. In the 41–60 min, following formalin injection while the animals displayed robust nociceptive flinching and lifting, the theta activity approached control levels. Furthermore, the theta peak frequency at peak of theta was higher than the corresponding values of sustained theta observed in correlation with the nociceptive behaviors; in contrast, high frequency theta rhythm was observed during formalin‐induced other moment‐to‐moment agitated behaviors. These findings favor the notion that in the formalin model the theta state of the hippocampus reflects a neural drive that is dissociated from the duration of nociceptive experience and is not selective to the typical nociceptive indices of lick, flinch, and lift of the injured paw.

Patterns of Hippocampal Neuronal Loss and Axon Reorganization of the Dentate Gyrus in the Mouse Pilocarpine Model of Temporal Lobe Epilepsy

The patterns of hippocampal neuronal loss and rewiring of the dentate gyrus (DG) were studied in the mouse model of temporal lobe epilepsy at 2 months after pilocarpine‐induced status epilepticus (PISE). NeuN immunocytochemistry showed two patterns of neuronal damage, i.e., type 1 with partial loss of pyramidal neurons in CA3 area and type 2 with almost compete loss of CA3 pyramidal neurons. Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA‐L) demonstrated that, at different rostrocaudal segments of the hippocampus, associational and commissural connections in the DG changed differently between mice with type 1 vs. type 2 neuronal loss. Calretinin (CR)‐immunopositive mossy cells in ventral hilus and its fibers in inner molecular layer of bilateral DG remained in mice with type 1 but almost disappeared in mice with type 2 neuronal loss, which was further supported by retrograde labeling with cholera toxin subunit B (CTB) showing colocalization of CTB with CR in the ventral hilus of bilateral DG in mice with type 1 neuronal loss, which was lost in those with type 2 neuronal loss. Furthermore, the sprouted PHA‐L‐immunopositive en passant and terminal boutons from the DG were found in CA1 area to contact with surviving calbindin‐, CR‐, and parvalbumin‐immunopositive neurons. The present study therefore suggests that different patterns of neuronal loss in CA3 area may be linked to different axon reorganizations in the DG.

Reticular stimulation evokes suppression of CA1 synaptic responses and generation of theta through separate mechanisms

The induction of hippocampal theta by reticular stimulation involves a relay to the hippocampus via the posterior hypothalamic–supramammillary region and then the medial septum. Interestingly, sensory‐ or behaviour‐induced theta is accompanied by suppression of hippocampal field CA1 synaptic responses. In the present study, performed on anaesthetized rats, we observed that reticular stimulation also induced a suppression of the CA1 pyramidal cell population spike and the corresponding dendritic field excitatory postsynaptic potential evoked by field CA3 stimulation. This suppression was observed at stimulation intensity below the threshold for generation of CA1 theta and was maximal at stimulation intensities at the threshold for theta. The frequency and amplitude of theta waves, by contrast, increased further with increasing reticular stimulation voltage. Neural inactivation by microinjection of the local anaesthetic procaine (20% w/v, 0.1–0.2 µL) or the inhibitory ligand gamma aminobutyric acid (0.8 m, 0.5 µL) in the posterior hypothalamic regions, especially the ipsilateral medial supramammillary region, or the medial septum attenuated both the suppression of CA1 pyramidal cell synaptic excitability and theta generation. However, the effects of microinjection on suppression and theta were not always in parallel. Furthermore, the effect of microinjection of gamma aminobutyric acid on reticularly elicited suppression was observed from relatively fewer sites in the posterior hypothalamus as compared with that on theta activation. These results suggest that reticular stimulation evokes an inhibition of CA1 pyramidal cell excitability that (i) is mediated, at least in part, via medial supramammillary and septal regions, but (ii) involves a separate neural pathway from theta generation.

Forebrain medial septum region facilitates nociception in a rat formalin model of inflammatory pain.

Summary The findings suggest that the medial septum is pro‐nociceptive and facilitates central processing of nociceptive information. The region may coordinate cortical and subcortical nociceptive responses. ABSTRACT The medial septum is anatomically and functionally linked to the hippocampus, a region implicated in nociception. However, the role of medial septum in nociception remains unclear. To investigate the role of the region in nociception in rats, muscimol, a GABA agonist, or zolpidem, a positive allosteric modulator of GABAA receptors, was microinjected into medial septum to attenuate the activity of neurons in the region. Electrophysiological studies in anesthetized rats indicated that muscimol evoked a stronger and longer‐lasting suppression of medial septal‐mediated activation of hippocampal theta field activity than zolpidem. Similarly, microinjection of muscimol (1 or 2 μg/0.5 μl) into the medial septum of awake rats suppressed both licking and flinching behaviors in the formalin test of inflammatory pain, whereas only the latter behavior was affected by zolpidem (8 or 12 μg/0.5 μl) administered into the medial septum. Interestingly, both drugs selectively attenuated nociceptive behaviors in the second phase of the formalin test that are partly driven by central plasticity. Indeed, muscimol reduced the second phase behaviors by 30% to 60%, which was comparable to the reduction seen with systemic administration of a moderate dose of the analgesic morphine. The reduction was accompanied by a decrease in formalin‐induced expression of spinal c‐Fos protein that serves as an index of spinal nociceptive processing. The drug effects on nociceptive behaviors were without overt sedation and were distinct from the effects observed after septal lateral microinjections. Taken together, these findings suggest that the activation of medial septum is pro‐nociceptive and facilitates aspects of central neural processing underlying nociception.