Intracellular correlates of morphine excitation in the hippocampal slice preparation

The influences of morphine and opioid peptides on hippocampal CA1 pyramidal cells were investigated using intracellular recordings from the in vitro slice preparation. Morphine applied to somal and basal dendritic areas of CA1 cells via a pressure ejection system confirmed a number of excitatory actions of opiates and opioid peptides in this brain region. These included an increase in the amplitude and duration of orthodromically (radiatum) elicited EPSPs and a decrease in amplitude of the following IPSP. The increase in EPSP amplitude was accompanied by a reduction in stimulus intensity necessary for eliciting the action potential. Morphine delivered to the slice in this manner induced synaptically elicited and spontaneous multiple spike burst discharges. In slice maintained in 10⁻⁴ M pentobarbital¹, morphine reversed the presumably GABA mediated long-duration depolarization following orthodromic stimulation. Finally, depending on the specific site of application (apical or basal dendritic region) of the opiate, morphine produced two different effects on the resting membrane potential and input resistance of CA1 pyramidal cells. These findings are discussed as to whether opiates act directly excitatory influences in the hippocampus, or via blockade of GABA mediated inhibitory mechanisms.     

Arginine-vasopressin inhibits centrally induced pressor responses by involving hippocampal mechanisms

Administration of arginine-vasopressin (AVP) or prolyl-leucyl-glycinamide (PLG) into a lateral cerebral ventricle reduced the magnitude of systolic blood pressure increase (pressor response) induced by electrical stimulation of the mesencephalic reticular formation (MRF) in urethane-anesthetized rats. Bilateral destruction of the dorsal hippocampus prevented the action of AVP on the pressor response. However, the effect of PLG was only slightly reduced by hippocampal lesion. Microinjection of AVP in the dentate area of the dorsal hippocampus mimicked the action of intracerebroventricularly administered peptides. The effect of a single injection of AVP lasted at least for 60 min. Neither hippocampal damage nor peptide administrations resulted in changes in mean arterial blood pressure (basal BP). Bradycardiac response accompanied the BP increase during MRF stimulation. Hippocampal damage or intracerebroventricular administration of AVP and PLG failed to affect the cardiac response. Injection of AVP into the hippocampus tended to reduce the magnitude of cardiac responses caused by MRF stimulation. It is suggested that the inhibition by AVP of a pressor response produced by MRF stimulation involves the dorsal hippocampus. The action of PLG or related peptides seems to be, at least in part, through mechanisms not involving the hippocampus.     

In vitro electrophysiological analysis of mature rat hippocampal transplants in oculo

We have investigated the maturation of isolated rat hippocampus grafted into the anterior chamber of the eye. Electrophysiological responses from transplants were compared to those recorded from the in vitro hippocampal slice preparation. Intracellular recording demonstrated that the passive membrane characteristics of intraocular hippocampal neurons were similar to those of the CA1 pyramidal cells in the in vitro slice preparation. However, the slow after-hyperpolarization which normally follows depolarization-induced action potentials was reduced or completely absent in the intraocular transplants, and the excitatory postsynaptic potential (EPSP) evoked by local stimulation was prolonged. The duration of the EPSP was reduced by perfusion with D-aminophosphonovaleric acid (2.5-50 microM), an N-methyl-D-aspartate receptor antagonist. Normal levels of glutamate decarboxylase (a marker for gamma-aminobutyric acidergic neurons) were found in the transplants, and responses to adenosine, bicuculline, and norepinephrine were similar in the in oculo transplants and in vitro slices. The data suggest that although many properties of hippocampal neurons are intrinsically determined, other aspects of the physiology of mature hippocampus either fail to develop, or develop abnormally in the absence of external inputs in oculo.     

Neurochemical anatomy of fetal hippocampus transplanted into large lesion cavities made in the adult rat brain.

The purpose of the present study was to determine whether neurochemicals normally found within neuron somata, fibers, and terminals of the hippocampal formation would also be present in transplanted hippocampal tissue that had developed in lesion cavities made in adult rat brains by aspiration of the hippocampus and overlying dorsolateral neocortex. Embryonic Day 15 or 16 rat brian tissue containing hippocampus with some medial pallial anlage was transplanted into the site of hippocampal aspiration lesions in adult male rats. One hundred ten to one hundred thirty-five days later the brains of these rats were sectioned and processed using the avidin-biotin-horseradish peroxidase immunocytochemical procedure to visualize choline acetyltransferase, met-enkephalin (MENK), neurotensin (NT), somatostatin, substance P, tyrosine hydroxylase (TH), or vasoactive intestinal polypeptide. Sections from two brains were stained using the thiocholine technique for visualization of acetylcholinesterase. All of these substances were found within cell bodies and/or fibers in the transplants. However, several abnormalities were noted. In addition to TH-immunoreactive fibers, TH-immunoreactive cell bodies were found in the transplants. Since TH is not expressed in mature hippocampal or cortical neurons this suggests that mechanisms for suppression of manufacture of this enzyme are lacking or inhibited in the transplants. Further, although all of the peptides were present either in fibers or in both cell bodies and fibers, the density of staining for NT and MENK was less than would be expected for normal hippocampus, and none of the cell bodies or fibers reacting for the peptides exhibited any apparent organization resembling that normally observed in hippocampus or cortex. However, some histological organization was present and the cholinergic markers were associated with this organization. These data suggest that some tropic and/or trophic factor such as nerve growth factor is present in the transplants to guide cholinergic innervation.     

Specific versus non-specific actions of opioids on hippocampal neurones in the rat brain

An investigation has been made into the pharmacological specificity of the actions of microelectrophoretically applied opioids on neurones in the rat hippocampus, a structure containing a low concentration of specific receptors for these substances.The majority of hippocampal neurones remained unaffected by morphine or enkephalin. Some neurones, however, displayed either inhibitory or excitatory responses to the opioids. Of the inhibitory effects, a few appeared to be specific, in that they could be antagonized by naloxone, but most of the other inhibitory responses were found to be potentiated by this drug. Similarly, naloxone not only failed to antagonize, but frequently potentiated the excitatory responses to the opioids.Further evidence for the predominantly non-specific nature of the responses of hippocampal neurones to opioids was provided by experiments with the stereoisomers levorphanol and dextrorphan. Neurones could be found which were either inhibited or excited by both enantiomers. Stereospecific responses, when observed, were inhibitory.Although non-specific, the excitatory effects of enkephalin and morphine on hippocampal neurones were greatly reduced in morphine tolerant/dependent rats. Indeed, in the hippocampus of these animals, the opioids had predominantly inhibitory effects which were potentiated, not antagonized, by naloxone.It is concluded that the low concentration of opiate receptors in the rat hippocampus renders neurones within this structure sensitive to a variety of non-specific opioid actions.     

The actions of cholecystokinin and related peptides on pyramidal neurones of the mammalian hippocampus

The presence of cholecystokinin octapeptide (CCK-8) immunoreactivity in the vicinity of the pyramidal neurones of the mammalian hippocampus has allowed us to investigate the central postsynaptic actions of CCK-8 and a number of related peptides, at a site thought to be innervated by a peptidergic pathway. Intracellular records from pyramidal cells of the CA1 region of the hippocampal slice preparation were used to determine changes in excitability and associated changes in membrane potential and resistance evoked by the pressure application of peptides into the cell body layer, from an independently mounted multibarrelled micropipette. The tetra- and octa-peptide C-terminal fragments of cholecystokinin evoked abrupt and rapidly reversible depolarizations which were accompanied by marked increases in excitability and a decrease in membrane input resistance. A comparison was made of the actions of these peptides with those of glutamate, released by iontophoresis from an adjacent barrel of the same multibarrelled pipette. The rate of onset of the cholecystokinin-evoked response was similar to that of the response evoked by glutamate. C-terminal fragments of gastrin (G-13 and G-14) and bombesin were also found to be excitatory to pyramidal neurones in the CA1 region. However, the nonsulphated form of CCK-8 was inactive, as was substance P, a peptide not present in the hippocampus.     

Alterations in opiate receptor binding in the hippocampus of aged Long-Evans rats

Quantitative in vitro autoradiography was used to examine [³H] -Ala², McPhe⁴, Gly-01⁵ enkephalin (DAGO) (μ-agonist) and [³H]diprenorphine (general opiate antagonist) binding sites in the hippocampal formation of young (6–8 months) and aged (25–28 months) Long-Evans rats. Age-related changes in these binding sites were restricted to specific regions but were not generally dependent on the ligand used. No reliable age-related changes in opiate binding were observed in the CA1 field or subicular region. In contrast, a decrease in the density of binding was found in both dorsal and ventral hippocampus within the CA3 field of aged brains. An age-related decrease in opiate binding within the dentate gyrus differed in its topography at dorsal and ventral levels of the hippocampus. A uniform decrease of opiate receptor binding was found throughout the dorsal dentate gyrus, while a more localized decrease of these sites occurred in hilar and granular layers of the ventral dentate gyros.These results indicate that a decrease of opiate binding in the hippocampal formation is largely localized to the CA3 region and dentate gyrus of aged rats. These findings are discussed with reference to age-related changes in hippocampal pathways containing opioid peptides. The implications for hippocampal opioid function in learning and age-related cognitive decline are also considered.     

Development of synapsin I and synapsin II in intraocular hippocampal transplants.

Previous studies have indicated that the appearance of synaptic vesicle-associated proteins known as the synapsins is one indicator of synapse formation. In this study, the levels and morphological distribution of synapsin I and synapsin IIa and IIb were studied in intraocular hippocampal transplants and in situ in the intact hippocampus. No detectable levels of either synapsin I or synapsin II were found in the fetal brain. The in situ levels of the synapsins exhibited parallel increases rapidly after birth, reaching peak levels at 8 weeks, after which a slight decline was noted in synapsin I and synapsin IIb. In hippocampal transplants, a comparable increase in the synapsins was seen during the first 8 weeks in oculo. It is likely that the synapse formation in the hippocampal transplants represents synapses from neurons within the transplant, as well as from various peripheral ganglia that send collaterals into the graft. Peripheral and central synapses express different synapsin I: synapsin IIa and IIb ratios. When the ratios of the synapsin proteins in hippocampal transplants were examined ratios essentially identical to those seen in the normal hippocampus were found, despite the numerous peripheral neurites innervating the grafts. Immunohistochemical studies supported the immunoblot data, showing no detectable immunofluorescence with synapsin antibodies in fetal or newborn hippocampal formation. The density of immunoreactive profiles increased substantially both in transplants and in the hippocampal formation in situ during the first 2 postnatal months. In conclusion, the present data demonstrate that hippocampal transplants in oculo can develop significant levels of the synapsins and that there is no time lag in development in these levels compared to the hippocampal formation in situ.     

Antidepressant-like effect of food-derived pyroglutamyl peptides in mice

The N-terminal glutamine residue, exposed by enzymatic cleavage of precursor proteins, is known to be modified to a pyroglutamyl residue with a cyclic structure in not only endogenous but also food-derived peptides. We investigated the effects of wheat-derived pyroglutamyl peptides on emotional behaviors. Pyroglutamyl leucine (pyroGlu-Leu, pEL) and pyroglutamyl glutaminyl leucine (pyroGlu-Gln-Leu, pEQL) exhibited antidepressant-like activity in the tail suspension and forced swim tests in mice. pEQL exhibited more potent antidepressant-like activity than pEL after i.p. and i.c.v. administration. pEQL exhibited antidepressant-like activity at a lower dose than Gln-Gln-Leu, suggesting that pyroglutamyl peptide had more potent activity. To examine whether pyroglutamyl peptides increased hippocampus neurogenesis, associated with the effects of antidepressants, we measured 5-bromo-2′-deoxyuridine (BrdU) incorporation. pEL and pEQL increased BrdU-positive cells in the dentate gyrus of the hippocampus. Intriguingly, pEL did not increase hippocampal mRNA and protein expression of brain-derived neurotrophic factor (BDNF), which is a factor associated with both neuropoietic and antidepressive effects. Thus, pyroglutamyl peptides may enhance hippocampal neurogenesis via a pathway independent of BDNF. We also confirmed that pEL and pEQL were produced in the subtilisin digest of major wheat proteins, glutenin and gliadin, after heat treatment. pEL and pEQL are the first peptides derived from wheat proteins to be shown to exhibit an antidepressant-like activity.     

Long-term effects of thyroid hormone deficiency on noradrenergic circuitry: electrophysiological changes in intraocular hippocampus-locus coeruleus double transplants

The effects of thyroid hormone deficiency on the noradrenergic innervation of hippocampus from locus coeruleus (LC) were examined using intraocular double transplants in albino rats. Fetal brainstem pieces containing the nucleus LC were transplanted to the anterior chamber of the eye of thyroidectomized and normal recipients and the brain grafts were allowed to mature for 8 weeks. Pieces of fetal hippocampal formation were introduced into the anterior eye chamber and placed in contact with the LC grafts or placed in previously operated eyes. As evidenced by high performance liquid chromatography, hippocampal transplants in contact with a brainstem graft gradually became hyperinnervated by noradrenergic fibers from these grafts. The levels of norepinephrine were lower in single control grafts and in double grafts in thyroidectomized animals than in control double grafts. Extracellular recordings of single neuronal activity were performed in hippocampal transplants in all 3 groups after 10-14 months in oculo. Superfusion with the α2- adrenergic agonist clonidine and the α-adrenergic antagonist phentolamine elicited significant increases in discharge rate of hippocampal neurons in control double transplants, but not in single hippocampal grafts or in double grafts in thyroidectomized hosts. The β-adrenergic antagonist timolol did not change the neuronal firing rate in any of the 3 groups. Superfusion with penicillin over single hippocampal transplants caused long-lasting increases in slow-wave activity. This increased bioelectric activity remained after the cessation of drug application. A similar increase in slow-wave activity was found in hyperinnervated control double transplants only when penicillin was combined with clonidine or phentolamine. However, the hippocampal portion of double grafts in thyroidectomized recipients readily responded to penicillin with seizures and/or interictal spiking. The data presented here suggest that chronic lack of thyroid hormones leads to significant disturbances of the central noradrenergic transmission in isolated LC-hippocampal circuits.     

Lateral habenula and hippocampal units: electrophysiological and iontophoretic study

In previous works we studied, on cats, the effects of lateral hobenula (LH) stimulation on hippocampal units. In particular, the results showed an excitation or an inhibition in relation to the stimulation frequency (0.5–3.0 Hz or 5.0–20 Hz, respectively). All the LH stimulation effects were antagonised by iontophoretic Intrahippocampal application of methysergide (MS). M this series of experiments It was possible to demonstrate, on rats, that LH stimulation causes an excitatory effect in a major number of hippocampal units in relation to the frequency increase. The inhibitory effect by iontophoretic serotonine application and the reversible blockade of habenular modulation after fontophoretic methysergide administration on hippocampal units suggest, on rats, the involvement of raphe. Such hypothesis, with anatomical evidences demonstrating an excitatory projection between LH and raphe, was confirmed by data concerning the effects of intraraphal NMDA iontophoretic application on hippocampal units (NMDA application for 30 S = excitation; NMDA administration for 10–15 MIN = inhibition). All the results suggest an habenular modulation of hippocampus through the Invohmment of the raphe in the context of which an interneurone Is Inhibitory on the efferent serotonergic raphe hippocampus pprojection. This hypothesis finds further support from MS blockade effect during Intreraphol NMDA iontophoretic administration.     

Influence of the hippocampus on interneurons of the rat prefrontal cortex

The hippocampus and prefrontal cortex (PFC), two structures implicated in learning and memory processes, are linked by a direct hippocampo-prefrontal pathway. It has been shown that PFC pyramidal cells receive monosynaptic excitatory inputs from the hippocampus and, in this study, we sought to determine the influence of the hippocampus on PFC interneurons in anesthetized rats. Extracellular recordings were coupled to juxtacellular injections of neurobiotin or biotinylated dextran amine to morphologically differentiate interneurons from pyramidal cells. In all cases, the action potentials of labeled interneurons were of shorter duration (< 0.70 ms) than those of identified pyramidal cells (> 0.70 ms). Single pulse stimulation of the hippocampal CA1/subiculum region induced an excitatory response in 70% of recorded interneurons in the prelimbic and medial-orbital areas of the PFC. In contrast to the one to two action potentials generated by pyramidal cells, an important group of interneurons fired a burst of action potentials in response to hippocampal stimulation. A large proportion of these excitatory responses was probably monosynaptic as their latency is consistent with the conduction time of the hippocampo-prefrontal pathway. In addition, when both a pyramidal cell and an interneuron were simultaneously recorded and both responded to stimulation, the interneuron consistently fired before the pyramidal cell. In conclusion, the hippocampus exerts a direct excitatory influence on PFC interneurons and is thus capable of feedforward inhibition of pyramidal cells. Hippocampal output is spatially and temporally focalized via this inhibitory process and consequently could facilitate the synchronization of a specific subset of PFC neurons with hippocampal activity.     

Stimulation of the perforant path alters hippocampal levels of opioid peptides, glutamine and GABA

This investigation demonstrates that stimulation of the perforant path under conditions which elicit wet dog shakes in rats produces a significant decrease in hippocampal levels of methionine-enkephalin, dynorphin A(1-8) and glutamine, and an increase in gamma-aminobutyric acid (GABA). Levels of these substances are not altered by stimulus parameters insufficient to elicit wet dog shakes. These results lend support to the notion that endogenous opioid peptides play a role in regulation of hippocampal excitability but may only be released under relatively intense stimulus conditions. The increase in GABA levels could be due to an increase in synthesis, an increase in reuptake or a reduction in release. The latter possibility is consistent with reports that iontophoretically applied enkephalin exerts its apparent excitatory effects via an inhibitory action on inhibitory neurons in the hippocampus.     

Hippocampal unit-behavior correlations during classical conditioning

The correspondence that develops over the course of classical conditioning between the temporal distribution of increased unit activity in the rabbit hippocampus and the amplitude-time distribution of the behavioral nictitating membrane response is analyzed. Results reveal a high degree of correspondence between neural and behavioral measures. The real time correlation between the within-trial probability of increased hippocampal unit discharge and amplitude-time course of the nictitating membrane response grows substantially with learning. Further analyses reveal that this apparent increase in correlation results from a growth in amount of hippocampal unit activity per se (i.e., a differentiation of the hippocampal unit response from background firing rates), rather than an increase in the correspondence between cellular and behavioral measures (i.e. a repatterning of hippocampal discharges to more accurately code spatio-temporal aspects of the behavioral response). These and other results indicate that the neuronal ‘temporal model’ of the behavioral response either develops within the hippocampus from the first few conditioning trials or develops first in entorhinal cortex to subsequently influence hippocampal discharge patterns. On the other hand, the increase in amount of hippocampal unit activity developing with conditioning appears to occur within the hippocampus.     

Glucagon-like peptide-1 modulates neuronal activity in the rat's hippocampus.

In order to assess effects of glucagon-like peptide-1 (GLP-1) in the brain excluding the hypothalamus, the effects of GLP-1 (7-36) amide, a naturally produced active fragment, on the electroencephalogram and hippocampal single unit activities of anesthetized male Wistar rats were examined. I.c.v. injection of GLP-1 (7-36) amide decreased the hippocampal theta wave duration. Juxtacellular administration of GLP-1 (7-36) amide first increased and then decreased single unit activities recorded in the hippocampal CA1, which effects were antagonized by exendin (9-39) amide, a GLP-1 receptor antagonist, or 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA type glutamate receptor antagonist. These results indicate that GLP-1 receptors exist in the hippocampus and are involved in modulating hippocampal activity through an increase in the release of excitatory amino acid transmitters.     

Effects of pentylenetetrazol kindling on glutamate receptor genes expression in the rat hippocampus

It has been hypothesized that changes in the excitatory amino acid receptor biosynthesis may be involved in the mechanism of kindling—an animal model of epileptogenesis. In order to test this hypothesis, we investigated the effects of pentylenetetrazol kindling on the expression of genes coding for NMDAR1 and GluR2 in the rat hippocampal formation. Pentylenetetrazol kindling decreased the hippocampal NMDAR1 mRNA level after 3 and 24 h; lowered the GluR2 flip level and elevated the flop mRNA one in the CA1 field and dentate gyrus after 3 and 24 h, respectively. A receptor autoradiography showed an increase in the [³H]MK-801 binding density in the hippocampus following both acute and repeated pentylenetetrazol administration. We conclude that an early occurrence of downregulation of the glutamate receptor gene expression may be an adaptive response of glutamate receptors to an oversupply of excitatory amino acids during repeated seizures.     

Mu-opiate receptors measured by positron emission tomography are increased in temporal lobe epilepsy

Neurochemical studies in animal models of epilepsy have demonstrated the importance of multiple neurotransmitters and their receptors in mediating seizures. The role of opiate receptors and endogenous opioid peptides in seizure mechanisms is well developed and is the basis for measuring opiate receptors in patients with epilepsy. Patients with complex partial seizures due to unilateral temporal seizure foci were studied by positron emission tomography using 11C-carfentanil to measure mu-opiate receptors and 18F-fluoro-deoxy-D-glucose to measure glucose utilization. Opiate receptor binding is greater in the temporal neocortex on the side of the electrical focus than on the opposite side. Modeling studies indicate that the increase in binding is due to an increase in affinity or the number of unoccupied receptors. No significant asymmetry of 11C-carfentanil binding was detected in the amygdala or hippocampus. Glucose utilization correlated inversely with 11C-carfentanil binding in the temporal neocortex. Increased opiate receptors in the temporal neocortex may represent a tonic anticonvulsant system that limits the spread of electrical activity from other temporal lobe structures.     

Grafts containing fetal hippocampal tissue reduce activity and improve passive avoidance in hippocampectomized or trimethyltin-exposed rats

Embryonic Day 16 or 17 rat tissue containing either hippocampus with some medial pallial anlage or cerebellar/alar plate anlage was transplanted to the site of the ablated hippocampus of otherwise normal adult rats or adult rats previously exposed to the neurotoxin trimethyltin. Ninety to one hundred five days later these rats were compared to control rats in acquisition of passive avoidance and in open field activity. Transplantation of both types of tissue produced behavioral recovery on both tasks in rats with hippocampal lesions that had not been exposed to trimethyltin. Only hippocampal transplants produced recovery of function in rats given trimethyltin. Although transplants of hippocampal tissue had an organotypic structure that was easily differentiated with cell and fiber stains from that of the cerebellar transplants, neither of these routine histological procedures nor immunocytochemical analysis revealed differences between transplants made into normal rats or toxicant-exposed rats. Either of two mechanisms may account for the ability of the transplants to produce behavioral recovery. These are reconstruction of damaged circuitry by the transplant and neurotrophic action of the developing transplant on the host brain. The second mechanism alone may be sufficient to restore function in brain-lesioned but otherwise normal rats. Therefore, either type of transplant is effective. Both mechanisms may be necessary for recovery in brain-lesioned, toxicant-exposed rats. Therefore, only transplants of tissue homotypic to the tissue removed from the brain are effective.     

Effects of atropine on hippocampal theta cells and complex-spike cells

The medial septal nuclei are essential for the naturally occurring hippocampal theta rhythm. Evidence that the rhythmic activity of the septum is carried via cholinergic afferents to the hippocampus has been: (a) the existence of a cholinergic septo-hippocampal projection, and (b) the sensitivity of one type of theta rhythm to antimuscarinic agents or cholinergic depletion. The muscarinic action of acetylcholine on pyramidal cells, however, is too slow to carry even a 4 Hz signal. Recent in vitro studies have confirmed a fast excitatory response by some hippocampal interneurons to muscarinic agonists. In urethane anesthetized rats, iontophoretic application of atropine to 17 hippocampal theta cells (presumed interneurons) during the theta rhythm, reduced their firing rates to an average of 24% of control rates. The effect of iontophoretic atropine application to 4 CA1 complex-spike cells (presumed pyramidal cells) was a selective elimination of their bursting activity with no significant effect on overall firing rate. The data suggest that: (1) interneuronal firing, during the hippocampal theta rhythm, is dominated by an excitatory cholinergic input and not by excitatory collaterals of pyramidal cells; and (2) somatic burst firing by CA1 pyramidal cells requires the presence of acetylcholine.