Host Hippocampus Research Articles

The grafted hippocampus: An epileptic focus

Field potentials and unitary activity were investigated in the grafted and the host hippocampi in freely moving rats and in vitro. The subcortical afferents and efferents of the hippocampus (fimbria-fornix, FF) were removed by aspiration. Solid pieces of hippocampal grafts derived from 15- to 16-day-old fetuses were placed in the lesion cavity in rats with unilateral FF lesions, and cell suspensions prepared from fetal hippocampi were grafted directly into the host hippocampi in animals with bilateral FF lesions. Reciprocal communication between the grafted and the host hippocampi was monitored with a 16-microelectrode probe from 7 to 10 months after grafting. The fluorescent retrograde tracer, Fluorogold, was used to examine graft-host projections and acetylcholinesterase staining to reveal host-derived fibers in the graft. The most typical neuronal pattern of the hippocampal graft was a highly synchronous population burst with concurrent EEG spike. The speed of propagation of the EEG spike within the graft and across the graft-host interface was either fast (>3 m/s) or slow (<0.5 m/s). Large amplitude, short duration EEG spikes usually propagated with a high speed, while smaller amplitude, wider spikes with broad population bursts spread at a lower velocity. The direction of propagation was usually uniform indicating that the population burst was triggered by a localized subgroup of highly excitable neurons (“focus”). Spontaneous seizures were also present in the solid graft which frequently invaded the host hippocampus. The incidence of EEG spikes was three times higher in rats with bilateral suspension grafts than in animals with FF lesion only. In about half of the grafted rats spontaneous behavioral seizures were also observed. Intracellular recordings from putative pyramidal cells in the graft and in the host revealed large amplitude (10–12 mV), spontaneously occurring EPSPs. IPSPs were difficult to detect even during depolarizations of up to 20 mV from rest. We suggest that the increased excitability of the hippocampal graft is due to the high incidence of recurrent excitatory collaterals terminating on or close to the somata of pyramidal neurons. Population bursts may spread fast via extensively arborizing axon collaterals or slowly by successively activating new sets of neighboring neurons. Spontaneous behavioral convulsions are explained by assuming that the grafted hippocampus serves as an epileptic focus which is capable of kindling the host brain by repeated seizure induction. These findings also draw attention to the need for chronic physiological studies of the transplanted neuronal tissue before possible application of the method in clinical settings for promoting recovery from disease or brain damage.

Long-term potentiation of evoked and spontaneous neuronal activity in the grafted hippocampus.

Adult rats with unilateral fimbria-fornix lesion received fetal hippocampal grafts into the lesion cavity. Seven to ten months after the transplantation the graft was examined for long-term potentiation (LTP) in response to host hippocampus and direct graft stimulation. High frequency tetanizing trains delivered to either the host hippocampus or the graft resulted in augmented field potentials and prolonged neuronal discharges in the graft lasting several hours. Very low currents (10-30 microA) were required to induce LTP by direct graft stimulation. In addition to the enhancement of evoked responses, the frequency of occurrence of spontaneously occurring EEG spikes and concurrent population neuronal bursts in the graft increased significantly after the tetanizing trains. These findings suggest that the physiological-biochemical mechanisms required for plastic changes of synaptic efficacy are present in the grafted hippocampus.

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats.

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.

Embryonic entorhinal transplants project selectively to the deafferented entorhinal zone of adult mouse hippocampi, as demonstrated by the use of thy-1 allelic immunohistochemistry. Effect of timing of transplantation in relation to deafferentation

The mouse Thy-1. 1/Thy-1.2 allelic marking system is used to show that transplanted embryonic entorhinal cortex can reinnervate adult host hippocampi. The projection is limited to the appropriate terminal zones-viz. the outer two-thirds of the stratum moleculare of the dentate gyrus, and the stratum lacunosum-moleculare of the hippocampus-and extends for up to about 2 mm into the denervated host terminal field. The reconstruction of the entorhinal projections to the host requires direct contact between the embryonic donor tissue and the denervated adult host terminal field, and is dependent upon removal of the ipsilateral host entorhinal area. In the absence of an overall deafferenting host entorhinal lesion the transplanted entorhinal area forms only small local projections which are confined to areas which would have been locally deafferented as a result of direct damage to the host entorhinal afferents (i.e. during their intrahippocampal course) by the hippocampal lesion caused at the time when the transplant was inserted. The correct relative timing of deafferentation and transplantation is vital for the formation of the transplant-to-host projection. The host dendrites can be made receptive to entorhinal transplant projections by removal of the host entorhinal area at the time of transplantation. When deafferentation is performed first and transplantation is delayed, it is found that the deafferented host dendrites retain this receptivity even when deafferentation has been performed as much as two months before transplantation. Reversing the order of transplantation and deafferentation, however, shows that the transplants have only a transient ability to project to the deafferented host territory. Thus, transplants inserted and allowed to become established for one week before host deafierentation make very much reduced projections to the host, and from two weeks onwards are incapable of any detectable response to subsequent removal of the host entorhinal area. Coextensive with the formation of transplant-to-host entorhinodentate projections, the host entorhinal lesion also induces an intensification of the acetylcholinesterase staining of the host septodentate afferents in the denervated outer dentate stratum moleculare. The findings demonstrate the accurate reconstruction of a lost projection in adult brain by transplanting the appropriate type of embryonic tissue, but the results of altering the relative timing of deafferentation and transplantation raise currently unsolved questions about the nature of the competitive interactions between transplant and host axons. Surprisingly, a delay allowing time for the lesion-induced sprouting of intact host axons into the denervated area does not seem able to prevent formation of transplant-to-host projections by a subsequent entorhinal transplant. However, unlike the host's own entorhinal area (which retains the ability to sprout to the unilaterally deafierented dentate gyrus throughout adult life), the ability of an embryonic entorhinal area transplant to project to the deafferented host hippocampus and dentate gyrus appears to be transient, and to disappear completely between one and two weeks after transplantation.

Alterations in excitatory and gabaergic inhibitory connections in hippocampal transplants

Solid pieces of embryonic hippocampal tissue were implanted in a cavity formed by aspiration of the fimbria-fornix and the overlying cingulate cortex in adult rats. Six to 8 months after the transplantation, chronic recording electrodes were implanted into the graft and the host hippocampi for the recording of electroencephalogram and unit activity in the freely moving animal. Irregularly occurring sharp waves or electroencephalogram spikes and concurrent synchronous discharge of large groups of neurons dominated the electrical activity of the grafts, in contrast to the situation in normal animals. Light microscopy and GABA immunocytochemistry in the grafts revealed the three major cell types of the hippocampal formation, i.e. pyramidal neurons, dentate granule cells and GABA-immunoreactive interneurons were present in the hippocampal grafts. At the ultrastructural level, however, significant alterations in connectivity were observed. The most striking finding was the absence or sparse occurrence of synapses on the axon initial segments of pyramidal neurons. The axon initial segments are normally densely covered by GABAergic synapses derived from a specialized type of interneuron, the chandelier or axo-axonic cell. On the other hand, numerous GABA-immunoreactive terminals were found in synaptic contact with somata of pyramidal neurons, suggesting that other types of GABAergic interneurons and their efferent connections may have developed in a normal manner. The cell bodies of pyramidal neurons received, in addition, several asymmetric synapses from GABA-negative terminals. These presumably excitatory synapses are not present on the somata of pyramidal cells in the normally developing hippocampus. We hypothesize that the somatic excitatory synapses originate, at least in part, from the axon collaterals of the neighbouring pyramidal cells in the graft. We suggest that the hyperexcitability of the neuronal circuitry within the graft is due to reduced inhibition (lack of axo-axonic synapses) coupled with increased collateral excitation of the pyramidal neurons.

Human amnion membrane matrix as a substratum for axonal regeneration in the central nervous system.

Human Amnion Membrane Matrix (HAMM) was used as a substratum for the regeneration of neuronal axons in the central nervous system. A large piece of HAMM was bound to nitrocellulose paper (NCP) as a supporting material, and cut into small strips. Aspirative lesions of the fimbria-fornix were made in adult rats leaving a cavity separating the septum from the hippocampus. In the same operation a small piece of HAMM-NCP was placed into the cavity with one end abutting the axotomized septum and the other end abutting the denervated hippocampus. At times between 2 weeks and 3 months after surgery the brains of the animals were examined histologically for 1) host response to the implant; 2) maintenance of HAMM-NCP in the originally implanted orientation; 3) growth of acetylcholinesterase (AChE)-positive fibers on the HAMM; 4) growth of the AChE-positive fibers into the denervated hippocampus; and 5) non-neuronal cells on the HAMM. The NCP remained in place over the 3-month period. In most cases the HAMM and NCP remained apposed and caused no greater reaction in the brain beyond that created in response to the aspirative lesion alone. AChE-positive fibers grew out from the septum onto the HAMM by 2 weeks, and by 8 weeks more extensive growth was observed on the HAMM. By 8-weeks, AChE fibers could be clearly seen coming off the membrane and entering the host hippocampus. By double-labelling for the basement membrane side of the HAMM (using anti-human laminin antibodies) and for cholinergic fibers (using AChE histochemistry) in the same sections, it was possible to see fibers clearly growing on the laminin-positive side of the HAMM but little or no growth on the opposing laminin-negative stromal side. The most important variables for extensive growth appeared to be the accurate placement of the implant and the amount of time following the lesions. The placement of the matrix on NCP prior to implantation in the brain made it easier to orient the membrane between the septum and the hippocampus, and seemed to assist in maintaining the membrane in that orientation. The HAMM represents a useful, regeneration-promoting grafting material for central nervous system repair studies.

Serotonin neurons grafted to the adult rat hippocampus. I. Time course of growth as studied by immunohistochemistry and biochemistry

The maturation and growth of fetal serotonergic raphe neurons have been studied immunohistochemically and biochemically between 1 week and 5 months after grafting to the hippocampal formation in 5,7-dihydroxytryptamine-pretreated adult rats. The average number of surviving neurons in each group was 1800, which is equivalent to approximately 20% of the potential number of serotonin neurons contained in the grafted cell suspension. The fetal raphe cells, which were taken from 12–14-day-old embryos, had developed strong serotonin immunoreactivity at 1 week after transplantation, and the number of serotonin cells present at 1 week was similar to that found at later time points. Fiber outgrowth was demonstrable already at 1 week but the serotonin-positive fibers were restricted to the areas close to the graft. Single fibers, however, could be traced for distances of up to 500–800μm into the host hippocampus and dentate gyrus. At later time points, the graft-derived serotonin-immunoreactive fiber network extended to cover the entire hippocampal formation. At the longest postoperative time point, 7 weeks and 5 months, some of the animals exhibited extensive hyperinnervation patterns throughout the dorsal parts of the hippocampus and the dentate gyrus. Consistent with these immunohistochemical observations, supranormal serotonin levels developed with time after transplantation in the grafted hippocampi from an average of 5% of normal at 1 week, to 28% of normal at 3 weeks, 146% of normal at 7 weeks, and 216% of normal at 5 months. Although the recovery of 5-hydroxyindoleacetic acid (5-HIAA) paralleled that of serotonin (5-HT), the increase in the metabolite concentrations was less than that of the amine, indicating a change in the turnover or metabolism of serotonin in the grafted neurons over time. Thus, the5-HIAA:5-HT ratio was higher than normal at 3 weeks post-grafting (when the host hippocampus was only partially reinnervated); it was similar to normal at 7 weeks, and it tended to be lower than normal in the hyperinnervated specimens at 5 months' survival. A regression analysis revealed a significant inverse correlation between the hippocampal 5-HT concentration and the5-HIAA:5-HT ratio in the graft-reinnervated hippocampal formation. In conclusion, the grafted serotonergic raphe neurons, in contrast to other types of aminergic neurons, exhibited a prominent tendency to form extensive hyperinnervation patterns in the previously denervated host target. Despite their ectopic placement, however, the grafted serotonergic neurons exhibited an active transmitter metabolism and the transmitter utilization rate appeared to be subjected to a tonic, possibly autoreceptor-mediated, regulatory control.

Human fetal basal forebrain neurons grafted to the denervated rat hippocampus produce an organotypic cholinergic reinnervation pattern

The septal/diagonal band (SDB) area, obtained from a 9- to 10-week-old abored human fetus, was grafted to the hippocampal formation of adult, immunosuppressed rats subjected to an aspirative lesion of the fimbria-fornix. Nineteen weeks after transplantation, microscopical analysis revealed large, partly acetylcholinesterase (AChE)-positive grafts in the hippocampus in 3 of the 5 recipients. The AChE-positive grafts gave rise to a reinnervation of the host hippocampus and an AChE-positive lamination of the different hippocampal subfields with the same characteristics as the normal septum-derived innervation. Immunological evaluation of host sera revealed that all rats were immunized by the graft. This indicates that grafted human cholinergic SDB neurons can respond to or interact with factors that regulate and guide the innervation of the rat hippocampus.

Transplanted raphe and hippocampal fetal neurons do not displace afferent inputs to the dorsal hippocampus from serotonergic neurons in the median raphe nucleus of the rat

To determine if fetal transplants can substitute for or suppress intrinsic serotonergic (5-HT) innervation, we studied the relationship between transplanted and the endogenous raphe neurons projecting to the hippocampus. Fetal raphe transplants produced a 5-HT hyperinnervation of dorsal hippocampus in adult rats. Yet, transplants of fetal raphe tissue did not affect the number of median raphe nucleus (MRN) neurons, approximately 300, which retrogradely transported HRP from the hippocampus. This provides evidence that transplanted 5-HT neurons can co-exist with intrinsic 5-HT nerve terminals in the target area for at least one month. In the second part of this study, fetal hippocampal tissue was transplanted into the host hippocampus. Intrinsic 5-HT immunoreactive fibers innervated the transplanted fetal tissue. Nevertheless, the number of MRN neurons innervating the host tissue as revealed by HRP retrograde transport remained unchanged. Changes in the innervation pattern and 5-HT level in the dorsal hippocampus occur following transplantation of fetal tissue. These changes are discussed and suggest that both the target tissue and afferent neurons readjust to accommodate extrinsic transplanted tissue.

Transmitter expression and morphological development of embryonic medullary and mesencephalic raphé neurones after transplantation to the adult rat central nervous system

Suspensions of cells taken from the mesencephalic or medullary raphé regions of the 13-14 day old embryonic rat brain were injected into the hippocampus of adult rats which had previously been denervated of its serotoninergic input by 5,7-dihydroxytryptamine. At periods of up to 14 months after implantation, the brains were taken for immunohistochemical analysis of 5-hydroxytryptamine (5HT)-, substance P (SP)- and thyrotropin releasing hormone (TRH)-like immunoreactivity. Surviving grafts were found in all animals. The implants derived from mesencephalic raphé contained neurones immunoreactive to 5HT, SP or both substances together. On average 19% of the potential number of mesencephalic 5HT neurones were found in the grafts. Outgrowth of 5HT-immunoreactive fibres was extensive, and displayed the typical pattern of 5HT innervation in the normal hippocampus-the densest plexuses were found in the dentate gyrus, with sparser networks in the CA1 and CA2 regions. SP-positive processes were principally found only in the graft itself. Transplants of medullary raphé cells contained 5HT-immunoreactive neurones, some of which also contained SP-and/or TRH-like immunoreactivity, thereby mirroring the situation found in the caudal raphé complex in situ. An average of 18% of the total available medullary serotoninergic neurones were found at each graft site. A rich outgrowth of 5HT-immunoreactive varicose processes was evident, with the same pattern as the 5HT innervation by the mesencephalic raphé grafts, and as in the normal hippocampus. SP- and TRH-positive fibres were essentially detectable only in the graft, but not in the host hippocampus. The present studies indicate that the milieu of the hippocampus does not preferentially attenuate the survival of the serotoninergic cells which do not normally project to it. Nor is the fibre outgrowth of these medullary raphé 5HT neurones significantly different from that of the mesencephalic raphé. However, the hippocampal environment may be responsible for the appearance of SP-LI in the otherwise apparently solely 5HT-containing mesencephalic raphé neurones, and for repressing the outgrowth of fibres containing TRH-, or SP-like immunoreactivity, regardless of their origin.

Behavioral deficits after intrahippocampal fetal septal grafts in rats with selective fimbria-fornix lesions.

Fetal septal transplants have been shown to promote behavioral recovery in young adult rats with aspiration fimbria-fornix lesions, rats with septal lesions and in intact aged rats. The present study examined the behavioral impact of intrahippocampal septal cell suspension transplants (T) in young female rats that had received, 10 days earlier, either medial fimbria lesions (Group FI.T), dorsal (subcallosal) fornix lesions (Group FO.T) or these two lesions together (Group FIFO.T). Relative to rats with lesions only (groups FI, FO and FIFO), grafted rats, irrespective of lesion locus, displayed unexpected impairments in (i) a serial alternation learning task, 5 weeks and 6 months after transplantation, and (ii) in a radial maze, 7 months after transplantation. In the first alternation test, Group FIFO showed impaired performance relative to Groups FI, FO and the sham-operated controls (Group S). In the second alternation test, Groups FO.T and FO showed impaired performance relative to Groups FI.T and FI, and only the performance of Group FI did not differ from that of Group S. In the radial maze, Groups FI, FO and FIFO all showed impaired performance relative to Group S. By contrast, there were no deleterious effects of lesions or of grafts in the acquisition and retention of a step-through passive avoidance task, 10 weeks after transplantation. Our findings on the effects of selective fimbria-fornix lesions did not confirm the report that rats with FI lesions but not those with FO lesions are unable to learn a serial alternation task, nor the report that FO lesions impair passive avoidance retention. Acetylcholinesterase (AChE) histochemistry revealed that grafts were present but graft-derived innervation of the host hippocampus varied from extensive to almost non-existent in all transplant groups. AChE-positivity in the dorsal hippocampus (DH) was not related to behavioral performance. However, the grafts often grew to a considerable size within the host brain and in many rats, especially those in Group FI.T, produced moderate to extreme damage of the host DH. There was a significant positive correlation between errors in the radial maze and graft-induced DH damage but no relationship between errors and graft size. The results indicate that, after partial lesions of the fimbria-fornix, intrahippocampal septal grafts survive well but are likely to damage recipient structures and result in behavioral impairments.

Hippocampal neurons grafted to newborn rats establish efferent commissural connections.

The formation of commissural projections from developing rat hippocampal neurons grafted to the hippocampus of newborn littermates was examined by retrograde axonal transport of the fluorescent dye Fluorogold (FG). At six weeks of age the recipients received two injections of FG in the hippocampus contralateral to the graft. This resulted in labelling of normal, commissurally projecting neurons in the dentate hilus and CA3 of the host hippocampus. Neurons in several transplants were also labelled, and based on morphology and location they were identified as CA3 and dentate hilus neurons.

Chapter 14 Regulation of acetylcholine muscarinic receptors by embryonic septal grafts showing cholinergic innervation of host hippocampus

Publisher Summary This chapter describes whether cholinergic innervation by embryonic septal or striatal grafts would result in the regulation of muscarinic receptors in the denervated hippocampus using quantitative autoradiography. The evidence indicates that the cholinergic input from the grafted embryonic septum also makes muscarinic synapses, as the electrophysiological responses are atropine sensitive and in aged rats transplantinduced behavioral recovery is atropine sensitive. Combined pharmacological and electrophysiological studies have shown that cholinergic synapses in the normal hippocampus are of the muscarinic type. The septo-hippocampal cholinergic projection is a particularly useful model for studying the capacity of the transplant to reinnervate the host region. Regulation of muscarinic sites after destruction of the cholinergic system innervating the cortex has been difficult to show. The results show an increase of muscarinic receptors that are shown as down regulated with appropriate cholinergic innervation by grafts of embryonic septal or striatal tissue. This work provides further evidence that the synaptic contacts made by these grafts are functional.

Interactions between grafted serotonin neurons and adult host rat hippocampus.

We have followed the development of physiological and functional properties of serotonin-containing raphe neurons grafted into an adult host hippocampus as a model system for graft-host interactions. These raphe cells have no clear identifying properties on the day of grafting: they develop them while growing in the host. Raphe neurons, recorded 1 month after grafting, possess adult normal physiological properties. These include high input resistance, slow membrane time constant, lack of inward rectification, a transient outward rectification, broad spikes having a Ca2+ component, lack of accommodation, and a large afterhyperpolarization. The graft is first innervated by host fibers and later projects to the host tissue. When stimulated, postsynaptic hyperpolarized responses are recorded in hippocampal neurons. In the freely moving rat, raphe grafts can restore sleep-wakefulness variations in an evoked population response of the hippocampus to afferent stimulation, which is eliminated by depletion of serotonin. These studies illustrate that grafted serotonin neurons develop functional relations with a host brain.

Transplants of cholinergic septal explants reinnervate adult rodent hippocampus

Embryonic septal-basal forebrain tissue was grown in expiant culture for 1, 4 or 5 days prior to transplantation to the hippocampus ot adult rats denervated of its septal input by a fomix/fimbria transaction. The expiant transplants were compared with transplants of septal cell suspensions. Analysis of fiber ingrowth by acetylcholinesterase (AChE) histochemistry at various timepoints post-transplantation shows little difference in the developmental time course or extent of cholinergic axon ingrowth into the host hippocampus between the suspension transplants and the expiant transplants. Septal expiants in culture for 5 days were as effective as those in culture for one day prior to transplantation in providing cholinergic reinnervation to the host hippocampus. Studies using antibodies to choline acetyltransferase (ChAT) and the fluorescent retrograde tracer, fast blue, confirmed that the AChE-positive cells in the transplants were cholinergic and that the innervation to the host hippocampus came from the transplanted cholinergic neurons. Thus the results demonstrate that embryonic cholinergic septal neurons can be maintained in short-term culture prior to transplantation without adversely affecting their ability to innervate an appropriate CNS target in vivo.

Restoration of rhythmic slow activity (θ) in the subcortically denervated hippocampus by fetal CNS transplants

Solid pieces of the fetal septal region (SG) or hippocampus (HPC) were implanted in a cavity formed by aspiration of the fimbriafornix (FF) and the overlying cingulate cortex on one side in adult rats. In other lesioned animals cell suspensions obtained from the fetal septal area (SS) or the locus coeruleus region of the brainstem (LC) were injected stereotaxically into the deafferented host hippocampus. Six to 9 months after transplantation the animals had chronic recording electrodes implanted into both hippocampi. EEG and unit activity were monitored during running in a wheel, drinking and immobility. Unilateral fimbria-fornix lesions abolished rhythmic slow activity (RSA or theta, θ) in the ipsilateral hippocampus and no recovery was seen up to 9 months later in either the control FF-lesioned animals or in the rats with LC suspension grafts. Recovery of RSA, however,was observed in all animals with solid septal grafts and in some rats with solid HPC grafts. Similar to normal rats, RSA was present only during running and absent during drinking and sitting still. Coherence measurements of RSA between the transplanted and intact hemispheres resulted in high values (0.70–0.95). Concurrent with RSA, interneurons and granule cells in the host hippocampus fired rhythmically at RSA frequency (6–9 Hz). The depth profile and the antero-posterior distribution of the power of RSA correlated with the amount and distribution of the graft-induced acetylcholinesterase-positive reinnervation of the host hippocampus. In contrast to the animals with solid septal grafts, placed within the FF lesion cavity, the rats with intrahippocampal septal suspension grafts displayed only short duration bursts of RSA, and mainly during immobility. Based on these findings it is suggested that at least a proportion of the RSA ‘pacemaker’ cells of the host septum survives the transection of the fimbria-fornix fibers and that a graft of fetal septal or hippocampal tissue implanted into the lesion cavity may be capable of relaying this pacemaker activity to the host hippocampus. This effect may be due to the ability of the grafted tissue to promote the regeneration of new, direct or indirect, septo-hippocampal connections across the lesion cavity.

Behavioral dependence of the electrical activity of intracerebrally transplanted fetal hippocampus

Adult rats with unilateral aspirative lesions of the fimbria-fornix and the overlying congulate cortex received implants of 17-day-old fetal hippocampal tissue in the lesion cavity, placed to form a tissue bridge across the cavity. From 6 to 8 months later they were equipped with chronic recording and stimulating electrodes in the transplant and the host brain. The dominant electrical pattern of the graft consisted of irregularly occurring sharp waves (SPW) or EEG spikes of 30–150 ms duration and concurrent synchronous neuronal bursts of large cell populations. SPWs occurred during all behaviors but their frequency was significantly lower during running than during drinking. Population bursts of neurons in the graft could be evoked by stimulating either the ipsilateral host hippocampus or the perforant path. Both complex spike cells and single spike cells could be recorded from the graft. The discharge frequency of single spike cells was considerably higher during running than drinking or immobility. Some of the single spike cells in the graft fired rhythmically at 6–9 Hz during running and walking, and phase-locked with the rhythmic slow EEG activity (theta, θ-EEG) recorded from the intact host hippocampus. Occasionally field θ-EEG was also present in the graft. These findings suggest that at least a portion of the graft neurons had come under the control of the host brain, and by way of the newly established host-graft connections the activity of some graft neurons was regulated in a near-normal manner.

Chapter 28 A functional analysis of connections between grafted septal neurons and host hippocampus

Publisher Summary This chapter discusses the efficacy of using neural grafting to restore physiological and behavioral functions following damage to the cholinergic septohippocampal system. It presents a study in which embryonic septal regions were injected into the hippocampi of fimbria–fornix (FF) transected rats. The grafts developed an extensive innervation of the host hippocampus and, when stimulated, produced a slow depolarizing response that was blocked by atropine and facilitated by physostigmine. There is evidence that the graft restored hippocampal theta rhythm, which had been eliminated by FF transactions or septal lesions. However, unlike unlesioned controls, theta in grafted animals is not correlated with movement. Performance in a water maze, which was markedly impaired by fornix transection, showed no improvement 6 months after transplantation, but was restored in septal lesioned, septal grafted rats. The results indicate that grafting may provide a useful tool for studying the action of neurotransmitter systems and their involvement in higher brain functions.

Transplantation of septal neurons maintained in long-term culture

Septal cells were maintained in culture for varying lengths of time and then transplanted to adult hippocampi from which the native septal input had been removed. Cells grafted after 1 week in culture survived transplantation to the brain and innervated the host hippocampus in a pattern comparable to the native cholinergic projection. Cells grafted after 2 weeks in culture did not survive in the brain, but did survive re-plating. In addition, the viability of cells transplanted from culture paralleled the viability of brain tissue taken from donors of equivalent ages, suggesting that changes which occur in culture and affect the viability of transplanted neurons may also occur during normal development.

Ultrastructural characterization of identified cholinergic neurons transplanted to the hippocampal formation of the rat.

The ultrastructural features of cholinergic neurons transplanted to the rat hippocampal formation were studied by using a monoclonal antibody to choline acetyltransferase (ChAT). Septal cell suspensions were prepared from E-18 rat embryos and injected into the hippocampus of host rats that had been previously subjected to a bilateral transection of the fimbria-fornix. Rats with fimbria-fornix lesions alone and unoperated rats served as controls and were examined to characterize the native hippocampal cholinergic system. Both unoperated controls and rats with fimbria-fornix lesions showed a sparse population of intrinsic ChAT-immunoreactive neurons that were most numerous in the subgranular zone, the hilus fascia dentata, and near the hippocampal fissure. ChAT-positive terminals from controls formed synapses on dendritic structures that were primarily symmetrical. ChAT-positive dendrites in controls received synaptic input from nonimmunoreactive axon terminals. In rats with septal transplants, ChAT-immunoreactive transplant neurons were found that were either bipolar or multipolar. Axons of transplanted neurons were unmyelinated and arose either from the cell body or a primary dendritic process where they gave off numerous collaterals. Terminals from transplant neurons formed synapses with many nonimmunoreactive neurons. In transplant animals, two main targets of ChAT-immunoreactive terminals were identified: The great majority of synapses were symmetrical junctions with dendritic spines and shafts. A number of terminals were found that appeared to be juxtaposed to nonimmunoreactive axon terminals, possibly forming symmetrical axo-axonic connections. In contrast, such axo-axonic contacts were not observed in the controls. It is concluded that transplanted cholinergic neurons may reinnervate the host hippocampus; however, this reinnervation is different from what is seen in the intact hippocampal formation.