Effects of cocaine-induced seizures during pregnancy in the rabbit.

The effects of chronic administration of cocaine to pregnant rabbits on maternal seizures and on pregnancy outcome were studied. Cocaine (2, 3 or 4 mg/kg/injection) or saline was administered, I.V., twice daily, from gestation Day 8 (G8) to G29. There were no significant differences in maternal weight gain or pregnancy outcome between saline control animals and animals given a cocaine dose of 2, 3 or 4 mg/kg/injection. Generalized tonic-clonic seizures (GTCSs) were occasionally elicited by the highest dose (4 mg/kg). There were significant individual differences in vulnerability to cocaine-elicited GTCSs in animals given 4 mg/kg/injection. Of this group, 18% were classified as having high vulnerability to seizures, and they experienced a range from 3 to 27 GTCSs. Postnatal mortality of their offspring was significantly increased. The incidence and temporal patterns of GTCSs elicited by chronic, I.V. cocaine in rabbits, at the doses used, are similar to those reported in human cocaine use. These GTCSs may involve different mechanisms from seizures elicited in other animal studies, in which high doses of cocaine are administered I.P. or S.C. Nevertheless, in our animal model, the GTCSs elicited by prenatal cocaine exposure had no detectable effects on pregnancy outcome (except in the highly vulnerable subgroup).

New evidence for neurotransmitter influences on brain development.

The early appearance of monoamine systems in the developing mammalian CNS suggests that they play a role in neural development. We review data from two model systems that provide compelling new evidence of this role. In one model system-in utero exposure to cocaine-specific and robust alterations are seen in dopamine-rich areas of the cerebral cortex, such as the anterior cingulate cortex: D1 receptor-G protein coupling is greatly reduced, the GABAergic system is altered and pyramidal dendrites undergo excessive growth. In a second model system-a transgenic mouse line in which the gene that encodes monoamine oxidase A (MAOA) is disrupted, resulting in excessively high 5-HT levels-barrels fail to form in the developing somatosensory cortex. Both models reveal the effects of very early manipulation of monoamines on forebrain development, and the long-term anomalies that persist into adulthood.

Cocaine administration in pregnant rabbits alters cortical structure and function in their progeny in the absence of maternal seizures.

Previous studies have reported that cocaine exposure in utero results in structural and functional alterations in the development of the anterior cingulate cortex (ACC). In the present study, the effects of maternal cocaine dosage and of cocaine-elicited maternal seizures on the progeny were studied. The incidence of maternal generalized tonic clonic seizures (GTCSs) elicited by cocaine was recorded. No GTCSs were elicited in pregnant rabbits by doses of 2 or 3 mg/kg of cocaine, but GTCSs were sometimes elicited by the highest dose (4 mg/kg per injection). We analyzed the offspring of cocaine-exposed and control animals using three assays of ACC development: (i) the structure of apical dendrites of pyramidal neurons, (ii) the distribution of a calcium binding protein (parvalbumin) in the dendrites of GABAergic neurons, and (iii) coupling of D1-like receptors and their G proteins. In all progeny of rabbits exposed to 3 or 4 mg/kg of cocaine during pregnancy, there was a significant change in the structure of apical dendrites, a significant increase in the number of dendrites of GABAergic neurons which were parvalbumin immunoreactive, and a significant reduction in D1/G protein coupling. In assays of apical dendrites, the effects on offspring of rabbits given 2 mg/kg cocaine were as pronounced as in offspring of rabbits given 3 or 4 mg/kg, but the effects on parvalbumin immunoreactivity and D1/G protein coupling were reduced at this low dose. Thus, previous findings of ACC developmental abnormalities in offspring of rabbits given a dose of 4 mg/kg were replicated, the effects were shown to be dose-related and to be independent of maternal seizures. A mechanism by which dysfunction of the D1 receptor system could mediate cocaine-associated changes in all three parameters of ACC structure and function is discussed.

Altered neuronal distribution of parvalbumin in anterior cingulate cortex of rabbits exposed in utero to cocaine.

Previous studies of rabbits exposed in utero to cocaine have revealed an increase in the number of neurons which are GABA immunoreactive in the anterior cingulate cortex (ACC), suggesting a cocaine-elicited modification in the balance of excitatory and inhibitory interactions. Of the major calcium binding proteins expressed by different subgroups of GABAergic neurons, parvalbumin has been observed in conditions involving excess excitation, and may serve to protect neurons from excitotoxicity. In the present study, we used immunocytochemistry to compare the effects of prenatal cocaine exposure on the postnatal development of parvalbumin immunoreactivity in interneurons of the visual cortex (VC) and ACC. We determined the number and laminar distribution of parvalbumin immunoreactive neurons, and we also assessed the distribution of parvalbumin immunoreactivity within primary, secondary and tertiary dendrites of neurons in these two cortical areas. In both ACC and VC, parvalbumin immunoreactive neurons were first observed around postnatal day 10 (P10) and their number increased rapidly from P10 to P20. At all ages studied (P10 to P60) there was no difference between cocaine-exposed and saline control animals in the number or laminar distribution of parvalbumin immunoreactive neurons in either cortical area. However, the distribution of parvalbumin immunoreactivity within dendrites revealed a significant difference between cocaine-exposed and saline control animals in ACC but not in VC. In ACC, at all ages studied, there was an increase in the number of primary, secondary and tertiary dendrites which were parvalbumin immunoreactive in cocaine-exposed animals compared with saline controls. This difference was most striking in secondary dendrites, and in laminae V and VI. The effect was observed at doses of 4 and 3 mg/kg per injection but not at 2 mg/kg per injection. In contrast to ACC, in VC there was no difference in the number of immunoreactive dendrites in cocaine-exposed animals compared with saline controls. These observations are consistent with the hypothesis that the ACC of rabbits exposed in utero to cocaine is characterized by altered excitatory/inhibitory interactions. ACC receives a dense dopaminergic input, but VC receives minimal dopaminergic innervation. Mechanisms by which the action of cocaine on the developing dopaminergic system may modify the balance of excitation and inhibition in ACC are discussed.

Altered expression of microtubule-associated proteins in cat trochlear motoneurons after peripheral and central lesions of the trochlear nerve.

Neurons lesioned in the peripheral nervous system (PNS) generally regenerate and survive, while neurons lesioned in the central nervous system (CNS) do not regenerate and often die. Investigators have traditionally compared the neuronal responses to PNS and CNS lesions in two separate populations of neurons. In this study, we compared the effects of PNS and CNS lesions on the expression of cytoskeletal proteins in a single neuronal population, the trochlear motoneurons of the cat. The trochlear nerve was lesioned either unilaterally in the PNS or bilaterally in the CNS (within the anterior medullary velum), and animals were allowed to survive 1, 2, or 4 weeks. Brain sections were reacted immunocytochemically using antibodies against microtubule -associated protein-2 (MAP-2) and a phosphorylated isoform of MAP1B, termed MAP1B-P. MAP-2 immunoreactivity (IR) was significantly decreased in the CNS-lesioned trochlear nucleus, compared to the lesioned and the unlesioned trochlear nucleus of PNS-lesioned animals. MAP1B-P IR was significantly increased in PNS- and CNS- lesioned trochlear axons, compared to axons in the unlesioned trochlear nerve of PNS-lesioned animals, and appeared in a small percentage of PNS- and CNS-lesioned cell bodies. These results support the growing body of evidence that MPA-2 can serve as a marker for cells that will eventually die following neuronal insult. The increased immunostaining of MAP1B-P in lesioned axons and its appearance in lesioned cell bodies are characteristic of the immature CNS and may reflect an initial recapitulation of early development, when the levels of this protein are high.

Normal development of tyrosine hydroxylase and serotonin immunoreactive fibers innervating anterior cingulate cortex and visual cortex in rabbits exposed prenatally to cocaine.

Anterior cingulate cortex develops abnormally in rabbits exposed to cocaine in utero but visual cortex is normal. The interactions of cocaine with the dopamine, norepinephrine and serotonin systems makes each a potential candidate for influencing these developmental effects. Here, we report no differences in the distribution and density of serotonin and tyrosine hydroxylase-immunoreactive fibers in the anterior cingulate cortex in postnatal rabbits exposed prenatally to cocaine or saline. Because the pattern and extent of cortical innervation by these systems appears normal, the data suggest that the effects of prenatal exposure to cocaine may involve changes in monoamine signaling distinct from morphological reorganization of the cortical afferents.

Properties of motor units after self-reinnervation of the cat superior oblique muscle.

1. The mechanical properties of motor units of the cat superior oblique muscle and axonal conduction velocities of trochlear motoneurons have been studied at several postoperative times after intracranial axotomy of the trochlear nerve. 2. Whole muscle twitch forces were generally within the normal range by approximately 4 mo postoperative, indicating that reinnervation is complete at this time. 3. Among animals studied 3.5-4.5 months after trochlear axotomy, average motor-unit tetanic forces were increased by a factor of approximately 2.5 compared with units studied in normal superior oblique muscle. Average motor-unit tetanic forces in animals studied 14.5-23 mo after axotomy were also increased relative to normal, but the difference was not significant. Among all reinnervated motor units, there was a tendency for increased twitch time-to-peak relative to control. Reinnervated motor-unit fatigue properties were similar to normal. 4. Average trochlear motoneuron conduction velocities for animals at all postoperative intervals remained significantly lower than the average conduction velocities from three of four normal animals. 5. Counts of Nissl-stained cell bodies in axotomized and control, contralateral trochlear nuclei showed that some cell loss had occurred, averaging approximately 17% 3.5-4.5 mo postoperative and 24% 14.5-23 mo postoperative. Associated with this loss was an increase (10%) of axotomized motoneuron soma cross-sectional area. 6. Muscle fiber cross-sectional areas (CSA) were measured in reinnervated superior oblique muscles and compared with CSAs from contralateral, control muscles. Average CSA was significantly decreased in all reinnervated muscles, with the relative decreases ranging from approximately 10 to 28%. 7. The results are discussed in terms of factors that determine motor-unit force; muscle fiber CSA, specific force, and innervation ratio. We conclude that the increases of average motor-unit force in short-term reinnervated superior oblique muscles are most likely related to polyneuronal innervation of muscle fibers and that the return of these forces to normal levels in long-term muscles is related to synapse elimination. Our results are compared with those of other self-reinnervation studies, and the potential role played by the time muscle remains denervated in determining the persistence of polyneuronal innervation after reinnervation is considered.

Intrauterine cocaine exposure of rabbits: persistent elevation of GABA-immunoreactive neurons in anterior cingulate cortex but not visual cortex.

The effects of prenatal cocaine exposure on the development of the rabbit cerebral cortex were studied. Two cortical areas were compared: primary visual cortex (VC) and anterior cingulate cortex (ACC). ACC was selected because behavioral deficits observed in cocaine-exposed infants suggest the involvement of ACC. In addition, ACC receives dense dopaminergic innervation and cocaine's action in inhibiting the re-uptake of dopamine is believed to underly the rewarding properties of cocaine. VC was selected as a control area because there is no evidence of behavioral deficits associated with visual perception in cocaine-exposed infants, and because VC receives minimal dopaminergic innervation. Two aspects of cortical development were studied: (i) cortical morphology, growth and cytoarchitectonic organization; and (ii) the development of the GABAergic neurotransmitter system. Measures of postnatal cortical growth, including cortical lamination, cell number and soma size, were compared in cocaine-exposed or control (saline) rabbits aged P5-P60. There was no difference between cocaine and saline animals in any of these parameters, and cortical cytoarchitecture appeared normal. However, despite the absence of major abnormalities in cortical development, we found that the number of GABA-immunoreactive neurons in cocaine-exposed animals was significantly higher than normal in ACC. This effect was highly consistent, was present in all laminae and at all ages studied, and persisted into maturity (P60). In contrast, in VC, the number of GABA-immunoreactive neurons in cocaine-exposed animals did not differ from normal. We suggest that increased GABA immunoreactivity may reflect a compensatory response to excessive excitatory input to ACC. A change in the balance of excitation and inhibition in ACC, reflecting 'noisy' or dysfunctional intracortical circuitry, may underly the emotional lability and attentional deficits characteristically described in infants exposed in utero to cocaine.

Regenerative axonal sprouting in the cat trochlear nerve.

Following peripheral trochlear nerve axotomy in the cat, the normal number of myelinated axons is restored despite significant motor neuron death, suggesting regulation of the number of myelinated axons in the regenerated nerve. In this study we used light and electron microscopy to examine the production and maintenance of axonal sprouts at different locations in the nerve and at different postoperative intervals. Despite proliferative sprouting and an overproduction of nonmyelinated axons in the regenerating trochlear nerve, the number of myelinated axons was strictly regulated. Only approximately 1,000 regenerated axons were eventually remyelinated, but many nonmyelinated axons were still present 6-8 months postaxotomy. Regenerated axons were remyelinated in a proximal-to-distal direction between 3 and 4 weeks postaxotomy. We also examined the maturation of regenerated myelinated axons by measuring axon diameter and myelin index (an expression of myelin thickness). Mean myelinated axon diameter remained significantly below normal in long-term regenerated nerves. Mean myelin index was not different from normal at 4 weeks postaxotomy but was significantly decreased at long postoperative intervals, reflecting a slightly thicker myelin sheath relative to the axon diameter. This relative increase in mean myelin thickness could serve to restore normal conduction velocity despite the decrease in mean axon diameter. We suggest that the regulation of the number of myelinated axons at the normal number despite cell death and the increase in mean myelin thickness may both be compensatory mechanisms that function to restore preoperative conditions and maximize functional recovery.

The rabbit as a model for studies of cocaine exposure in utero.

The rabbit has been used to model the potential effects of in utero exposure to cocaine on fetal and postnatal development. Special advantages of this animal model include the fact that cocaine can be easily administered intravenously, thus mimicking crack cocaine use by pregnant women. Results indicate that at the dosage used (8 mg/kg of body weight, given intravenously daily) gross teratologic defects do not develop. Cocaine-exposed pregnant does not differ from controls in weight gain or in the number of live kits delivered. Cocaine-exposed kits do not differ from controls in survival or in postnatal weight gain. The importance of this rabbit model is that offspring that have been exposed to these doses of cocaine in utero have a variety of abnormalities in structure and function of the central nervous system in the absence of any major teratologic defects.

Axotomy-induced changes in cytochrome oxidase activity in the cat trochlear nucleus.

Following a unilateral section of the trochlear nerve, the effects of axotomy on cytochrome oxidase levels in the trochlear nucleus were studied. Cytochrome oxidase levels in the axotomized nucleus were significantly lower than in the control nucleus. The maximal decrease was observed at 2 weeks. Following partial restoration during weeks 3 and 4, cytochrome oxidase levels stabilized at levels only slightly below normal. Since a significant number of trochlear motoneurons die following axotomy, the restoration of cytochrome oxidase levels close to normal suggests that the surviving neurons may compensate for an increased load with a permanent increase in oxidative metabolism.

The effects of epileptic cortical activity on the development of callosal projections.

The effect of epileptic neural activity on the postnatal development of the corpus callosum was studied. Epileptiform activity was induced in the visual cortex of postnatal rabbits by continuous infusion of penicillin. Callosal projections of the occipital cortex were studied in rabbits aged at least 4 weeks. In these penicillin-exposed rabbits, the visual callosal projections extended through most of area 17 in a projection pattern characteristic of neonatal rabbits, rather than being restricted to a narrow callosal zone at the lateral border of area 17, as they usually are by this age. The results indicate that epileptic cortical activity stabilizes immature callosal projections which are normally eliminated during development. The maintenance of such immature and non-specific projections in the mature CNS may interfere with normal cortical functions and could underlie the cognitive deficits which have been observed following childhood epilepsy.

Calcitonin gene-related peptide increases following axotomy of trochlear motoneurons.

Neuropeptides have long been considered to act as neurotransmitters or neuromodulators, but they may also contribute to a variety of regulatory and trophic neuronal functions. In the present study, we determined the effects of axotomy on the levels of the neuropeptide calcitonin gene-related peptide (CGRP) in trochlear motoneurons (TMNs) of adult cats. The number of neurons with detectable CGRP immunoreactivity, and the intensity of their CGRP immunoreactivity, increased dramatically 1 week postaxotomy, gradually returned toward normal levels, but remained significantly higher than normal 12 weeks postaxotomy--a time when axonal regeneration and functional reinnervation of the superior oblique muscle should be complete. Our observation that CGRP levels in TMNs increase after axotomy suggests a role for this peptide in the response of motoneurons to injury and in regeneration. In addition, since many axotomized TMNs die, we suggest that the maintenance of high CGRP levels even after regeneration is complete may reflect an increased load on those TMNs that survive.

Visual callosal projections in the adult ferret.

The laminar and tangential organization of visual callosal projections of areas 17 and 18 were investigated in the adult ferret, using histochemical methods to visualize axonally transported horseradish peroxidase (HRP). Normal adult ferrets were given injections of HRP throughout one visual cortex or had gelfoam soaked in HRP applied to the transected corpus callosum. The ferret callosal cell distribution has a greater tangential extent in area 18 than in area 17. In addition, the radial organization of callosal cells in areas 17 and 18 differs: three times as many infragranular cells are present in area 18 than in area 17, although the number of supragranular cells is similar for both areas 17 and 18. Since the projections of alpha retinal ganglion cells are reported to be exclusively contralateral in the ferret (Vitek et al., 1985), callosal projections may make a major contribution to the binocularity of neurons in area 18.

Transcallosally evoked responses in the visual cortex of normal and monocularly enucleated rabbits.

In visual cortex of normal adult rabbits, callosal projections are restricted to a 2 mm wide band at the area 17/18 border. In adult rabbits which are monocularly enucleated (ME) on the day of birth, the callosal zone extends 4 mm into the medial region of area 17 in the cortex ipsilateral to the remaining eye. In this study, the function of these anomalous callosal projections in ME rabbits was investigated using electrophysiological techniques. A microelectrode was placed in the visual cortex ipsilateral to the enucleated eye at the 17/18 border, bipolar stimulating electrodes were placed in a homotopic location in the contralateral cortex, and averaged evoked responses (AERs) to stimulation were recorded. The stimulating electrodes were then moved mediolaterally in 1 mm steps, and the AERs were recorded for each location of the stimulating electrodes. In the normal rabbit, a maximal short latency evoked response was recorded when the stimulating electrodes were at a location homotopic to the recording electrode. When the stimulating electrodes were moved a distance of 1 mm or more from this optimal position, this short latency response was either absent or dramatically decreased in amplitude, reflecting the precise topographic pattern of the normal callosal projection. In contrast, in ME rabbits, a consistent response was evoked at the 17/18 border when the stimulating electrodes were moved as much as 3 mm medial to the homotopic position. Since antidromically activated responses and both pre- and postsynaptic orthodromically activated responses contribute to the AER, recordings were also made from single cells in some animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of monocular enucleation on visual topography in area 17 in the rabbit.

The effects of neonatal monocular enucleation on the topographic representation of the ipsilateral visual field in the visual cortex of the rabbit were investigated, using electrophysiological recordings of multi-unit activity in area 17. Topography of receptive fields was determined in normal adult rabbits, adult rabbits monocularly enucleated on the day of birth and adult rabbits monocularly enucleated as adults. In normal rabbits and in adult enucleates, the projection from the ipsilateral eye is represented by a strip of cortex extending approximately 2 mm from the 17/18 border. In neonatal enucleates, the width of the area of cortex in which the projection from the ipsilateral eye is represented was approximately twice as large as normal. Visual topography was normal in the superior-inferior axis but was distorted in the nasotemporal axis. Our data suggest that the abnormal topography observed in the visual cortex of neonatally enucleated rabbits may play a major role in shaping the abnormal visual callosal projections observed in these animals. In addition, our data indicate that, following neonatal monocular enucleation, developmental abnormalities in the topography of geniculocortical projections can occur independently of any alteration in the retinogeniculate projection patterns.

Organization of callosal connections in the visual cortex of the rabbit following neonatal enucleation, dark rearing, and strobe rearing.

The organization of visual callosal projections was studied in (1) normal adult rabbits; (2) adult rabbits which had undergone monocular enucleation (ME) or binocular enucleation (BE) at birth; and (3) adult rabbits which had been deprived of normal visual experience during development by dark rearing (DR) or strobe rearing (SR). Previously published observations (Murphy and Grigonis, Behav Brain Res 30:151, 1988) on callosal organization in adult rabbits in which retinal ganglion cell activity was eliminated during development by intraocular tetrodotoxin (TTX) injections, are also summarized for comparison with these data. The tangential extent of the callosal cell zone was significantly larger than normal in DR, TTX, and ME rabbits, was unchanged in BE rabbits, and was significantly reduced in SR rabbits. An analysis of the laminar distribution of the callosal cells revealed a significant increase in the percentage of callosal cells in lamina IV in ME, DR, and TTX animals. Measurements of density of callosal cells showed a significant increase in the density of the callosal projection in ME and SR rabbits and a decrease in density in BE rabbits compared with normal. The data suggest that the mechanisms involved in the development of the tangential and laminar organization of the callosal cell zone are different. In addition, the data suggest that the mechanisms involved in the maintenance of callosal projections are different from the mechanisms involved in the elimination of callosal projections during development. The effects of these developmental manipulations on callosal organization in other mammals are reviewed and compared with the effects in rabbits. The data suggest that species differences in the degree of maturity of the visual system at birth and in the extent of callosal development at the time of eye opening, may underlie species differences in the effects of these manipulations on the organization of visual callosal projections during development.

Regeneration and soma size changes following axotomy of the trochlear nerve.

The effects of CNS and PNS axotomy of the IVth nerve on cell death, soma size, axon size, and axon number were investigated. In adult cats, the IVth nerve was axotomised by using four surgical paradigms: (1) peripheral IVth nerve crush, (2) peripheral IVth nerve cut, (3) peripheral IVth nerve resection, and (4) a CNS IVth nerve cut in the velum. The extent of cell death resulting from each surgical paradigm was determined. Following axotomy distal to the decussation of the IVth nerves, cell death was least after nerve crush, intermediate after nerve cut, and maximal after resection of 5-7 mm of the nerve. Following axotomy at the decussation--a CNS lesion--most cells died but some successful regeneration was observed. Soma size measurements following a short-term survival (3 days to 4 weeks) before the regenerating axons reached their target muscle revealed that somas of axotomised cells underwent hypotrophy within 1 week of axotomy and then gradually increased in size. They re-attained normal size by 4 weeks postoperative when regenerating axons first reach their target. Following a long-term survival (greater than 2 months), somas were significantly hypertrophied, and the degree of hypertrophy was inversely related to the extent of cell survival up to a limit of 40% soma size increase. Counts and measurements of axons revealed that mean axon diameter of regenerated axons was much smaller than normal 3 months after axotomy, increased during the third to sixth postoperative months, but then showed no subsequent increase and remained below normal. In animals with cell death varying from 10% to 70%, the number of axons in the nerve was maintained constant at approximately 1,000. These data indicate that there is a mechanism for the production and maintenance of the appropriate number of regenerative axonal branches following axotomy. In animals in which cell death exceeded 70%, the number of axons was controlled by a maximum ratio of 3 to 4 axon branches per surviving cell. The results suggest that axon number is strongly influenced by the target muscle and that hypertrophy of regenerated cells is related to the number of axonal sprouts each cell has to produce and support in order to re-establish the preoperative number of axons in the regenerated trochlear nerve.